ABSTRACTS New biology from ancient enzymes 2 A prebiotic RNA … · 2013-05-30 · the last common ancestor and hence its study can provide insight to early events in the origin of

ABSTRACTS

1 New biology from ancient enzymes

Paul Schimmel*

The Scripps Laboratories for tRNA Synthetase Research, TheScripps Research Institute, 10650 North Torrey Pines Road, LaJolla, CA 92037, USA*Email: [email protected], Phone: (858) 784-8970,Fax: (858) 784-8990

The aminoacyl tRNA synthetases arose early in evolutionto establish the genetic code during translation. Longthought of as cytoplasmic enzymes with a single definedfunction, new studies have demonstrated their roles innuclear and extracellular signaling pathways, where theyregulate angiogenesis, inflammation, mTor signaling,tumorigenesis, and more. These novel functions are typi-cally associated with novel domains added to highereukaryote tRNA synthetases, and specific resected formsthat are generated by alternative splicing and natural prote-olysis. The tRNA synthetases are now seen as central“nodes” that use their novel domains to connect with mul-tiple-cell signaling pathways through a variety of interact-ing partners. These partners include nuclear proteins,extracellular receptors, cytoplasmic proteins, and cellularRNAs. This new biology from tRNA synthetases is anendless frontier.

ReferencesGou, M., & Schimmel, P. (2013). Essential nontranslational

functions of tRNA synthetases. Nature Chemical Biology,9, 145–153.

Yao, P., & Fox, P. L. (2013). Aminoacyl-tRNA synthetases inmedicine and disease. EMBO Molecular Medicine. epubdate: 2013/02/22.

2 A prebiotic RNA apparatus functionswithin the contemporary ribosome

Miri Krupkin, Ella Zimmerman, Anat Bashan andAda Yonath*

Department of Structural Biology, Weizmann Institute, Rehovot,Israel*Email: [email protected], Phone: +972-8-9343028

Ribosomes, the universal cellular machines, possessspectacular architecture accompanied by inherent mobil-ity, allowing for their smooth performance as polymer-ases that translate the genetic code into proteins. The sitefor peptide bond formation is located within a universalinternal semi-symmetrical region, which was identifiedwithin all contemporary ribosomes. The high conserva-tion of this region implies its existence irrespective ofenvironmental conditions and indicates that it may repre-sent an ancient RNA molecular apparatus. Hence, wenamed it the “proto-ribosome”. This prebiotic pocket-likeRNA entity is suggested to be capable to accommodatesubstrates whose stereochemistry enables the creation ofchemical bonds. It could have evolved from an earliercatalytic RNA entity that we named the “pre-proto-ribo-some”, presumed to be a molecular machine capable ofperforming various essential tasks in the RNA world,which was snatched by the amino acid invaders for pro-ducing proteins.

ReferenceKrupkin, M., et al. (2011). A vestige of a prebiotic

bonding machine is functioning within the contemporary ribo-some. Philosophical Transactions of the Royal Society B: Bio-logical Science, 366, 2972–2978. doi: 10.1098/rstb.2011.0146.

Journal of Biomolecular Structure and Dynamics, 2013Vol. 31, No. S1, 1–139, http://dx.doi.org/10.1080/07391102.2013.786317

Copyright � 2013 Taylor & Francis

3 Origins and evolution of the translationmachinery

George E. Foxa*, Quyen Trana, Mario Rivasb andVictor Stepanova

Department Biology & Biochemistry, University of Houston,Houston, TX 77204-5001; UNAM, Mexico City, Mexico*Email: [email protected], Phone: (713)-743-8363,Fax: (713) 743-8351

The protein synthesis machinery largely evolved prior tothe last common ancestor and hence its study can provideinsight to early events in the origin of life, including thetransition from the hypothetical RNA world to living sys-tems as we know them. By utilizing information from pri-mary sequences, atomic resolution structures, andfunctional properties of the various components, it is pos-sible to identify timing relationships (Hsiao et al., 2009;Fox, 2010). Taken together, these timing events are usedto develop a preliminary time line for major evolutionaryevents leading to the modern protein synthesis machinery.It has been argued that a key initial event was the hybrid-ization of two or more RNAs that created the peptidyltransferase center, (PTC), of the ribosome (Agmon et al.2005). The PTC, left side of figure, contains a characteris-tic cavity/pore that serves as the entrance to the exit tunneland is thought to be essential to the catalysis (Fox et al.,

2012). This cavity is distinct from typical RNA pores(right side of figure) in that the nitrogenous bases facetowards the lumen of the pore and thus are available forhydrogen bonding interactions. In typical RNA pores, thebases carefully avoid the lumen region. In support ofAgmon et al. 2005), it is argued that this key differencereflects the fact the pore was created by an early hybridiza-tion event rather than normal RNA folding.

This work was supported by the NASA Center forRibosome Evolution and Adaptation at the GeorgiaInstitute of Technology (NNA09DA78A), A NASAEarth and Space Science Fellowship (10-Planet10R-0025) to QT, and a NASA Planetary Biology Intern-ship award to MR.

ReferencesAgmon, I., Bashan, A., Zarivach, R., & Yonath, A. (2005). Sym-

metry at the active site of the ribosome: Structural and func-tional implications. Biology Chemistry, 386, 833–844.

Fox, G. E., Tran, Q., & Yonath, A. (2012). An exit cavity wascrucial to the polymerase activity of the early ribosome.Astrobiology, 12, 57–60.

Fox, G. E. (2010). Origin and evolution of the ribosome. ColdSpring Harbor Perspectives in Biology, 2, a003483.

Hsiao, C., Mohan, S., Kalahar, B. K., & Williams, L. D.(2009). Peeling the onion: Ribosomes are ancient molecularfossils. Molecular Biology Evolution, 26, 2415–2425.

4 Towards non-enzymatic RNA replication

Jack W. Szostak*

Department of Molecular Biology, Center for Computationaland Integrative Biology, Massachusetts General Hospital, 185Cambridge St, Boston, MA 02114, USA*Email: [email protected],Phone: (617) 726-5981, Fax: (617) 726-6893

The direct path from prebiotic chemistry to the RNAWorld requires a plausible route for the synthesis of

activated ribonucleotides and RNA templates, along witha means for the complete replication of potentially usefulRNA sequences. However, many apparent roadblocksmake non-enzymatic RNA replication look quitedifficult, if not impossible. These problems include theslow rate, low accuracy and poor regioselectivity of non-enzymatic template copying, the hydrolysis of activatedmonomers and absence of good re-activation chemistry,the difficulty of strand separation after template copying,the rapidity of strand reannealing, the absence of primersin any realistic replication scenario, and the apparentincompatibly of RNA copying chemistry (which requires

2 Journal of Biomolecular Structure and Dynamics Vol. 31, Supplement, 2013

a high Mg2+ concentration) with fatty acid-based proto-cell membranes, which are destroyed by low Mg2+

concentrations. I will discuss recent progress from mylaboratory on four of these issues. We have found thatfunctional RNAs such as aptamers and ribozymes cantolerate moderate levels of 2′–5′ linkages without greatloss of activity. It therefore appears that the presenceof 10–25% of such linkages in the products ofnon-enzymatic copying would not prevent the evolutionof functional RNAs. Furthermore, 2′–5′ linkages can behelpful, as they decrease the melting temperature ofRNA duplexes enough to allow strand separation tooccur under geophysically plausible conditions. Recently,we have found that small chemical changes to the nucle-obases can greatly increase the fidelity of non-enzymatictemplate copying, and we have found conditions thatrender RNA copying chemistry compatible with vesicleintegrity, thereby allowing RNA copying to occur insidefatty acid-based model protocell membranes. I will dis-cuss potential approaches to solving the remaining issuesthat stand in the way of complete RNA replication. If allof the problems with RNA replication can be overcome,it should be possible to construct functioning protocellsin the laboratory.

This research has been supported by NSF, NASA andHHMI.

ReferencesSzostak, J. W. (2012). The eightfold path to non-enzymatic

RNA replication. Journal of Systems Chemistry, 3, 2.Englehart, A. E,, Powner, M. W., & Szostak J. W. (in press).

Functional RNAs exhibit tolerance for non-heritable 2′–5′vs. 3′–5′ backbone heterogeneity. Nature Chemistry.

5 Efficient and high-fidelity copying of anRNA-like model prebiotic system

Shenglong Zhanga,b,c, Sergei M. Gryaznovd andJack W. Szostaka,b,c*aHoward Hughes Medical Institute, Massachusetts GeneralHospital, 185 Cambridge Street, Boston, Massachusetts 02114,USA; bHarvard Medical School, Massachusetts GeneralHospital, 185 Cambridge Street, Boston, Massachusetts 02114,USA; cDepartment of Molecular Biology and Center forComputational and Integrative Biology, Massachusetts GeneralHospital, 185 Cambridge Street, Boston, Massachusetts 02114,USA; dGeron Corporation, 230 Constitution Drive, MenloPark, CA 94025, USA*Email: [email protected],Phone: (617) 726-5980, Fax: (617) 643-3328

Nonenzymatic RNA replication would provide animportant bridge to the RNA world. However, the

demonstration of efficient and high-fidelity copyingchemistry remains a great experimental challenge. Itrequires an efficient mechanism that can lead to botha high rate of polymerization and a high degree offidelity in the copying chemistry. Previous experimentsconcerning nonenzymatic template-directed synthesis ofRNA with activated monomers have led to the copy-ing of short RNA templates, but these reactions aregenerally slow (taking days to weeks) and highlyerror-prone. Therefore, the ability to efficiently andaccurately copy arbitrary template sequences remainsfrustratingly out of reach. N3′-P5′-linked phosphorami-date DNA is a highly reactive model for self-replicat-ing genetic materials and has been used for studiesof nonenzymatic RNA self-replication. It is also anexcellent RNA mimic, due to its similar overallduplex structure, rigidity, and level of hydration (Ter-eshko, Gryaznov, & Egli, 1998). Our experimentsshow that the high reactivity imparted by the pres-ence of an amino nucleophile allows rapid and effi-cient copying of all four nucleobases on bothhomopolymeric and mixed templates. On the otherhand, G:T wobble pairing leads to a high error rate.We have, therefore, investigated the use of the modi-fied nucleobase, 2-thio T (Ts) (Sintim & Kool, 2006),to suppress formation of the G:T wobble base-pair.Our results illustrate that the 2-thio modification canboth increase polymerization rate and enhance fidelityin this self-replicating N3′-P5′-DNA system. Theseresults suggest that this simple nucleobase modifica-tion may have played a role in primordial RNA (orproto-RNA) replication. In addition to suppressing theG:T mismatch, an additional benefit gained from itsstronger base-pairing with A is that it also reducesA:C mismatch formation. Thus, simple modificationsof nucleobases might provide a means of suppressingmismatches to yield better fidelity. Taken together, ourresults show that a high rate of polymerization and ahigh degree of fidelity are not mutually exclusive, butcan be achieved simultaneously in nonenzymaticcopying of N3′-P5′-linked phosphoramidate DNA. Thestructural similarity of NP-DNA to RNA suggests thatthese results could be translated to an RNA-only sys-tem.

ReferencesSintim, H. O., & Kool, E. T. (2006). Enhanced base pairing

and replication efficiency of thiothymidines, expanded-sizevariants of thymidine. Journal of the American ChemicalSociety, 128, 396–397.

Tereshko, V., Gryaznov, S., & Egli, M. (1998). Consequencesof replacing the DNA 3′-oxygen by an amino group: High-resolution crystal structure of a fully modified N3’ → P5’phosphoramidate DNA dodecamer duplex. Journal of theAmerican Chemical Society, 120, 269–283.

Book of Abstracts. Albany 2013: The 18th Conversation 3

6 Crystal structure studies of RNAmolecules containing 2′–5′-linkages

Jia Sheng, Li Li, Aaron E. Engelhart andJack W. Szostak*

Department of Molecular Biology, Massachusetts GeneralHospital, Department of Genetics, Harvard Medical School,Howard Hughes Medical Institute, Boston, MA 02114, USA*Email: [email protected],Phone: (617) 726-5980, Fax: (617) 643-3328

RNA can play dual roles as a carrier of genetic infor-mation and as a catalyst of specific reactions, and itmay have been the first biopolymer to have emergedon the early earth. The non-enzymatic replication ofRNA was likely a key step in the evolution of simplecellular life from prebiotic chemistry. In the currentmodel of template-directed polymerization of activatedmonomers, the chemical copying of RNA always gener-ates a mixture of 3′–5′ and 2′–5′ backbone linkages dueto the similar nucleophilicity and orientation of the 2′and 3′ hydroxyl groups on the ribose. This lack of reg-iospecificity has been regarded as a central problem forthe evolution of functional RNAs, since the resultingbackbone heterogeneity was expected to disrupt theirfolding, molecular recognition and catalytic propertiesof functional RNAs such as ribozymes. However, arecent study from our lab has demonstrated that RNAswith a certain percentage of 2′–5′ linkages can stillretain RNA functions, for example, in a FMN-bindingaptamer and a hammerhead ribozyme system. Moreinterestingly, it has been known for a long time that2′–5′ linkages can reduce the melting temperature ofRNA duplexes, making it easier to separate the strands.Although the detailed mechanism is still not clear, con-sidering that strand separation is another unsolved bigproblem for non-enzymatic RNA replication, this fea-ture may actually afford a selective advantage toduplexes exhibiting backbone heterogeneity. In addition,previous studies have revealed that 2′–5′ linkages in aRNA duplex are more easily hydrolyzed compared tonormal 3′–5′ linkages. Thus, there is a selective advan-tage for the evolution of homogeneous RNA systemswith more accurate replication. Altogether, the coexis-tence of 2′–5′ and 3′–5′ linkages may be a central fea-ture that allowed RNA to play a central role in theoriginal stage of life. In this work, we will present sev-eral X-ray crystal structures of RNA duplexes and anaptamer that contain 2′–5′ linkages. These structureshelp us to understand how RNA can adjust its structureto accommodate the backbone heterogeneity.

7 Imperfect RNA synthesis via modelprebiotic reactions and consequences forfunctional RNAs

Aaron E. Engelhart, Matthew W. PownerJack W. Szostak*

Department of Molecular Biology, Center for Computationaland Integrative Biology, Massachusetts General Hospital,185 Cambridge St., Boston, MA 02114, USA*Email: [email protected],Phone: (617) 726-5980, Fax: (617) 643-3328

Contemporary life synthesizes RNA of homogeneouslength and regioisomer composition via sophisticatedenzymatic catalysis. Before such catalysts existed,RNA could have been produced only via simpler,non-enzymatic means, which model prebiotic systemshave shown produce pools of products that are similar,but varied (e.g. in regioisomer composition). Recently,we have demonstrated that functional RNAs (ribo-zymes and aptamers) containing mixed-regioisomerbackbones (i.e. 2′–5′ vs. 3′–5′ linkages) retain function.This observation, coupled with the well-known factthat mixed-regioisomer RNAs exhibit depressed melt-ing temperatures relative to native RNA, suggests thatmixed-regioisomer backbones could actually be adap-tive in an RNA (or pre-RNA) world. In this poster,we will show our recent work with functional RNAsrepresentative of those produced in non-enzymaticpolymerization reactions and their behaviours as cata-lysts and receptors.


ReferencesEngelhart, A. E., Powner, M. P., & Szostak, J. W. (in press).

Functional RNAs exhibit tolerance for non-heritable 2′-5′ vs.3′-5′ backbone heterogeneity. Nature Chemistry.

Trevino, S. G., Zhang, N., Elenko, M. P., Luptak, A., & Szostak,J. W. (2011). Evolution of functional nucleic acids in the pres-ence of nonheritable backbone heterogeneity. Proceedings ofNational Academy of Science of USA, 108, 13492–13497.

8 Using pseudorotation as a reactioncoordinate in free energy simulations ofnucleic acids

Li Li and Jack W. Szostak*

Center for Computational and Integrative Biology,Massachusetts General Hospital, Boston, MA 02114*Email: [email protected],Phone: (617) 726-5980, Fax: (617) 643-3328

Backbone sugar groups are central components of nucleicacids. The conformations of the ribose/deoxyribose can beelegantly described using the concept of pseudorotation(Altona and Sundaralingam, 1972), and are dominated bythe C2′- and C3′-endo conformers. The free energy barrierof the transition between these two major puckeringmodes can be probed by NMR relaxation experiments(Johnson and Hoogstraten, 2008), but an atomic picture ofthe transition path per se is only available for several trun-cated nucleoside analogues (Brameld & Goddard III,1999). Here, we implemented a new free energy simula-tion method for Molecular Dynamics simulations usingpseudorotation as the reaction coordinate (Cremer andPople, 1975). This allowed us to compute the free energylandscape of a complete pseudorotation cycle. The freeenergy landscape revealed not only the relative stability ofC2′- and C3′-endo conformers, but also the main transitionpath and its free energy barrier. As a validation of our newapproach, we calculated free energy surface of the pseudo-rotation of guanosine monophosphate. The free energysurface revealed that the C2′-endo conformation is�1 kcal/mol that is more stable and the free energy barrierfor the transition is 4.5–5 kcal/mol. These are in excellentagreement with previous NMR measurements (Zhanget al., 2012; Röder et al., 1975). We have further appliedthis method to other systems that are important in pre-bio-tic chemistry, including an RNA duplex with unique 2′, 5′-phosphodiester linkages.

This research is supported by the Howard HughesMedical Institute.

ReferencesAltona, C., & Sundaralingam, M. (1972). Journal of the

American Chemical Society, 94, 8205–8212.

Brameld, K. A., & Goddard III, W. A. (1999). Journal of theAmerican Chemical Society, 121, 985–993.

Cremer, D., & Pople, J. (1975). A. Journal of the AmericanChemical Society, 97, 1354–1358.

Johnson, J. E., & Hoogstraten, C. G. (2008). Journal of theAmerican Chemical Society, 130, 16757–16769.

Röder, O., Lüdemann, H.-D., & von Goldammer, E. (1975).European Journal of Biochemistry, 53, 517–524.

Zhang, N., Zhang, S., & Szostak, J. W. (2012). Journal of theAmerican Chemical Society, 134, 3691–3694.

9 Prebiotic RNA synthesis: significance ofmineral salts in montmorillonite-catalyzed reactions

Prakash C. Joshi*, Michael F. Aldersley and James P. Ferris

The New York Center for Astrobiology and Department ofChemistry & Chemical Biology, RPI, Troy, NY 12180*Email: [email protected], Phone: (518) 276-8494,Fax: (518) 276-4887

The dual properties of RNA as an enzyme catalyst and itsability to store genetic information suggest that early lifewas based on RNA, and DNA and protein evolved from it.Our lab has demonstrated synthesis of long RNAoligomers by Na+-montmorillonite-catalyzed reactions of5′-end-activated mononucleotides (Joshi et al., 2009). TheNa+-montmorillonite not only catalyzes the prebiotic syn-thesis of RNA but also facilitates homochiral selection(Joshi et al., 2011, 2013). The montmorillonite-catalyzedreactions of 5′-phosphorimidazolide of adenosine werefurther investigated to study the effect of salts. These reac-tions were found to be dependent on the nature of mineralsalts present. While montmorillonite (pH 7) produced onlydimers in water, addition of sodium chloride (1M)enhanced the chain length of oligomers to 10-mers asdetected by HPLC. Magnesium chloride produced a simi-lar effect but the presence of both sodium chloride andmagnesium chloride did not produce any difference in theoligomer chain length. The effect of monovalant cations inRNA synthesis was of the following order: Li+ >Na+

>K+. A similar effect was observed with the anions,enhancing catalysis in the following order: Cl�>Br�> I�.Inorganic salts that tend to salt out organic compoundsfrom water and salts which show salt-in effects had noeffect in the oligomerization process, indicating that themontmorillonite-catalyzed RNA synthesis is not affectedby hydrophobic or hydrophilic interactions. A 2.3-folddecrease in the yield of cyclic dimer was observed uponincreasing the sodium chloride concentration from 0.2Mto 2.0M. Inhibition of cyclic dimer formation is essentialfor increasing the yield of linear dimers as well as theoverall chain length. The results of this study show thatthe presence of salts is essential in prebiotic RNA synthe-sis catalyzed by clay minerals.


This research has been supported by NASAAstrobiology Institute grant NNA09DA80A.

ReferencesJoshi, P. C., Aldersley, M., Delano, J., & Ferris, J. P. (2009).

Mechanism of montmorillonite catalysis in the Formationof RNA oligomers. Journal of the American ChemicalSociety, 131, 13369–13374.

Joshi, P. C., Aldersley, M. F., & Ferris, J. P. (2011). Homochiralselectivity in RNA synthesis on montmorillonite-catalyzedreactions of D L-purine and pyrimidine nucleotides. Originsof Life and Evolution of Biosphere, 41, 213–236.

Joshi, P. C., Aldersley, M. F., & Ferris, J. P. (2013). Progressin Demonstrating Homochiral selection in Prebiotic RNASynthesis. Advances in Space Research, 51, 772–779.

10 The montmorillonite-catalyzedsynthesis of RNA dimer

Michael F. Aldersley*, Prakash C. Joshi andJames P. Ferris

Department of Chemistry and Chemical Biology, RensselaerPolytechnic Institute, Troy, NY 12180, USA*Email: [email protected], Phone: (518) 276-4080,Fax: (518) 276-4887

A synthesis has been developed, providing nucleotidedimers comprising natural or unnatural nucleosideresidues. A ribonucleoside 5′-phosphorimidazolide isadded to a nucleoside adsorbed on montmorillonite at neu-tral pH with the absence of protecting groups. Approxi-mately 30% of the imidazolide is converted into each 2′-5′dimer and 3′-5′ dimer with the rest hydrolyzed to the 5′-monophosphate. Experiments with many combinationshave suggested the limits to which this method may beapplied, including heterochiral and chimeric syntheses.This greener chemistry has enabled the synthesis of dimersfrom activated nucleotides themselves, activated nucleo-tides with nucleosides, and activated nucleotides withnucleotide 5′-monophosphates. Both homo- and heterochi-ral combinations of reagents have been tried. The mont-morillonite-catalyzed oligomerization of 5′-activatednucleotides leads to oligomers up to 50 residues in length(Huang & Ferris, 2007) using the excellent catalyst Vol-clay®. However, all oligomers must necessarily begin asdimers, so we considered it important to study in detail theformation of these products under prebiotic conditions.Then, a meaningful comparison could be drawn betweenour syntheses and the formation of long oligomers that ispart of our studies of the origins of life. In the synthesis oftrimers from these dimers, we looked for alternative syn-thetic methods via a 5′-phosphate dimer with activatednucleotides as well as 5′-hydroxy nucleotide dimers withthe same reactant. The method has shown promise in tar-geting trimer synthesis and the procedure lends itself to

the development of combinatorial libraries. The use ofenzymatic hydrolysis has played a crucial role in thiswork, facilitating product identity across the spectrum ofproducts prepared. The yields of the corresponding homo-chiral and heterochiral dimers from A and U will requirecareful modeling of the reactants in their interactions withboth the clay and one another to locate the source of thesimilarities and differences. The lack of reactivity ofarabino- and xylo-nucleosides also poses interesting struc-tural, modeling, and origins of life issues. Results withclays that catalyze long oligomer formation only poorlyreveal that they too catalyze these dimer syntheses, albeitless well than Volclay®.

This research was supported by NASA AstrobiologyInstitute grant NNA09DA80A.

ReferencesHuang, W., & Ferris, J. P. (2007). One-step, regioselective syn-

thesis of up to 50-mers of RNA oligomers by montmoril-lonite catalysis. Journal of the American Chemical Society,128, 8914–8919.

Joshi, P. C., Aldersley, M. F., Zagorevskii, D. V., & Ferris, J. P.(2012). Nucleosides, Nucleotides and Nucleic Acids, 31,536–566.

11 Findings and hurdles on the pathleading from formamide to thespontaneous generation of RNA

Samanta Pino, Raffaele Saladino, Giorgia Botta,Giovanna Costanzo and Ernesto Di Mauroa*aDipartimento di Biologia e Biotecnologie “Charles Darwin”,“Sapienza”, Università di Roma, P.leAldo Moro, 5, 00185Rome, Italy; bDipartimento di Agrobiologia ed Agrochimica,Università della Tuscia, via San Camillo De Lellis, 01100,Viterbo, Italy; cIstituto di Biologia e e Patologia Molecolari,CNR, P.le Aldo Moro, 5, 00185 Rome, Italy*Email: [email protected],Phone: (+39) 06-49912880, Fax: (+39) 06-49912500

Our laboratories analyze the synthetic reactions leadingfrom formamide, NH2COH to prebiotically relevantcompounds in the presence of catalysts. We havedescribed the formation of all the biological nucleic


bases of carboxylic acids of two aminoacids, and ofcondensing agents in the presence of catalysts of terres-trial origin (Saladino et al., 2012) and of one meteorite.Heat-dependent synthetic reactions from NH2COH leadto the synthesis of acyclonucleosides, not (yet?) to thatof nucleosides [hurdle # 1]. Nucleosides are phosphory-lated in the presence of NH2COH and a phosphatesource yielding cyclic nucleotides as well. (Costanzoet al., 2007). 3′,5′-cyclic GMP nonenzymaticallypolymerizes up to at least 25mers, as shown by PAGE,MALDI ToF, 31P-NMR, specific RNAse and inhibitorsanalyses (Costanzo et al., 2012).The reaction is stimu-lated by 1,8-diazabicycloundec-7-ene and dimethylform-amide. 3′,5′-cUMP does not polymerize spontaneously[hurdle # 2], 3′,5′-cAMP polymerizes very poorly [hur-dle # 3]. We will discuss data on the polymerization of3′,5′-cCMP and on a ribozyme activity exerted by olig-omers neosynthesized from cyclic nucleotides. Thisapproach finds its larger perspective in the evolutionaryscenario depicted by Trifonov (2009).

ReferencesCostanzo, G., Saladino, R., Botta, G., Giorgi, A., Scipioni, A.,

Pino, S., & Di Mauro, E. (2012). Generation of RNA mole-cules by base catalyzed click-like reaction. ChemBioChem,13, 999–1008.

Costanzo, G., Saladino, R., Crestini, C., Ciciriello, F., & DiMauro, E. (2007). Nucleoside phosphorylation by phos-phate minerals. Journal of Biological Chemistry, 282,16729–16735.

Saladino, R., Botta, G., Pino, S., Costanzo, G., & Di Mauro, E.(2012). Genetics first or metabolism first? The formamideclue Chemical Society Reviews, 41, 5526–5565.

Trifonov, E. N. (2009). Origin of the genetic code and of theearliest oligopeptides. Research in Microbiology, 160, 481–486.

12 Reconstructing the RNA world

James Attwater, Aniela Wochner and Philipp Holliger*

MRC Laboratory of Molecular Biology, Francis Crick Avenue,Cambridge Biomedical Campus, Cambridge, CB2 0QH, UK*Email: [email protected]

A critical event in the origin of life is thought to havebeen the emergence of an RNA molecule capable ofself-replication as well as mutation, and hence evolutiontowards ever more efficient replication. As this primor-dial replicase appears to have been lost in time, we usesynthetic biology to build modern-day “Doppelgangers”of the ancestral replicase to reconstruct and study theirproperties in an effort to learn more about life’s firstgenetic system. I will discuss our progress in theengineering and evolution of RNA polymerase ribo-zymes as well as the potential role that structured mediasuch as the eutectic phase of water–ice may have playedin the emergence of RNA self-replication.

This research was supported by the MRC (U105178804)and a Junior Research Fellowship from Homerton CollegeCambridge (JA).

ReferencesAttwater, J., Wochner, A., Pinheiro, V.B., Coulson, A., &

Holliger, P. (2010). Ice as a protocellular medium for RNAreplication. Nature Communications, 1, 76. doi: 10.1038/ncomms1076

Wochner, A., Attwater, J., Coulson, A., & Holliger, P. (2011).Ribozyme-catalyzed transcription of an active ribozyme.Science, 332, 209–212.


13 RNA fitness landscapes

Irene A. Chen*

Department of Chemistry and Biochemistry, University ofCalifornia, Santa Barbara, CA 93106, USA*Email: [email protected], Phone: (805) 893-8364,Fax: (805) 893-4120

The origin of life is believed to have progressedthrough an RNA World, in which RNA acted as bothgenetic material and functional molecules. Understand-ing early evolution requires systematic knowledge ofthe relationship between RNA sequence and functionalactivity. In particular, knowing the structure of the fit-ness landscape of RNA is critical in estimating theprobability of the emergence of functional sequencesand the role of historical accident during evolution.Much theoretical work has been devoted to fitness land-scapes, but experimental maps have been relatively lim-ited. We use in vitro selection on a pool of short RNAsequences that nearly saturates sequence space to recon-struct the form of a comprehensive fitness landscape.We also study mutations during non-enzymatic poly-merization to understand how early RNA replicatorswould ‘move’ in sequence space.

14 What RNA world ?? Ancestralpolypeptides likely participated inthe origins of translation

Charles W. Carter, Jr.*, Li Li, S. Niranj Chandrasekaran,Katiria Gonzales Rivera and Martha L. Collier

Biochemistry and Biophysics, UNC, Chapel Hill, NC, USA*Email: [email protected], Phone: 919 966-3263,Fax: 919 966-2852

A widespread consensus holds that protein synthesisaccording to a genetic code was launched entirely bysophisticated RNA molecules that played both codingand functional roles. This belief persists, unsupportedby phylogenetic evidence for ancestral ribozymes thatcatalyzed either amino acid activation or tRNAaminoacylation. By contrast, we have adduced strongexperimental evidence that the most highly conservedportions of contemporary aminoacyl-tRNA synthetases(aaRS) accelerate both reactions well in excess ofrates achieved by RNA aptomers derived from combi-natorial libraries and of rates required for primordialprotein synthesis. Such ancestral enzymes, or“Urzymes”, characterized for Class I (TrpRS (Phamet al., 2010, 2007) and LeuRS (Collier et al., 2013);130 residues) and Class II (HisRS; 120–140 residues;

(Li et al., 2011)) synthetases generally havepromiscuous amino acid specificities, whereas ATPand cognate tRNA affinities are within an order ofmagnitude of those for contemporary enzymes. Thesecharacteristics match or exceed expectations for theprimordial catalysts necessary to launch protein synthe-sis. Structural hierarchies in Class I and II aaRS alsoexhibit plateaus of increasing enzymatic activity, sug-gesting that catalysis by peptides similar to the Alephmotif identified by Trifonov (Sobolevsky et al.) mayhave been both necessary and sufficient to launch pro-tein synthesis. Sense/antisense alignments of TrpRS andHisRS Urzyme coding sequences reveal unexpectedlyhigh middle-base complementarity that increases inreconstructed ancestral nodes (Chandrasekaran et al.),consistent with the proposal of Rodin and Ohno (Rodin& Ohno, 1995). Thus, these ancestors were likelycoded by opposite strands of the same gene, favoringsimultaneous expression of aaRS activating both hydro-phobic (core) and hydrophilic (surface) amino acids.Our results support the view that aaRS coevolved withcognate tRNAs from a much earlier stage than thatenvisioned under the RNA World hypothesis, and thattheir descendants make up appreciable portions of theproteome.

This research has been supported by NIGMS 78227.

ReferencesChandrasekaran, S. N., Yardimci, G. G., Erdogan, O., & Carter

C. W. Jr. (under revision). Statistical evaluation of theRodin-Ohno hypothesis: Sense/antisense coding of ancestralclass I and II aminoacyl-tRNA synthetases. MolecularBiology and Evolution.

Collier, M. L., Ibba, M., & Carter, C. W., Jr. (in prepa-ration). LeuRS Urzyme: A second class I aminoacyl-tRNA synthetase urzyme. Journal of BiologialChemistry.

Li, L., Weinreb, V., Francklyn, C., & Carter, C. W., Jr. (2011).Histidyl-tRNA synthetase urzymes: Class I and II amino-acyl-tRNA synthetase urzymes have comparable catalyticactivities of cognate amino acid activation. JournalBiological Chemistry, 286, 10387–10395.

Pham, Y., et al. (2007). A minimal TrpRS catalytic domainsupports sense/antisense ancestry of class i and iiaminoacyl-tRNA synthetases. Molecular Cell, 25, 851–862.

Pham, Y., et al. (2010). Tryptophanyl-tRNA synthetase urzyme:A model to recapitulate molecular evolution and investigateintramolecular complementation. Journal of BiologicalChemistry, 285, 38590–38601.

Rodin, S. N., & Ohno, S. (1995). Two types of aminoacyl-tRNA synthetases could be originally encoded by comple-mentary strands of the same nucleic acid. Origins of Lifeand Evolution of Biospheres, 25, 565–589.

Sobolevsky, Y., Frenkel, Z. M., & Trifonov, E. N. (2007).Combinations of ancestral modules in proteins. Journal ofMolecular Evolution, 65, 640–650.


15 Combinatorial chemistry in theprebiotic environment

David Deamer*

Department of Biomolecular Engineering, University ofCalifornia, Santa Cruz, 95064, CA, USA*Email: [email protected]

The pathway leading to the origin of life presumablyincluded a process by which polymers were synthesizedabiotically from simpler compounds on the early Earth,then encapsulated to form protocells. Previous studieshave reported that mineral surfaces can concentrate andorganize activated mononucleotides, thereby promotingtheir polymerization into RNA-like molecules. However,a plausible prebiotic activation mechanism has not beenestablished, and minerals cannot form cellular compart-ments. We are exploring ways in which nonactivatedmononucleotides can undergo polymerization andencapsulation. We found that small yields of RNA-likemolecules are synthesized by a condensation reactionwhen mixtures of amphiphilic lipids and mononucleo-tides are exposed to cycles of dehydration and rehydra-tion. The lipids concentrate and organize the monomerswithin multilamellar liquid-crystalline matrices that self-assemble in the dry state. The chemical potential driv-ing the polymerization reaction is supplied by the anhy-drous conditions in which water becomes a leavinggroup, with heat providing activation energy. Signifi-cantly, the polymeric products are encapsulated in tril-lions of microscopic compartments upon rehydration.Each compartment is unique in its composition andcontents, and can be considered to be an experiment ina natural version of combinatorial chemistry that wouldbe ubiquitous in the prebiotic environment. A success-ful experiment would be a compartment that capturedpolymers capable of catalyzing their own replication. Ifthis can be reproduced in the laboratory, it would repre-sent a significant step toward understanding the originof cellular life.

16 Mechanical energy, protein motion,and the possible origin of lifebetween mica sheets

Helen Greenwood Hansma*

Department of Physics, University of California, SantaBarbara, CA, USA*Email: [email protected]

The origins of life require reliable energy sources. Onefeasible energy source has not been considered untilrecently. This is mechanical energy-work (Hansma,

2010, 2012). The spaces between moving muscovitemica sheets are the environment in which mechanicalenergy is hypothesized to have been involved in theorigins of life. Mechanical energy from moving micasheets has two main sources: (1) The open-and-shutmotions of mica sheets in response to water movementsin and out between the sheets, and (2) Thermal cycles ofday and night acting on bubble ‘defects’ between micasheets. This mechanical energy is hypothesized to havebeen involved in the formation (and breaking) of cova-lent bonds, the rearrangement of polymers and molecularaggregates, and the budding-off of protocells, in the ear-liest form of cell division. Furthermore, it is hypothe-sized that the mechanical energy from mica sheetsmoving open-and-shut is the source of the commonopen-and-shut motions of enzymes, originating from aprotobiotic era when mechanical energy was plentifuland chemical energy was not yet available.

ReferencesHansma, H. G. (2010). Possible origin of life between mica

sheets. Journal of Theoretical Biology, 266, 175–188.Hansma, H. G. (2012). Possible origin of life between mica sheets:

Does life imitate mica? Journal of Biomolecular Structure andDynamics. doi:10.1080/07391102.2012.718528.

17 Origin of life by metabolic avalanchebreakthrough in an iron–sulfurworld

Günter Wächtershäuser*

Munich, Germany/Chapel Hill, NC, USA*Email: [email protected], Phone: 919 942-5943,Fax:919 929 9383

Origin-of-life research is polarized on two levels. Onthe methodological level, we distinguish two heuris-tics: (a) the conventional heuristic of parallel backextrapolations into the chaos of a prebiotic broth and(b) the heuristic of convergent biochemical retrodictionof a ‘pioneer organism’. On the theoretical level, wedistinguish two testable, explanatory theories: (1) Thegenetics-first RNA world theory postulates an originby polymer replication, that is, by random aqueouspolycondensations in a slowly accumulating, cold pre-biotic ocean. The biochemical system is truncated toC–H–N–O–P–Mg. Explanatory power is meek andexperiments can do no more than rationalize partialaspects of the low-probability overall scheme. (2) Themetabolism-first Fe–S world theory postulates an ori-gin by carbon fixation, that is, by directional aqueous,synthetic redox reactions in a hot volcanic-hydrother-mal fluid flow. The reactions are driven by the chem-


ical potential of quenched volcanic gases and cata-lyzed by transition metal centers of crustal minerals.The full biochemical system C–H–N–O–S–Se–P–Mg–Fe–Co–Ni–W is assumed. Experiments have demon-strated reductive carbon fixation pathways to a widepanel of highly functionalized organic compounds (e.g. thioesters, hydroxyl/amino acids, peptides). The pio-neer organism reproduces and evolves by ligandeffects of its organic products. Specifically, organicproducts of a reaction turn into activity-enhancingligands of catalytic transition metals. By ligand feed-back effect, an organic product enhances a catalyst ofthe pathway, whence it derives (metabolic reproduc-tion). By ligand feed-forward effect, an organic prod-uct enhances a catalyst for transforming itself (oranother organic reaction product) into a new organicproduct (metablic innovation = evolution). Any feed-forward effect weakens itself by weakening the path-way, whence it derives, unless it is accompanied bysufficiently strong feedback effects. From the point ofview of a pre-established metabolic network, a feed-back into a peripheral branch pathway is detrimentalby weakening its mother network (virulyst effect).This metabolic virus effect has to be compensated bysufficient positive feedback into the mother network(vitalizer effect). These simple considerations lead usto two consequences: (1) at least some organic prod-ucts should become enhancing ligands for two ormore transition metal catalysts, and (2) at least sometransition metal centers should catalyze two or moresynthetic steps. These two conditions amount to self-accelerating metabolic expansions (avalanche break-through). By these ligand effects, the origin of life ina Fe–S world is a unique, complexity-increasing,intrinsically synthetic, and directional process, preor-dained in the universal laws of chemistry and defi-nitely knowable.

ReferencesHuber, C., & Wächtershäuser, G. (1997). Activated acetic acid

by carbon fixation on (Fe, Ni)S under primordial condi-tions. Science, 276, 245–247.

Huber, C., & Wächtershäuser, G. (1998). Peptides by activationof amino acids on (Fe, Ni)S surfaces: Implications for theorigin of life. Science, 281, 670–672.

Huber, C., & Wächtershäuser, G. (2006). α-Hydroxy and α-amino acids under possible hadean, volcanic origin-of-lifeconditions. Science, 324, 630–632.

Huber, C., Kraus, F., Hanzlik, M., Eisenreich, W., & Wächtershä-user, G. (2012). Elements of metabolic evolution. Chemistry:A European Journal, 18, 2063–2080.

Wächtershäuser, G. (2006). From volcanic origins of chemoau-totrophic origin of life to bacteria, archaea and eukarya.Philosophical Transactions of the Royal Society B London,361, 1787–1808.

Wächtershäuser, G. (2007). On the chemistry and evolution ofthe pioneer organism. Chemistry & Biodiversity, 4, 584–602.

18 Possible extraterrestrial life: aquantum-chemical look on the siliconanalogs of carbon biomolecules

Maxim S. Kondratyev*, Artem V. Kabanov,Alexander A. Samchenko, Vladislav M. Komarov andNikolay N. Khechinashvili

Institute of Cell Biophysics Russian Academy of SciencesPushchino, 142290 Russia*Email: [email protected], Phone: +7 4967 739 404,Fax: +7 4967 330 509

The uniqueness of life on our planet has been an impor-tant topic of discussion in scientific literature for manydecades. The most particular findings are in the fields ofthe structure of biomolecules and the mechanisms of theirconformational and chemical transfers since they underlieall the biospheric processes of our planet. The com-pounds based on carbon are the subject of study oforganic chemistry, which has an appropriate thoroughlydeveloped classification of such substances; a number ofapproaches have been proposed for the analysis of com-position and structure of the organic compounds, and atheoretical basis has been created, which describes thecharacter of various chemical bonds involving carbonatoms. At the same time, since quite a while, there is awidely discussed hypothesis (Alison, 1968) concerningthe possibility of existence of compounds, which are sim-ilar to organic, but are based on silicon atoms. Even ininterstellar medium, among all the diversity of moleculesdetected, 84 are based on carbon, and 8 on silicon (Lazio,2000), including four hybrid types, i.e. containing bothsilicon and carbon. According to approximate evalua-tions, the contents ratio of carbon to silicon in the spaceequals to 10:1, though the Earth’s crust consists of 87%of silicon in the form of oxides. In the Periodic Table, sil-icon is situated in the same group IV, like carbon. Thesetwo elements are largely similar in the structure of theirvalent electronic shells, and their noteworthy that previ-ously it was stated (Lazio, 2000) that silicon-containingcompounds are not as diverse in structure as carbon com-pounds. Despite having higher mass and radius, theatoms of silicon form double and triple covalent bonds(Wang et al., 2008). Therefore, the issue concerning theexistence of silicon structures similar to carbon biomole-cules, as well as the question of hypothetical “biochemi-cal” processes involving non-carbonic analogs ofaminoacids, carbohydrates, proteins, lipids, and other bio-molecules, is still a matter of discussion in scientific and


popular science literature. It is particularly notable thatthe modern methods of computational chemistry allowcarrying out the estimating calculations of the structureand dynamics of such compounds, which is quite similarto the known approaches of substance modeling de novoin drug design. For instance, first by calculations (Nag-ase, Kudo, & Aoki, 1985), and later on experimentally(Abersfelder, White, Rzepa, & Scheschkewitz, 2010),aromaticity of cyclic carbohydrate-like derivatives of sili-con was studied. In the present study, we used quantum-chemical semiempirical PM3 and ab initio B3LYP/6-311G(d,p) level of theory to investigate the peculiaritiesof several structural and thermodynamic parameters ofmolecules, which can be assumed as complete siliconanalogs of carbonic L-amino acids and other biomole-cules, so-called bricks of life: carbohydrates, nitrogenousbases, fatty acids, as well as vitamins and caffeine. Thequantum-mechanical calculations that we made displayedthat the molecules of silicon amino acids possess higherthermodynamic stability compared to carbon analogs.Thereby, silicon amino acids have a similar conformationfreedom, increased values of dipole moment, as well asmore pronounced electron-donor characteristics. Siliconanalogs of carbohydrates, fatty acids, and nitrogenousbases are as well considered as heavier thermodynami-cally stable compounds, having special features in 3D-organization and worth further experimental study. Thepresent work also deals with the question of the existenceand stability of “alpha-helices” composed of siliconamino acids, because in the molecules of Si-analogs ofaspartate and glutamate, we have discovered effective for-mation of intramolecular hydrogen bond (due to the sidechain), which is highly important for Pauling–Coreyalpha helix formation in natural L-amino acids (Kon-dratyev, Kabanov, & Komarov, 2010). Our estimationsshow that an “alpha helix” composed of 10 silicon ala-nine analogs is more stable in isolated state than a linearform of such macromolecule, which was not observed fora molecule of the same composition having a carbonbackbone.

ReferencesAbersfelder, K., White, A. J. P., Rzepa, H. S., &

Scheschkewitz, D. (2010). A tricyclic aromatic isomer ofhexasilabenzene. Science, 327, 564–566.

Alison, A. (1968). Possible forms of life. Journal of the BritishInterplanetary Society, 21, 48.

Kondratyev, M. S., Kabanov, A. V., & Komarov, V. M. (2010).Modeling of helix formation in peptides containing asparticand glutamic residues. Computer Research and Modeling,2, 83–90Russian.

Lazio J. (2000). Why do we assume that other beings must bebased on carbon? Why couldn’t organisms be based onother substances? (Report based on a lecture by AlainLeger (IAS) at the SPIE Astronomical Telescopes andInstrumentation 2000 Conference).

Nagase, S., Kudo, T., & Aoki, M. (1985). Hexasilabenzene(Si6H6). An ab initio theoretical study of its aromaticityand relative stability. Journal of the Chemical Society,Chemical Communications, 16, 1121–1122.

Wang, Y., Xie, Y., Wei, P., King, R. B., Schaefer, H. F.III, von,P., … Robinson, G. H. (2008). A stable silicon (0) compoundwith a Si=Si double bond. Science, 321, 1069–1071.

19 Quantum chemical studies on theformamide-based origin of life

Judit E. Šponera*, Arnošt Mládeka, Martin Ferusb,Svatopluk Civišb and Jiří Šponera

aInstitute of Biophysics, Academy of Sciences of the CzechRepublic, Královopolská 135, Brno CZ-61265, Czech Republic;bJ. Heyrovský Institute of Physical Chemistry, Academy ofSciences of the Czech Republic, Dolejškova 3, Prague 8,18223, Czech Republic*Email: [email protected], Phone: 420 541 517 146,Fax: 420 541 211 293

Recently, we have applied quantum chemical calcula-tions to supplement the experimentally available infor-mation on the prebiotic synthesis of nucleic acids witha molecular-level insight into the structure, energy, elec-tronic structure, and spectroscopic properties of thecompounds involved in these processes. In particular, inthe presentation, we will focus on our studies related tothe formamide-based origin of life. We will discuss themechanism of the formamide-based synthesis of nucleo-bases suggested by Saladino et al. (2007). We showthat the computed activation energy of the experimen-tally proposed pathway is noticeably higher than that ofthe HCN-based synthetic route, but it is still feasibleunder the experimental conditions of the Saladino syn-thesis. Our calculations thus demonstrate that the exper-imentally suggested reaction path without theinvolvement of aminoimidazole–carbonitrile intermedi-ates is also a viable alternative for the nonaqueous syn-thesis of nucleobases (Šponer et al., 2012). Further, wehave used quantum chemical calculations to interpretthe results of high-resolution infrared spectra obtainedfor an icy mixture of formamide and an FeNi meteor-itic material treated with high-energy laser pulses (Feruset al., 2012). In this joint experimental-theoretical study,we have identified a new reaction channel for the for-mation of nucleobases from formamide ices, whichmight be highly relevant for the formation of nucleo-bases under a high-energy impact event, e.g. an atmo-spheric breakup of an icy extraterrestrial body. We havealso shown that the theoretically proposed reactionroute is highly realistic, because we could detect someof the theoretically proposed key intermediates usinghigh-resolution IR spectroscopy. Thus, formation ofnucleobases from formamide in a high-energy impact


event may give new, astrobiological dimensions to theincreasingly popular idea of formamide-based life origi-nally suggested by Saladino et al., 2007.

ReferencesFerus, M., Civiš, S., Mládek, A., Šponer, J., Juha, L., &

Šponer, J. E. (2012). On the road from formamide ices tonucleobases: IR-spectroscopic observation of a directreaction between cyano radicals and formamide in a high-energy impact event. Journal of the American ChemicalSociety, 134, 20788–20796.

Saladino, R., Crestini, C., Ciciriello, F., Costanzo, G., & DiMauro, E. (2007). Formamide chemistry and the originof informational polymers. Chemistry & Biodiversity, 4,694–720.

Šponer, J. E., Mládek, A., Šponer, J., & Fuentes-Cabrera, M.(2012). Formamide-based prebiotic synthesis of nucleo-bases: A kinetically accessible reaction route. Journal ofPhysical Chemistry A, 116, 720–726.

20 Supramolecular polymerization ofnucleobase-like monomers in water

Isaac Gállegoa*, Brian J. Caffertya, Michael C. Chena,Katherine I. Farleya, Ramon Eritjab andNicholas V. Huda

aDepartment of Chemistry and Biochemistry, Parker H. PetitInstitute for Bioengineering and Bioscience, Georgia Instituteof Technology, Atlanta, GA 30332-0400, USA; bInstitute forResearch in Biomedicine, Parc Científic de Barcelona,Barcelona 08028, Spain*Email: [email protected], Phone: 404-385-1166

Elucidating the physiochemical principles that governmolecular self-assembly is of great importance forunderstanding biological systems and may provideinsight into the emergence of the earliest macromoleculesof life, an important challenge facing the RNA Worldhypothesis. Self-assembly results from a delicate balancebetween multiple noncovalent interactions and solvent

effects, but achieving efficient self-assembly in aqueoussolution with synthetic molecules has proven particularlychallenging. Here, we demonstrate how two physicalproperties – monomer solubility and large hydrophobicsurfaces of intermediate structures – are key elements toachieving supramolecular polymers in aqueous solution(Cafferty et al., 2013). Applying these two principles, wereport the highly cooperative self-assembly of twoweakly interacting, low molecular weight monomers[cyanuric acid and a modified triaminopyrimidine] into awater-soluble supramolecular assembly (see schemebelow). The observed equilibrium between only twoappreciably populated states – free monomers and supra-molecular assemblies – is in excellent agreement withthe values previously determined for the free energy ofhydrogen bonding (Klostermeier & Millar, 2002), π� πstacking (Frier et al., 1985), and the calculated freeenergy penalty for the solvation of hydrophobic struc-tures in water (Chandler, 2005). The similarity of themolecules used in this study for the nucleobases foundin contemporary nucleic acids and the demonstration thatthese monomers assemble while the natural nucleobasesdo not, suggests that the first informational polymersmay have emerged from a similar self-assembly process,if the nucleobases were different then they are today(Hud et al., 2013).

This research has been supported by NSF and the NASAAstrobiology Program under the NSF Center for ChemicalEvolution [CHE-1004570], and Consejo Superior deInvestigaciones Cientifíficas (CSIC) [MEC, SAB2010-0163].

ReferencesCafferty, B. J., et al. (2013). Journal of the American Chemical

Society, 135, 2447–2450.Chandler, D. (2005). Nature, 437, 640–647.Frier, S. M., et al. (1985). Biochemistry, 24, 4533–4539.Hud, N. V., et al. (2013). Chemistry & Biology, Submitted.Klostermeier D., & Millar D. P. (2002). Biochemistry, 41,

14095–14102.


21 Ribosomal evidence suggests thatarchaea evolved after fungi

William L. Duax*, David Dziak and Jordan Tick

Hauptman Woodward Medical Research Institute, Buffalo, NY14203, USA*Email: [email protected], Phone: (716) 898-8616,Fax:(716) 898-8660

We are exploring the potential to trace species evolu-tion with the ribosomal proteins (RibPs) present inbacterial, eukaryotic, and archaeal ribosomes and tocompare the independent trees for consistency. Thecomplete genomes of over 8400 bacteria, eukaryota,and archaea are presently in the SwissPro/TrEMBL(SPT) database. A search of SPT using a vectordesigned with ScanProsite formats (V1) finds andaligns 8405 sequences (5312 bacterial, 2905 eukaryotic,and 169 archaeal) that are homologous with bone fidebacterial S19 ribosomal proteins(S19s). When the 8405sequences are perfectly aligned, 15 residues are con-served at 90% identity and 40 are conserved at 70%identity. We are not aware of any previous publicationreporting sequence alignment of 8400 members of anysingle family including all bacteria, eukaryota andarchaea, for which complete genomes have beenpublished.

A Pro and a Gly separated by 11 residues are 100%conserved in the 8405 S19s. In the position immedi-ately before the fully conserved Gly, two residues(Asp and Asn) are present in 98.3% of the 8405sequences. The Asp residue is found almost exclu-sively in 2190 gram-positive bacteria. The Asn resi-due is found in 3065 gram-negative bacteria, 123Archaea, 1939 eukaryotes, and 64 specific species ofgram-positive bacteria. There is biochemical evidencefor the existence of distinct mitochondrial, chloroplast,and cytosolic ribosomes and reports that plants haveall three forms and mammals only two. Reliable dataconcerning how individual ribosomal proteins differ indifferent types of ribosomes are meager. Examinationof the eukaryotic S19s reveals the existence of threedistinct types. Two of the distinctly different typesare found in most fungi, three of the types are foundin some viridiplante, and only one type is found inmetazoa and archaea. We demonstrate the sequence

homology between the mitochondrial form found infungi and plants and the S19 proteins of alpha prote-obacteria; between the chloroplast S19s and the S19sof cyanobacteria; and among the cytosolic S19s foundonly in fungi, metazoa, archaea, and in some viridi-plantae. Our findings suggest that most archaeal spe-cies appeared after a gene duplication event infungi that correlates with the origin of the cytosolicribosome.

22 Text segmentation approach revealssimple repeat “fossils” in genomicsequences

Zakharia M. Frenkela,b* and Edward N. Trifonovb

aDepartment of Software Engineering, ORT Braude College,Karmiel, Israel; bGenome Diversity Center, Institute of Evolution,University of Haifa, Haifa, Israel*Email: [email protected],Phone/Fax: (972)-4-8288096

A novel concept, on mechanisms of evolution of genesand genomes, formulated recently in Frenkel andTrifonov, 2012; Koren and Trifonov, 2011, and sug-gested by results of earlier works, starting from Ohno,1972, is that the sequences evolve largely by localevents of tandem repeats expansion and subsequentmutational changes in the repeats. According to thisview, frequently occurring segments of tandemly repeat-ing codons manifest the immediate memory about therecent expansion events. The main arguments in favorof this hypothesis are: (1) Codons of GCC, GCA, andGAA expanding families detected in triplet expansiondiseases are dominant, making 46.6% of all observedrepeats in mRNA, (2) Those codons, which are morefrequently found in tandem, are also generally more fre-quent in the regions with no repeats, (3) Sequence seg-ments up to 300 nucleotides in size, starting andending with the same triplet, have substantially elevatedcontent of the border triplet itself and of the pointmutation derivatives of this triplet, and (4) More than40% of natural sequences have both the dominantcodon, and one of its first derivatives, on the top of thecodon frequency list (instead of expected, random case,15%). We applied a clustering (text segmentation) algo-rithm for rigorous mapping of the original, now hidden,triplet expansions in genomic sequence. A significantdifference between the natural sequences and the corre-sponding shuffled sequences is detected. The naturalfragments are longer and more similar to the putativerepeat sequences. More than 35% of bacterial genomicsequences detectably “remember” their ancient expan-


X(0,100)X(6)[RGWCKT]X(5)PX(3)[GARDENS]

X(4)[VIL][HYF][DNSTQVKA]GX(7)

[LIVMP]X(5)[GDNFSYAR]XX[LFI][GASR]

[DEA][FYME]X(0,30)V1

sion history. A significant difference betweensegmentations of genomic sequences from differenttaxonomic classes was detected. The developed toolopens the possibility to investigate the influence of theancient triplet expansion events on modern protein 3Dstructure.

ReferencesFrenkel, Z. M., & Trifonov, E. N. (2012). Origin and evolution of

genes and genomes. Crucial role of triplet expansions. Journalof Biomolecular Structure & Dynamics, 30, 201–210.

Koren, Z., & Trifonov, E. N. (2011). Role of everlasting tripletexpansions in protein evolution. Journal of Molecular Evo-lution, 72, 232–239.

Ohno, S. (1972). In Smith, H. H. (ed), Evolution of GeneticSystems.

23 The phylogenomic roots of modernbiochemistry, translation, and thegenetic code

Gustavo Caetano-Anollés*

University of Illinois at Urbana-Champaign, Urbana,IL 61801, USA*Email: [email protected], Phone: (217) 333-8172,Fax: (217) 333-8046

The origin and evolution of modern biochemistry is acomplex problem that has puzzled scientists foralmost a century. In my laboratory, we have dissectedthe emergence of the very early macromolecules thatpopulated primordial cells using ideographic (histori-cal, retrodictive) approaches. Deep evolutionary signalswere retrieved from a census of molecular structures

and functions in thousands of nucleic acids and mil-lions of proteins using powerful phylogenomic meth-ods. These clock-like signals revealed that modernbiochemistry resulted from gradual coevolution andaccretion of molecular parts and molecules. This wasmade evident in the study of aminoacyl-tRNA synthe-tase (aaRS) enzymes and the ribosomal ensemble.aaRSs coevolved with tRNAs, as catalytic aaRSdomains and acceptor arm tRNAs accreted domains,and RNA substructures. Similarly, the ribosome origi-nated in its central ratchet mechanism and expandedby coevolving rRNA–protein interactions (Figure 1).Remarkably, while the first biochemical functions weremetabolic, the translation, the genetic code, and theribosome appeared quite late as ‘exacting’ mechanismsthat enhanced protein folding speed and flexibility,benefiting the search for new molecular functions.Our timelines reveal that translation unfolded onlyafter the rise of viruses but prior to the appearanceof diversified archaeal microbes. Remarkably, its debutcoincided with the rise of nucleotide and amino acidbiosynthetic pathways.

ReferencesDebes, C., Wang, M., Caetano-Anollés, G., & Gräter, F.

(2013). Evolutionary optimization of protein folding. PLOSComputational Biology, 9, e1002861.

Harish, A., & Caetano-Anollés, G. (2012). Ribosomal historyreveals the origin of modern protein synthesis. PLOS ONE,7, e32776.

Nasir, A., Kim, K. M., & Caetano-Anollés, G. (2012). Giantviruses coexisted with the cellular ancestors and representa distinct supergroup along with superkingdoms Archaea,Bacteria and Eukarya. BMC Evolutionary Biology, 12,156.


24 Evolutionary dynamics of inteins inbacteria

Olga Novikova*, Samantha Merwin, Natalya Topilinaand Marlene Belfort

Department of Biological Sciences and RNA, InstituteUniversity at Albany, Albany, NY 12222, USA*Email: [email protected], Phone: (518) 437-4445,Fax: (518) 437-4445

Inteins are protein sequences that autocatalyticallysplice themselves out of protein precursors – analogousto introns – and ligate the flanking regions into afunctional protein. Inteins are present in all three king-doms of life, but have a sporadic distribution. Theyare found predominantly in proteins involved in DNAreplication and repair such as helicases. The distribu-tion of inteins suggests an adaptive function. The evo-lutionary forces which shaped the observed distributionof inteins are generally unknown. Some authors viewinteins only as the selfish elements and argue that fre-quent horizontal transfer is behind inteins sporadic dis-semination (Gogarten et al., 2002). On the other hand,the ancient nature of the inteins and the process ofgain/loss could lead to the scattered distribution ofinteins among species (Pietrokovski, 2001). It is neces-sary to note that the exclusively selfish nature ofinteins is questionable; recent findings support thehypothesis of possible functional roles of inteins inprotein regulation (Callahan et al., 2011). Moreover,both hypotheses were built on a limited number of the

intein representatives. The amount of genomic dataavailable for bacteria is enormous and in silico analy-sis for diverse inteins is warranted. We decided to takeadvantage of these microbial genomic data and per-formed comprehensive mining for the inteins using abioinformatic pipeline. Altogether, 1757 species wereanalysed from 19 major phyla yielding more than4500 intein-like sequences. The majority of these bac-terial inteins were not described previously. Approxi-mately 55% of the inteins were found in polymerases,helicases, or recombinases (Figure 1). Phylogeneticanalysis indicated the complex evolutionary dynamicsof inteins which includes horizontal transfers, highevolutionary rates coupled with recurrent gains, andlosses. The preponderance of inteins in helicases andreductases is being investigated in terms of functionalrelevance.

This research has been supported by NIH grantGM44844.

ReferencesCallahan, B. P., Topilina, N. I., Stanger, M. J., Van Roey, P., &

Belfort, M. (2011). Structure of catalytically competentintein caught in a redox trap with functional and evolution-ary implications. Nature Structural and Molecular Biology,18, 630–633.

Gogarten, J. P., Senejani, A. G., Zhaxybayeva, O., Olendzenski,L., & Hilario, E. (2002). Inteins: structure, function, andevolution. Annual Review Microbiology, 56, 263–287.

Pietrokovski, S. (2001). Intein spread and extinction inevolution. Trends in Genetics, 17, 465–472.

25 Why no group II introns in nucleargenomes? translational repression,RNA-RNA interactions andmis-compartmentalization of mRNAs

Guosheng Qu, Xiaolong Dong,Venkata R. Chalamcharla, Sheila Lutz M. Joan Curcioand Marlene Belfort*

SUNY at Albany, Department of Biology, Albany, NY 12222,USA*Email: [email protected], Phone: (518) 437-4445,Fax: (518) 437-4445

Group II introns are commonly believed to be theprogenitors of spliceosomal introns. However, the nota-ble absence of group II introns from nuclear genomesbegs examination. We have shown that althoughnuclear expression of a group II intron-containing pre-mRNA in yeast results in efficient transcription andprotein-dependent splicing, RNA transcripts suffer non-sense-mediated decay and translational repression (Cha-lamcharla et al. 2010). To demonstrate the possiblemechanism for the translational repression, we haveinvestigated cellular dynamics of group II intron-con-taining RNAs, from transcription to cellular localiza-


tion, by utilizing multi-disciplinary techniques. Special-ized 5’-RACE indicates that group II intron-containingRNA and its spliced product are both 5’-capped and3’-polyadenylated and are most eukaryotic RNA poly-merase II transcripts. Additionally, primer extensionassay showed that RNA transcription and splicing bothinitiate from the correct sites. Importantly, molecularpull-down experiments reveal that spliced RNA and itsprecursor in cell lysates can be co-isolated by affinityresins specialized for the precursor, suggesting interac-tions between RNAs or RNA-protein complexes.Finally, fluorescence microscopy and immuno-precipita-tion analyses both demonstrate that the unspliced pre-cursor and spliced RNA are significantly co-localizedwith processing bodies and stress granules, the twomain cellular compartments known to retain untrans-lated or aberrant RNAs in eukaryotic cells. Consistentwith these observations, 5’-3’ truncated intron-contain-ing RNA species were detected by primer extension.We present a model whereby mRNA–pre-mRNA asso-ciation and mis-compartmentalization of mRNPs resultin translational repression of mRNAs that containedgroup II introns, and that may have resulted inexpunging of such introns from nuclear genomes.

This research has been supported by NIH.

ReferenceChalamcharla, V. R., Curcio, M. J., & Belfort, M. (2010).

Nuclear expression of a group II intron is consistent withspliceosomal intron ancestry. Genes & Development, 24,827–836.

26 Inversion approach to the origin oflife: theoretical notions andexperimental data

Vladimir N. Kompanichenko*

Institute for Complex Analysis, Russian Academy of Science,Birobidzhan 679016, Russia*Email: [email protected], Phone: (42622) 24013,Fax: (42622) 61362

The essence of the inversion concept of the origin oflife can be narrowed down to the following theses: (1)thermodynamic inversion is the key transformation ofprebiotic microsystems leading to their transition intoprimary forms of life; (2) this transformation mightoccur only in the microsystems oscillating around thebifurcation point under far-from-equilibrium conditions.The transformation consists in the inversion of the bal-ance “free energy contribution entropy contribution” (as

well as “information contribution informational entropycontribution”), from negative to positive values. At theinversion moment, the microsystem radically reorga-nizes in accordance with the new negentropy (i.e. bio-logical) way of organization. According to this concept,the origin-of-life process on the early Earth took placein oscillating hydrothermal medium. The process wastaking two successive stages: (1) spontaneous self-assembly of initial three-dimensional prebiotic microsys-tems composed mainly of hydrocarbons, lipids, andsimple amino acids, or their precursors, within the tem-perature interval of 100–300 °C (prebiotic stage); (2)nonspontaneous synthesis of sugars, ATP, and nucleicacids started at the inversion moment under the temper-ature 70–100 °C (biotic stage). Macro and microfluctu-ations of thermodynamic and physicochemicalparameters able to sustain this way of chemical conver-sion have been detected in several contemporary hydro-thermal systems (Kompanichenko, 2012). Conditions inpotential hydrothermal medium for the origin of lifewere explored on the examples of several hydrothermalsystems in Kamchatka peninsula. Temperature of waterin hot springs ranges from < 60 to 98 °C, in the boreholes water-steam temperature varies from < 100 to239 °C, and pressure from < 1 to 35 bars at the well-heads; pH is within the interval 2.5–9.0. Pressure moni-toring at the depth 950meters in the borehole No. 30(Mutnovsky field) has revealed high-amplitude (up to1–2 bars) irregular macrofluctuations and low-amplitudequite regular microoscillations of pressure (amplitudes0.1–0.3 bars). Hydrocarbons, lipid precursors, and sim-ple amino acids are available in the fluid. The lifelesscondensate of water-steam mixture (temperature 108–175 °C) contains aromatic hydrocarbons, n-alkanes,ketons, alcohols, and aldehydes. In addition to those,cycloalkanes, alkenes, dietoxyalkanes, naphtenes, fattyacids, ethyl ethers of fatty acids, and monoglycerideshave been detected in hot solutions inhabited by ther-mophiles and hyperthermophiles (temperature 70–98 °C). According to Mukhin et al. (1979), glycine of prob-ably abiotic origination was detected in lifeless conden-sate.

This research has been supported by the grants RFBRNo. 12-05-98517 and FEB RAS 12-I-П28-05.

ReferencesKompanichenko, V. N. (2012). Inversion concept of the origin

of life. Origins of Life and Evolution of Biospheres, 42,153–178.

Mukhin, L. M., Bondarev, V. B., & Vakin, E. A. (1979).Amino acids in the thermae of Southern Kamchatka.Doklady AN USSR, 244, 974–977 (in Russian).


27 Structural analyses by NMR revealinsights into the enzymaticmechanism of tRNA t6A modificationbiosynthesis

Kimberly A. Harrisa,b*, Benjamin G. Bobaya,Yann Bilbillea and Paul F. Agrisb

aNorth Carolina State University, Raleigh, NC, USAbUniversity at Albany-SUNY, Albany, NY, USA*Email: [email protected]

Post-transcriptional modification is an inherent featureof RNA maturation and is particularly abundant intransfer RNA (tRNA) where over 90 distinct modifi-cations are found across all phylogenetic groups. Ofthese, the universally conserved N6-threonylcarbamoyl-adenosine (t6A) modification 3′-adjacent to the antico-don is essential in translational fidelity for severaltRNA species by ensuring accurate codon recognition.Recent studies of the t6A biosynthesis pathway inbacteria have identified four proteins that are neces-sary and sufficient for in vitro formation: YrdC,YgJD, YjeE and YeaZ. As this enzyme complex iscomprised of essential proteins, two of which areunique to bacteria, YeaZ and YjeE, it is a potentialtarget for anti-microbial development. Thus, a detailedunderstanding of the interactions within this pathwayholds significant value but has yet to be accom-plished; therefore, further structural characterisationand biochemical analyses are necessary. Of particularinterest is YrdC, a universally conserved, monomeric21 kDa protein that is believed to be the central com-ponent of this complex. Using quantitative methods,we have shown that YrdC binds to all componentsrequired for t6A biosynthesis: ATP, L-threonine andthe unmodified target tRNA. Additionally, we havedeveloped a novel application of saturation transferdifference NMR for detecting protein–RNA interac-tions and used it to observe the interaction of YrdCwith a 17-nt tRNALys anticodon domain truncation. Inorder to map these binding interfaces under biologicalconditions, high-resolution structures are vital; there-fore, the solution structure of E. coli YrdC was deter-mined by NMR. Several key residues have beenidentified by 15N-HSQC titrations and molecular dock-ing simulations were performed to map ligand-bindingsites and protein–protein interaction interfaces.

This work is supported by NSF grant MCB1101859.

28 The study of tRNA modifications bymolecular dynamics

Xiaoju Zhanga, Ross Walkerb and David H. Mathewsa*aDepartment of Biochemistry and Biophysics and Center forRNA Biology, University of Rochester Medical Center, Rochester,New York, USA; bSan Diego Supercomputer Center, Universityof California San Diego, La Jolla, California, USA*Email: [email protected],Phone: (585)-275-1734, Fax: (585)-275-6007

Modified nucleosides are prevalent in tRNA. Experi-mental studies reveal that modifications play animportant role in tuning tRNA activity. In this study,molecular dynamics (MD) simulations were used toinvestigate how modifications alter tRNA structureand dynamics. The X-ray crystal structures of tRNA-Asp, tRNA-Phe, and tRNA-iMet, both with and with-out modifications, were used as initial structures for333-ns time-scale MD trajectories with AMBER. Foreach tRNA molecule, three independent trajectory cal-culations were performed. Force field parameters werebuilt using the RESP procedure of Cieplak et al. for17 nonstandard tRNA residues. The global root-mean-square deviations (RMSDs) of atomic positions showthat modifications only introduce significant rigidity totRNA-Phe’s global structure. Interestingly, regionalRMSDs of anticodon stem-loop suggest that modifiedtRNA has more rigid structure compared to theunmodified tRNA in this domain. The anticodonRMSDs of the modified tRNAs, however, are higherthan those of corresponding unmodified tRNAs. Thesefindings suggest that rigidity of the anticodon arm isessential for tRNA translocation in the ribosomecomplex, and, on the other hand, flexibility of antico-don might be critical for anticodon–codon recognition.We also measure the angle between the 3D L-shapedarms of tRNA; backbone atoms of acceptor stem andTψC stem loop are selected to indicate one vector,and backbone atoms of anticodon stem and D stemloop are selected to indicate the other vector. Bymeasuring the angle between two vectors, we findthat the initiator tRNA has a narrower range of hingemotion compared to tRNA-Asp and tRNA-Phe, whichare elongator tRNA. This suggests that elongatortRNAs, which might require significant flexibility inthis hinge to transition from the A–to-P site in theribosome, have evolved to specifically accommodatethis need.


29 Accelerating nucleic acid designusing pre-selected sequences

Stanislav Bellaousov and David H. Mathews*

Department of Biochemistry, University of Rochester,601 Elmwood Avenue, Box 712, Rochester, NY 14642, USA*Email: [email protected], Phone: (585)275-1734, Fax: (585)-275-6007

Nanoscale nucleic acids could potentially be designed tobe catalysts, pharmaceuticals, or probes for detectingpathogens. We hypothesize that designing nucleic acidmolecules from pre-selected sequences, rather than fromrandom sequences, would increase the speed of design-ing large molecules and also increase the accuracy ofdesign. Helices should be formed in the optimal foldingfree energy change range, have maximal structureprobability, and minimal ensemble defect. Loops shouldbe composed of sequences with the lowest ensemble freeenergy change. All sequences should have low tendencyto cross- and self-hybridize. These features are observedin RNA sequences with known structure.We demonstratethat preselected sequences and accelerate the design ofstructures that are mimics of biologically relevant struc-tures. This is implemented as a new structure designcomponent of RNAstructure (http://rna.urmc.rochester.edu/RNAstructure.html). This work is a collaborationwith Celadon Laboratories, Inc. (http://www.celadonlabs.com/).

30 Simulation of RNA tandem GA basepairs provides insights about theforces behind conformationalpreference

David H. Mathews and Asaminew H. Aytenfisu*

Department of Biochemistry & Biophysics and Center for RNABiology, University of Rochester Medical Center, Office:3-8807, 601 Elmwood Avenue, Box 712, Rochester, NY 14642,USA

*Email: [email protected], Phone: (585) 275-1748,Fax: (585) 275-6007

Conformational changes are important for RNA function.We used molecular mechanics with all-atom models tounderstand conformational preference in RNA tandemguanine–adenine (GA) base pairs. These tandem GAbase pairs play important roles in determining the stabil-ity and structural dynamics of RNA tertiary structures.Previous solution structures showed that these tandemGA base pairs adopt either imino (cis-Watson-Crick/cis-Watson-Crick interaction) or sheared (trans-Hoogsteen/trans-Hoogsteen interaction) pairing depending upon thesequence and orientation of the adjacent base pairs. In oursimulations, we modeled (GCGGACGC)2 (Wu and Turner1996) and (GCGGAUGC)2 (Tolbert et al., 2007), experi-mentally preferred as imino and sheared, respectively.Besides the experimentally preferred conformation, weconstructed models of the nonnative conformations bychanging cytosine to uracil or uracil to cytosine. We usedexplicit solvent molecular dynamics and free energy cal-culation with umbrella sampling to measure the freeenergy deference of the experimentally preferred confor-mation and the nonnative conformations. A modificationto ff10 was required, which allowed the guanine bases’amino group to leave the base plane (Yildirim et al.,2009). With this modification, the RMSD of unrestrainedsimulations and the free energy surfaces are improved,suggesting the importance of electrostatic interactions byG amino groups in stabilizing the native structures.

ReferencesTolbert, B. S., Kennedy, S. D., Schroeder, S. J., Krugh, T. R.,

& Turner, D. H. (2007). NMR structures of (rGCU-GAGGCU)2 and (rGCGGAUGCU)2: Probing the structuralfeatures that shape the thermodynamic stability of GApairs. Biochemistry, 46, 1511–1522.

Wu, M., & Turner, D. H. (1996). Solution structure of r(GCGGACGC)2 by two-dimensional NMR and the iterativerelaxation matrix approach. Biochemistry, 35, 9677.

Yildirim, I., Stern, H. A., Sponer, J., Spackova, N., &Turner, D. H. (2009). Journal of Chemical Theory andComputation, 5, 2088–2100.


31 Discovery of novel ncRNA byscanning multiple genome alignments

Yinghan Fua, Zhenjiang Xua, Zhi J. Lub, Shan Zhaoa andDavid H. Mathewsa,c*aDepartment of Biochemistry & Biophysics and Center forRNA Biology, University of Rochester Medical Center,Rochester, NY, USA; bSchool of Life Sciences, TsinghuaUniversity, Beijing, China; cDepartment of Biostatistics &Computational Biology, University of Rochester MedicalCenter, Rochester, NY, USA*Email: [email protected], Phone: (585)275-1734, Fax: (585)-275-6007

Recently, non-coding RNAs (ncRNAs) have beendiscovered with novel functions, and it has beenappreciated that there is a pervasive transcription.Therefore, de novo computational ncRNA detectionthat is accurate and efficient is desirable. The purposeof this study is to develop a ncRNA detection methodbased on structural conservation. A new method calledMultifind, based on Multilign (Xu & Mathews, 2011),was developed. It uses an algorithm that predicts com-mon structures among multiple sequences and esti-mates the probability that input sequences are ncRNAusing a classification support vector machine (SVM).Multilign uses Dynalign (Mathews & Turner, 2002),which folds and aligns two sequences simultaneouslywithout requiring any sequence identity; its structureprediction quality will therefore not be affected byinput sequence diversity. Benchmarks showed, Multi-find performs better than RNAz on testing sequencesextracted from Rfam database (Gardner et al., 2011),especially on sequences that are more diverse. For denovo ncRNA discovery in genomes, Multifind had anadvantage in low similarity regions of genome align-ments. Multifind takes about 48 hours to finish scan-ning the whole yeast genome alignment and RNAztakes about 4 hours, therefore, its computationalrequirements do not present a barrier for most of theusers.

The program was implemented in C++ and is included inRNA structure package (Reuter & Mathews, 2010):http://rna.urmc.rochester.edu.

ReferenceXu, Z. J., & Mathews, D. H. (2011). Multilign: An algorithm

to predict secondary structures onserved in multiple RNAsequences. Bioinformatics, 27, 626–632.

Mathews, D. H., & Turner, D. H. (2002). Dynalign: An algo-rithm for finding the secondary structure common to twoRNA sequences. Journal of Molecular Biology, 317, 191–203.

Gardner, P. P., et al. (2011). Rfam: Wikipedia, clans and the “dec-imal” release. Nucleic Acids Research, 39, D141.

Reuter, J. S., & Mathews, D. H. (2010). RNAstructure: Soft-ware for RNA secondary structure prediction and analysis,BMC Bioinformatics, 11.

32 High-resolution cryo-electronmicroscopy structure of theTrypanosoma brucei ribosome

Yaser Hashema*, Amedee desGeorgesa*, Jie Fub,Sarah N. Bussc, Fabrice Jossinetd, Amy Jobee,Qin Zhangf, Hstau Y. Liaob, Robert A. Grassuccia,Chandrajit Bajajf, Eric Westhof d,Susan Madison-Antenuccic and Joachim Franka,e

aHHMI, Department of Biochemistry and MolecularBiophysics, Columbia University, 650 W 168[th] St., New York,NY 10032, USA; bDepartment of Biochemistry and MolecularBiophysics, Columbia University, New York, NY, USAWadsworth Center, Albany, NY, USA; cInstitut de BiologieMoléculaire et Cellulaire (CNRS), Strasbourg 67084, France;dDepartment of Biological Sciences, Columbia University, NewYork, NY, USA; eDepartment of Computer Science, Universityof Texas, Austin, TX, USA*Email: [email protected], Phone: 212.305.9547,Fax: 212.305.9500, *[email protected],Phone: 212.305.9524, Fax: 212.305.9500

Ribosomes, the protein factories of living cells, translategenetic information carried by messenger RNAs intoproteins, and are thus involved in, virtually, all aspectsof cellular development and maintenance. The few avail-able structures of the eukaryotic ribosome reveal that itis more complex than its prokaryotic counterpart owingmainly to the presence of eukaryote-specific ribosomalproteins and additional ribosomal RNA insertions, calledexpansion segments. The structures also differ amongspecies, partly in the size and arrangement of theseexpansion segments. Such differences are extreme inkinetoplastids. Here, we present a high-resolution (�5Å)cryo-electron microscopy structure of the ribosome ofTrypanosoma brucei, the parasite that is transmitted bythe tsetse fly and that which causes African SleepingSickness. The atomic model reveals the unique featuresof this ribosome, characterized mainly by the presence ofunusually large eukaryotic expansion segments and ribo-somal protein extensions leading to the formation of fouradditional inter-subunit bridges. We also find additionalrRNA insertions, including one large rRNA domain thatis not found in other eukaryotes. Furthermore, the struc-ture reveals the five cleavage sites of the kinetoplastidlarge ribosomal subunit rRNA chain, which is known tobe cleaved uniquely into six pieces and suggests thatthe cleavage is important for the maintenance of the T.brucei ribosome in the observed structure. We discussseveral possible implications of the large rRNA expan-sion segments for the translation regulation process, and


we show a possible link between the protein translationinitiation process and expansion segments 6 and 7 on thesmall ribosomal subunit. Our structure could serve as abasis for future experiments aimed at understanding thefunctional importance of these kinetoplastid-specificribosomal features in protein translation regulation,

an essential step towards finding effective and safekinetoplastid-specific drugs.

ReferenceHashem Y. et al. (2013). Nature. doi:10.1038/nature11872

33 High-resolution cryo-EM structure ofthe Trypanosoma brucei 80S: aunique eukaryotic ribosome

Amy Jobea*, Yaser Hashema, Amedee des Georgesa,Jie Fub, Sarah N. Bussc, Fabrice Jossinetd, Qin Zhange,Hstau Y. Liaob, Bob Grassuccic, Chandrajit Bajaje,Susan Madison-Antenuccic, Eric Westhofd andJoachim Franka,b,f

aHHMI, Department of Biochemistry and Molecular Biophysics,Columbia University, New York, NY, USA; bDepartment ofBiochemistry and Molecular Biophysics, Columbia University,New York, NY, USA; cDivision of Infectious Diseases, WadsworthCenter, New York State Department of Health, Albany, NY, USA;dArchitecture et Réactivité de l’ARN, Université de Strasbourg,Institut de Biologie Moléculaire et Cellulaire (CNRS),Strasbourg, France; eDepartment of Computer Science, Institutefor Computational Engineering and Sciences, University ofTexas, Austin, TX, USA; fDepartment of Biological Sciences,Columbia University, New York, NY, USA*Email: [email protected]

Eukaryotic 80S ribosomes of known structure are farmore complex than their 70S bacterial counterparts.Those from Saccharomyces cerevisiae, Tetrahymenathermophila, and Triticum aestivum, for example, bearinsertions of ribosomal RNA (rRNA) called expansionsegments (ES) and additional ribosomal proteins. Theribosomes of the kinetoplastid Trypanosoma brucei,

though, are especially fascinating: structurally andtheir other kinetoplastids’ ribosomes bear very largeESs, as well as smaller ESs, and protein extensions.Additionally, T. brucei ribosomes require novel proteinfactors for maturation, although they do not requireseveral eukaryotic initiation factors or a recycling fac-tor. As a species, T. brucei is fascinating not only interms of structure, but also in terms of gene expres-sion and even public health: the species is responsiblefor the incurable, terminal human African Trypanoso-miasis (sleeping sickness); and during post-transcrip-tional regulation, a single common RNA segmentcalled a splice leader is trans-spliced onto the 5′ endsof many of T. brucei’s mRNAs. The purpose of thissplice event in translation is unknown. Here, we pres-ent a high-resolution structure of the T. brucei ribo-some which contributes a great deal to addressing theabove unknowns. We have employed map segmenta-tion, homology modeling, ab initio rRNA modeling,and Molecular Dynamics Flexible Fitting (MDFF) tomodel the ribosome’s atomic structure. The positionsand structures of the ribosome’s novel ESs and pro-tein extensions were previously unknown, but ourstructure reveals the precise spatial contexts of thesecomponents. With this information in hand, we canbegin to decipher T. brucei’s unusual translationalrequirements.


34 Cyo-EM visualization ofMycobacterium 70S ribosome revealsunique structural components at thefunction sites

Jayati Sengupta* and Manidip Shasmal

Structural Biology & Bio-Informatics Division, CSIR-IndianInstitute of Chemical Biology, 4, Raja S.C Mullick Road,Kolkata 700032, India*Email: [email protected], Phone: 91-33-24995802,Fax: 91-33-24735197

The 3D structures of prokaryotic and eukaryoticribosomes by crystallography and electron microscopyhave revealed that they share an evolutionarily conservedcore (Schmeing & Ramakrishnan, 2009), but each of theribosomes contains its own set of specific proteins (orextensions of conserved proteins) and expansionsegments of rRNAs (Melnikov et al., 2012). How thesedifferences correlate to function still remains largelyunknown. A 3D cryo-EM map of the 70S ribosome fromMycobacterium smegmatis (Msm70S) unveiled strikingnew structural features (Shasmal & Sengupta, 2012). Thecore of the Msm70S shows overall similarity with thecore of the Escherichia coli 70S ribosome while contain-ing additional mass in the periphery and solvent exposedsides. Some of the Mycobacterium ribosomal proteinsare significantly bigger as compared to the E. coli coun-ter parts. The rRNAs also contain extra helices, alsorevealed by their secondary structures. Most of the addi-tional density of the Msm70S can be largely attributed tothe extra helices present in the rRNAs, and extradomains of homologous proteins. One of the most nota-ble features appears in the large subunit near L1 stalk asa structure forming a long helix with its upper endlocated in the vicinity of the mRNA exit channel (whichwe term the ‘steeple’). We propose that the prominenthelical structure in mycobacterium 23S rRNA partici-pates in modulating different steps of translation, espe-cially the E site tRNA exit mechanism and propagationof mRNA 5′ end.

This work was supported by the CSIR-Indian Institute ofChemical Biology, Kolkata, India.

ReferencesMelnikov, S., Ben-Shem, A., Garreau de Loubresse, N., Jenner,

L., Yusupova, G., & Yusupov, M. (2012). One core, twoshells: bacterial and eukaryotic ribosomes. Nature Struc-tural & Molecular Biology, 19, 560–567.

Schmeing, T. M. & Ramakrishnan, V. (2009). What recent ribo-some structures have revealed about the mechanism oftranslation. Nature, 461, 1234–1242.

Shasmal, M. & Sengupta, J. (2012). Structural diversity in bac-terial ribosomes: Mycobacterial 70S ribosome structurereveals novel features. PLoS ONE, 7, e31742.

35 The energy landscapes of ribosomefunction

Jeffrey K. Noela, Karissa Y. Sanbonmatsub andPaul C. Whitfordc*aCenter for Theoretical Biological Physics, Rice University,6100 Main, Houston, TX 77005-1827, USA; bTheoreticalBiology and Biophysics Group, Los Alamos NationalLaboratory, Los Alamos, NM 87545, USA; cDepartmentof Physics, Northeastern University, 360 Huntington Ave,123 Dana Research Center, Boston, MA 02115, USA*Email: [email protected], Phone: (617) 373-2952,Fax: (617) 373-2943

The vast information available about ribosome structureand dynamics makes it the ideal system for systematicallyinvestigating the physical–chemical properties that lead tobiological function. To provide a comprehensive frame-work for understanding the relationship between the struc-tural, energetic, diffusive, and kinetic aspects of large-scale collective dynamics, we use explicit-solvent simula-tions of complete ribosomes (2–3 million atoms), in addi-tion to molecular models that employ simplifieddescriptions of the energetics (40,000–150,000 atomsper simulation). From multi-hundred nanosecond explicit-solvent simulations, we have established a quantitativerelationship between tRNA accommodation and its associ-ated free-energy barriers. For translocation, we have per-formed a 1.4-microsecond explicit-solvent simulation,from which we identified specific coordinate-spaces thatcapture the diffusive aspects of large-scale ‘ratcheting’ and‘swivel’ rearrangements, thus providing physics-basedcoordinates for describing the free-energy landscape oftranslocation. To complement explicit-solvent calcula-tions, we use all-atom simulations that employ simplifiedenergetic models, with which we partition the contributionof molecular flexibility and energetics during accommoda-tion and translocation. With these simpler models, the roleof electrostatic screening and the responsiveness of theenergy landscape to external perturbations are now being


elucidated, which may aid in the design of novel antibiot-ics. Building on the principles of energy landscape theory,these calculations provide a comprehensive theoreticalfoundation with which experimental kinetics, single-mole-cule measurements, structural/mutational data, and theo-retical calculations may be consistently interpreted andanalyzed.

36 Identification and characterization ofsmall subunit ribosomalintermediates

Neha Gupta, Keith Connolly and Gloria M. Culver*

Departments of Biology and of Biochemistry and Biophysics,Center for RNA Biology: From Genomes to Therapeutics,University of Rochester, Rochester, NY, USA*Email: [email protected],Phone: (585) 276-3602, Fax: (585) 275-2070

Bacterial ribosome biogenesis is poorly understood espe-cially in terms of the role of ribosomal RNA (rRNA) mat-uration in vivo. A major problem in addressing thesequestions are asynchronous biogenesis, a large populationof mature particles and the lack of techniques to isolatedin vivo formed ribosome biogenesis intermediates. Ourgroup has taken multiple approaches to allow study ofribosome biogenesis in Escherichia coli. We have usedgenetic manipulation to discover that for specific biogene-sis factors, there is a delicate balance that is necessary forviability. Additionally, we have pioneered an affinity puri-fication approach to allow for isolation of in vivo formedintermediates. Data will be present on our findings for therole of rRNA maturation in biogenesis, subsequent ribo-some function, and cell viability. Our findings may resultin identification of novel targets for antimicrobial develop-ment.

This work is supported by a grant from NIHGMSGM624312.

37 A comparative study of ribosomalproteins: linkage between amino aciddistribution and ribosomal assembly

Brittany Burton Lotta, Takuya Nakazatob,c* andYongmei Wanga,b†

aDepartment of Chemistry, The University of Memphis,Memphis, TN 38152, USA; bDepartment of Bioinformatics,The University of Memphis, Memphis, TN 38152, USAcDepartment of Biological Sciences, The University ofMemphis, Memphis, TN 38152, USA*Email: [email protected], Phone: (901) 678-5539,Fax: (901) 678-4457, †[email protected], Phone: (901)678-2629, Fax: (901) 678-3447

Assembly of the ribosome from its protein and RNAconstituents has been studied extensively over the past50 years, and here we utilize a comparative analysisapproach to relate the composition of ribosomal pro-teins (r-proteins) to their role in the assembly process.We computed the amino acid distributions for the30S subunit r-protein sequences from 560 bacterialspecies and compared this composition to those ofother house-keeping proteins from the same species.We found that r-proteins have a significantly highercontent of positively charged residues (Lysine, K, andArginine, R) than do nonribosomal proteins (10% forR and 11% for K in r-proteins, vs. 4.7% R and5.9% K in non-ribosomal proteins), which is consis-tent with prior knowledge of net positive charges car-ried by r-proteins (Baker et al., 2001; Klein et al.,2004; Burton et al., 2012). Furthermore, these tworesidues are also highly represented at contact sitesalong the protein/RNA interface (contact enrichmentfactor (CEF) > 1). These results provide further evi-dence of the importance of electrostatic interactionsbetween the positively charged proteins and negativelycharged ribosomal RNA (rRNA) during ribosomeassembly. Other highly represented contact residuesinclude polar and aromatic residues, which are likelyto interact with rRNA via hydrogen bonds and basestacking interactions, respectively. Interestingly, theproportion of K residues generally decreases with r-protein size, reflecting a negative correlation betweenprotein lengths and the proportion of K (Spearman’srank correlation, ρ=�0.802, p= 2.60e� 5). We sug-gest that this trend helps the smaller r-proteins, whichexperience higher translational entropy than large pro-teins, overcome the increased free energy barrier dur-ing assembly. When the r-protein sequences werecategorized according to the species’ optimal growthtemperature, we found that thermophiles showincreased R, Isoleucine (I), and Tyrosine (Y) content,whereas mesophiles have increased proportions of Ser-ine (S) and Threonine (T). These results reflect onetypical distinction between thermophiles and meso-philes (Kumar and Nussinov 2001), yet these differ-ences in amino acid distributions do not extend totheir respective contact sites. That is, the makeup ofthermophilic and mesophilic r-protein contact residuesare not significantly different (p > 0.01). This indicatesthat, while the percent compositions of amino acidsrelating to qualities such as thermostability and pro-tein folding are expected to vary with environmentaltemperature, the distributions of residues in contactwith rRNA are comparable for all bacterial species.From this, we conclude that the electrostatic interac-tions that guide ribosome assembly are independentof temperature.


ReferencesBaker, N. A., D. Sept et al. (2001). Electrostatics of nanosys-

tems: application to microtubules and the ribosome.Proceedings of National Academy of Sciences of the UnitedStates of America, 98, 10037–10041.

Burton, B., Zimmermann, M. T., et al. (2012). A computa-tional investigation on the connection between dynamicsproperties of ribosomal proteins and ribosome assembly.Plos Computational Biology, 8, e1002530.

Klein, D. J., Moore, P. B., et al. (2004). The roles of ribosomalproteins in the structure assembly, and evolution of the largeribosomal subunit. Journal of Molecular Biology, 340, 141–177.

Kumar, S. & Nussinov, R. (2001). How do thermophilicproteins deal with heat? Cellular and Molecular LifeSciences, 58, 1216–1233.

38 A network of interactions within thesupraspliceosome

Joseph Sperlinga and Ruth Sperlingb*aDepartment of Organic Chemistry, The Weizmann Institute ofScience, Rehovot 76100, Israel; bDepartment of Genetics, TheHebrew University of Jerusalem, Jerusalem 91904, Israel*Email: [email protected], Phone: (972) 2-6585162,Fax: (972) 2-5617920

Pre-mRNA splicing of RNA polymerase II transcripts isexecuted in the cell nucleus within a huge (21 MDa)and highly dynamic RNP machine – the supraspliceo-some. Supraspliceosomes harbor all five spliceosomal UsnRNPs, as well as protein splicing factors, and theyare functional in splicing. Supraspliceosomes also har-bor other components of pre-mRNA processing, suchas the RNA-editing enzymes ADAR1 and ADAR2,cap-binding proteins, and 3'-end processing components.The supraspliceosome is composed of four nativespliceosomes, each resembling the spliceosome assem-bled in vitro, and they are connected by the pre-mRNA(Sperling et al., 2008). The structure of the nativespliceosome, at a resolution of 20Å, was determinedby cryo-EM (Azubel et al., 2004). A unique spatial

arrangement of the spliceosomal U snRNPs within thenative spliceosome emerged from in silico studies(Frankenstein et al., 2012). The model localizes the fiveU snRNPs mostly within the large subunit of the nativespliceosome, requiring only minor conformationchanges, and the components of the active core of thespliceosome are found sheltered deep within the largespliceosomal cavity. We use gold-tagged probes andcryo-EM to experimentally confirm this model. Thesupraspliceosome structure likely provides a platformfor regulation of RNA-processing activities (e.g. alterna-tive splicing). Indeed, all the phosphorylated splicingregulators SR proteins are predominantly found associ-ated with supraspliceosomes. Furthermore, both the con-stitutively and alternatively spliced mRNAs of testedendogenous human Pol II transcripts are predominantlyfound in supraspliceosomes, and changes in alternativesplicing occur within them (Sebbag-Sznajder et al.,2012). These findings substantiate the role of thesupraspliceosome as the nuclear pre-mRNA processingmachine.

This research has been supported by the US NIH, grantGM079549 to R.S. and J.S., and the Helen and MiltonKimmelman Center for Biomolecular Structure andAssembly at the Weizmann Institute to J.S.

ReferencesAzubel, M., Wolf, S. G., Sperling, J., & Sperling, R.

(2004). Three-dimensional structure of the native spliceo-some by cryo-electron microscopy. Molecular Cell, 15,833–839.

Frankenstein, Z., Sperling, J., Sperling, R., & Eisenstein, M.(2012). A unique spatial arrangement of the snRNPs withinthe native spliceosome emerges from in silico studies.Structure, 20, 1097–1106.

Sebbag-Sznajder, N., Raitskin, O., Angenitzki, M., Sato, T. A.,Sperling, J., & Sperling, R. (2012). Regulation ofalternative splicing within the supraspliceosome. Journal ofStructural Biology, 177, 152–159.

Sperling, J., Azubel, M., & Sperling, R. (2008). Structure andfunction of the pre-mRNA splicing machine. Structure, 16,1605–1615.


39 In-silico study of the arrangement ofthe snRNPs within the nativespliceosome

Ziv Frankensteina, Joseph Sperlingb, Ruth Sperlingc andMiriam Eisensteind*aDepartment of Structural Biology, Weizmann Institute ofScience, Rehovot 76100, Israel; bOrganic Chemistry, WeizmannInstitute of Science, Rehovot 76100, Israel; cDepartment ofGenetics, The Hebrew University of Jerusalem, Jerusalem91904, Israel; dChemical Research Support, Weizmann Instituteof Science, Rehovot, 76100, Israel*Email: [email protected], Phone: 972-89343031, Fax: 972-89343361

The elaborate process of transforming the informationcoded in the DNA to protein molecules is performedby several large and intricate molecular machines:RNA polymerase II transcribes the coded genes topre-mRNAs, the spliceosome processes the pre-mRNAs, eliminating noncoding introns and producingfunctional mRNAs, and the ribosome translates thegenetic code embedded in the mRNAs and catalyzesthe synthesis of proteins. The spliceosome is a hugemega-Dalton ribonucleoprotein (RNP) assembly. Elec-tron microscopy structures of the native spliceosomeand of several spliceosomal subcomplexes, such as thespliceosomal U snRNPs, are available but the spatialarrangement of the latter within the native spliceosomeis not known. We developed fitEM2EM computationaltools (Frankenstein et al., 2008), that match and docklow resolution structures. Next, we represented eachspliceosomal subcomplex by an ensemble of normal-modes conformers and designed a new “conformerselection” procedure that efficiently fitted the thousandsof conformers into the native spliceosome envelope.Despite the low resolution limitations, we obtainedonly one model that complies with the available bio-chemical data. Our model localizes the five smallnuclear RNPs (snRNPs), mostly within the large sub-unit of the native spliceosome, requiring only minorconformation changes. The remaining free volume pre-sumably accommodates additional spliceosomal compo-nents. Moreover, the ample free volume suggests thatstructural modulations of the snRNPs can be toleratedwhile keeping the integrity of the spliceosome assem-bly. The constituents of the active core of the spliceo-some are juxtaposed in our model, forming acontinuous surface deep within the large spliceosomalcavity. This cavity emerges as the site of mRNA bind-ing and splicing; its depth provides a sheltered envi-ronment for the splicing reaction (Frankenstein et al.,2012). To experimentally localize U snRNPs withinthe native spliceosome and validate the model, we usegold nanoclusters of 1.5 nm in diameter, covalently

attached to antisense oligodeoxynucleotides, each com-plementary to one of the spliceosomal U snRNAs.

This research has been supported by the US NIH, grantGM079549 to R.S. and J.S., and the Helen and MiltonKimmelman Center for Biomolecular Structure andAssembly at the Weizmann Institute of Science.

ReferencesFrankenstein, Z., Sperling, J., Sperling, R., & Eisenstein, M.

(2008). FitEM2EM – Tools for low resolution study of mac-romolecular assembly and dynamics. PloS ONE, 3, e3594.

Frankenstein, Z., Sperling, J., Sperling, R., & Eisenstein, M.(2012). A unique spatial arrangement of the snRNPs withinthe native spliceosome emerges from in-silico studies.Structure, 20, 1097–1106.

40 Probing single molecule RNA foldingusing temperature and force

William Stephensona, Rachel Santiagob, Sean Kellerb,Scott Tenenbauma,c, Michael Zukerc,d andPan T.X. Lib,c*aCollege of Nanoscale Science and Engineering, Universityat Albany, SUNY, 257 Fuller Rd., Albany, NY 12222, USA;bDepartment of Biological Sciences, University at Albany,SUNY, 1400 Washington Ave, Albany, NY 12222, USA;cThe RNA Institute, University at Albany, SUNY, 1400Washington Ave, Albany, NY 12222, USA; dDepartment ofMathematics, Rensselaer Polytechnic Institute, 110 8[th]Street, Troy, NY 12180, USA*Email: [email protected], Phone: (518) 591-8879,Fax: (518) 442-4767

Nucleic acids can be unfolded either by temperature,such as in UV melting, or by mechanical force usingoptical tweezers. In UV melting experiments, the fold-


ing free energy of nucleic acids at mesophilic tempera-tures are extrapolated from unfolding occurring at ele-vated temperatures. Additionally, single moleculeunfolding experiments are typically performed only atroom temperature, preventing calculation of changes inenthalpy and entropy. Here, we present temperature-controlled optical tweezers suitable for studying foldingof single RNA molecules at physiological temperatures.Constant temperatures between 22 and 37 °C are main-tained with an accuracy of 0.1 °C, whereas the opticaltweezers display a spatial resolution of �1 nm overthe temperature range. Using this instrument, we mea-sured the folding thermodynamics and kinetics of a20-base-pair RNA hairpin by force-ramp and constantforce experiments. Between 22 and 37 °C, the hairpinunfolds and refolds in a single step. Increasing temper-ature decreases the stability of the hairpin and thusdecreases the force required to unfold it. The equilib-rium force, at which unfolding and refolding rates areequal, drops �1 pN as temperature increases every 5 °C. At each temperature, the folding energy can bequantified by reversible work done to unfold the RNAand from the equilibrium constant at constant forces.Over the experimental temperature range, the foldingfree energy of the hairpin depends linearly on tempera-ture, indicating that ΔH is constant. The measuredfolding thermodynamics are further compared with thenearest neighbor calculations using Turner’s parametersof nucleic acid folding energetics.

41 The essential adenosine stacking in atwo-base-pair minimal kissingcomplex

William Stephensona, Papa Nii Asare-Okaib,Alan A. Chenc, Scott Tenenbauma,d,Angel E. Garciad,e, Daniele Fabrisb,c,d andPan T.X. Lic,e*aCollege of Nanoscale Sciences and Engineering, University atAlbany, SUNY, 1400 Washington Ave, Albany, NY 12222;bDepartment of Chemistry, University at Albany, SUNY, 1400Washington Ave, Albany, NY 12222; cDepartment of BiologicalSciences, University at Albany, SUNY, 1400 Washington Ave,Albany, NY 12222; dThe RNA Institute, University at Albany,SUNY, 1400 Washington Ave, Albany, NY 12222; eDepartmentof Physics and Center for Biotechnology and InterdisciplinaryStudies, Rensselaer Polytechnic Institute, 110 8[th] Street, Troy,NY 12180*Email: [email protected], Phone: (518) 591-8879,Fax: (518) 442-4767

A stable RNA helix requires at least three base pairs.Surprisingly, a tertiary kissing complex formed betweentwo GACG hairpin loops contains only two GC pairs.

In the NMR structure of this complex, the two flankingadenosines stack on the kissing GC pair. This observa-tion raised a possibility that the 5’-dangling adeninescontribute to the formation and stability of the kissinginteraction. To test this hypothesis, we took atwo-pronged approach to examine the effects of variousmutational and chemical modifications of the flankingadenosines on the folding of the kissing complex.Using mass spectrometry, we studied formation of kiss-ing dimers formed by different hairpins. Using opticaltweezers, we monitored mechanical unfolding of intra-molecular kissing complex at single-molecule level. Inboth experiments, replacing adenine with uridine abol-ished the kissing interaction, suggesting that a minimalkissing complex must contain two GC pairs flanked byinter-strand stacking adenines. The stabilizing effect bythe adenines can be explained by the fact that thestacking purine nucleobases shield the hydrogen bondsof the adjacent GC pairs, preventing them from fraying.Unlike in the context of secondary structure, the 5’-unpaired adenines in the tertiary structure are structur-ally constrained in a way that allows for effectivestacking onto the adjacent base pairs.

42 Computer folding of RNAtetraloops? Are we there yet?

Petra Kührováa*, Pavel Banáša, Robert B. Bestb,Jiří Šponerc and Michal Otyepkaa,c

aFaculty of Science, Department of Physical Chemistry andRCPTM, Palacky University Olomouc, 17. listopadu 12, 77146 Olomouc, Czech Republic; bLaboratory of ChemicalPhysics, National Institute of Diabetes and Digestive andKidney Diseases, National Institutes of Health, Bethesda, MD20892-0520; cInstitute of Biophysics, Academy of Sciences ofthe Czech Republic, Kralovopolska 135, 612 65 Brno, CzechRepublic*Email: [email protected], Phone: (+420) 585 634 766,Fax: (+420) 585 634 761

The tetraloops (Tls) are basic and unusually stablebuilding blocks of RNA structure that often participatein a variety of biochemical processes – includingnucleation in RNA, folding and formation of tertiarycontacts. Moreover, they are known to play roles intranscription and translation as well as serving as rec-ognition sites for RNA-binding proteins. Understandingthe folding of small RNA hairpins is a critical firststep in understanding the folding of larger RNA mole-cules. In present study, we investigate the folding andunfolding of two RNA Tls at the atomic level basedon replica exchange molecular dynamics (MD) simula-tions. Using the most recent reparametrisation of the


Amber family of RNA force fields (ff99bsc0 OL3

(Banáš et al. 2010, Zgarbová et al. 2011)), we have,for the first time, folded tetraloop structures to within2 Å all-atom RMSD of the native structure involvingall signature interactions of native fold, starting fromfully unfolded conformations. The most native-likestructures reported by previous studies were only �4Åfrom native; due to force field deficiencies, Tl struc-tures degraded even in short MD simulations initiatedfrom folded (Banáš et al. 2010). We described thefolding pathway, which is very similar for both tetralo-ops, and it is comparable with experimental results.Further, we can use our results to address severalquestions related to the biological function of tetralo-ops. For example, an open question is whether theantibiotic restritocin induces the conformational changeof the GNRA TL or binds to a transiently unstructuredGNRA TL. We observed the misfolded state of UUCGTl recently suggested by mutation experiments, whichmost likely acts as a kinetic trap during UUCG fold-ing. We can also relate our results to ultrafast spec-trosopy experiments that identified some features ofmisfolded states of GNRA TLs reminiscent of the mis-folded-compact GAGA structure identified in thisstudy. Nevertheless, our simulations show that despiteREMD technique being powerful tool which significantincreases the sampling, obtaining an equilibriumbetween the unfolded, misfolded and folded states isstill very challenging.

The authors gratefully acknowledge the support by theOperational Programme Research and Development forInnovations – European Regional Development Fund(project CZ.1.05/2.1.00/03.0058), the OperationalProgramme Education for Competitiveness – EuropeanSocial Fund (project CZ.1.07/2.3.00/20.0017), and Inte-gration of Regional Centre of Advanced Technologies

and Materials into International Networks of Nanotech-nological and Optical Research (project CZ.1.07/2.3.00/20.0058).

ReferencesBanáš, P., Hollas, D., Zgarbová, M., Jurečka, P., Orozco, M.,

Cheatham, T. E., … Otyepka, M. (2010). Performance ofmolecular mechanics force fields for RNA simulations.Stability of UUCG and GNRA hairpins. Journal of Chemi-cal Theory and Computation, 6, 3836–3849.

Zgarbová, M., Otyepka, M., Šponer, J., Mládek, A., Banáš, P.,Cheatham, T. E., & Jurečka, P. (2011). Refinement of theCornell et al. nucleic acid force field based on referencequantum chemical calculations of torsion profiles of theglycosidic torsion. Journal Chemical Theory Computation,7, 2886–2902.

43 Why G? Aggregation and gelationof GMP with XMP: the plot thickens

Linda B. McGown*, Lauren Cassidy and Akshar Gupta

Department of Chemistry and Chemical Biology, New YorkCenter For Astrobiology, Rensselaer Polytechnic Institute, Troy,NY 12180, USA*Email: [email protected], Phone: (518) 276-3681,Fax: (518) 276-4887

GMP alone, among the individual ribonucleotides,exhibits a reversible self-aggregation through hydrogenbonding to form tetrads that are the building blocks ofhigher order structures. These “G-tetrads” can furtherassociate through π–π stacking to form chiral, colum-nar aggregates and, at higher monomer concentrations,lyotropic liquid crystalline phases. This alternate path-way for GMP should compete with its incorporation


into oligonucleotides, which is why it is difficult tosynthesize or amplify highly G-rich RNA or DNAwith good efficiency in the absence of natural proteins,such as helicases, that function to unwind the strands.Given this competing pathway for GMP, we can ask ifit came to be one of the four ribonucleotides in mod-ern RNA in spite of, or because of, its unique proper-ties. Our hypothesis is that the competition betweenreversible aggregation and covalent polymerization direc-ted RNA toward sequences that were best suited to lifeon early earth. We find support in the observation that thesame interactions that promote self-assembly of mono-meric GMP also promote folding of G-rich RNA andDNA sequences to form inter- and intramolecular G-quadruplex structures. Such sequences are prevalentthroughout the biological world and are thought to serveimportant functions related to genomic stability and generegulation. G-quadruplex structures are also commonmotifs in aptamers, which are combinatorially derivedDNA or RNA sequences that exhibit highly selective,high-affinity binding to molecular and macromoleculartargets. An important consideration for GMP aggregationin a prebiotic RNA World scenario is the effect of otherXMP on GMP self-assembly. In this talk, we will focuson the properties of solutions containing mixtures ofGMP with AMP, CMP, and UMP. The results show thateach nucleotide exerts a different influence on the self-assembly of GMP, raising interesting questions about sce-narios on prebiotic Earth that would be consistent withabiotic RNA polymerization.

This research is supported by NASA NAI through theNew York Center for Astrobiology at Rensselaer Poly-technic Institute (Grant NNA09DA80A).

44 Circular dichroism (CD) studies ofthe interaction betweenG-quadrule xes/I-motif with saffronsecondary active metabolites

S. Zahra Bathaie*, Reyhane Hoshyar andNasim Shahhamzeie

Department of Clinical Biochemistry, Faculty of MedicalSciences, Tarbiat Modares University, P.O. Box: 14115-111,Tehran, Iran*Email: [email protected]

Telomeric DNA contains some unique secondary struc-tures, such as G-quadruplex and I-motif. These struc-tures may be stabilized or changed by binding tospecific proteins or small molecules. In continuation ofour previous studies on the interaction between crocinand crocetin, as the natural C20 carotenoids, and

picrocrocin and safranal, as the natural monoterpenealdehydes, obtained from saffron and DNA (Bathaieet al., 2007; Hoshyar et al., 2008), herein, we reportthe in vitro effect of these saffron metabolites on thementioned structures (Hoshyar et al., 2012). Circulardichroism (CD) data indicate that crocetin has higheraffinity for these structures. Safranal and crocin inducelittle change in the I-motif and G-quadruplex, respec-tively. The molecular docking confirms the experimen-tal data and indicates the minor groove binding ofligands with G-quadruplex. Effects of these ligands onthe stability of these structures is studied using someother techniques and determination of thermodynamicparameters.

ReferencesBathaie, S. Z., Bolhasani, A., Hoshyar, R., Ranjbar, B.,

Sabouni, F., & Moosavi-Movahedi, A. A. (2007). DNA andCell Biology, 26, 533–540.

Hoshyar, R., Bathaie, S. Z., & Ashrafi, M. (2008). DNA andCell Biology, 27, 1–9.

Hoshyar, R., Bathaie, S. Z., Kyani, A., & Mousavi, M. F.(2012). Nucleoside. Nucleotide, Nucleic Acids, 31,801–812.

45 Electrochemical investigation of theinteraction between G-quadruplexand some ligands usingMWCNT modified carbon pasteelectrode

S. Malelahia, S. Zahra Bathaieb*, M.F. Mousavia andH. Ilkhania

aFaculty of Basic Science, Department of Chemistry, TarbiatModares University, Tehran, Iran; bFaculty of Medical Science,Department of Clinical Biochemistry, Tarbiat ModaresUniversity, Tehran, Iran*Email: [email protected]

The G-quadruplex is an unusual DNA secondary struc-ture based on the Hoogesteen G–G base pairing,which stabilizes in the presence of certain metal cat-ions, mainly alkali ions such as K+ (Elahi, Bathaie,Mousavi, Hoshyar, & Ghasemi, 2012). In continuationof our previous studies on the interaction betweenDNA and small molecules (Ghanbari, Bathaie, Mous-avi, 2008; Heli, Bathaie, & Mousavi, 2004), morerecently, interaction of G-DNA with polyamines andethidium bromide was studied (Elahi et al., 2012). Inthe present study, we investigated the interaction ofcarotenoids (crocin and crocetin) and monoterpenealdehydes (safranal) of saffron with G-DNA. TheMWCNT was ultrasonically treated in 35% solution ofnitric acid for 6 h to be functionalized with carboxylic


acid groups. The carbon paste electrode was preparedby mixing graphite powder with paraffin at the ratio65:35. The resulting paste was packed into Teflontube. The outer layer of the electrode was made ofcarbon paste modified by the addition of modifiedMWCNT at the concentration of 10% (w/w). G-DNAwas covalently immobilized onto the CPE modifiedwith MWCNT (MWCNT/CPE) in the presence ofEDC/NHS. Interaction of G-DNA with the namedligands was investigated by cyclic voltammetry anddifferential pulse voltammetry using the electroactivecomplex [Ru(NH3)6]

3+ (RuHex). The results showedthat crocetin has more affinity than crocin and safranalfor binding to G-DNA. Binding constants for theseinteractions are 4.4� 104, 1.4� 104, and 1.6� 104M�1

for crocetin, crocin and safranal, respectively.

ReferencesElahi, M. Y., Bathaie, S. Z., Mousavi, M. F., Hoshyar, R.,

& Ghasemi, S. (2012). Electrochimica Acta, 82,143–151.

Ghanbari, K., Bathaie, S. Z., & Mousavi, M. F. (2008). Biosen-sors & Bioelectronics, 23, 1825–1831.

Heli, H., Bathaie, S. Z., & Mousavi, M. F. (2004). Electro-chemistry Communications, 6, 1114–1118.

46 Effect of guanine substitutions inhuman telomeric G3(T2AG3)3 DNAsequence

Martin Tomaškoa, Michaela Vorlíčkováa*,Miroslav Fojtaa and Janos Sagib

Institute of Biophysics, Academy of Sciences of the CzechRepublic, Brno, Czech Republic; Rimstone Laboratory, RLI,Cheshire, CT, USA*Email: [email protected], Phone: +420 5 4151 7188,Fax: +420 5 4124 0497

In addition to the well-known Watson–Crick doublehelix, DNA can form other structures. One of them is afour-stranded quadruplex, formation of which was alsoacknowledged in in vivo conditions. It was suggestedthat the presence of quadruplexes in e.g. telomeric regionhas a significant biological importance. We have studiedstructural properties of the human telomeric quadruplexformed by G3(T2AG3)3 and related sequences, in whicheach guanine base was one-by-one replaced by adenine.In the next step, we have studied sequences, in whichtwo, or even four guanines were replaced by adenine.These sequences were studied in the presence of sodiumor potassium ions. Using CD spectroscopy, UV thermalstability measurements, and polyacrylamide gel electro-

phoresis we found that none of the substitutions hinderedthe formation of the antiparallel quadruplex formed bythe unsubstituted sequence in sodium solutions.However, the effect of substitution differed depending onthe position of the guanine replaced. The middle quartetof the antiparallel basket scaffold was the most sensitiveand led to the least stable structures. With othersequences, the effect of substitution depends on the posi-tion and also on the syn/anti glycosidic bond orientationof the appropriate guanosine in the original quadruplexstructure. In the case of the multiple A for G substitu-tions, the G3(T2AG3)3 quadruplex was most destabilizedby the G:G:A:A tetrad, in which the adenosines substi-tuted syn guanosines. Interestingly, unlike withG3(T2AG3)3, no structural transitions were observed withthe A-containing analogs of the sequence when sodiumions were replaced by potassium ions. The basic quadru-plex topology remained antiparallel for all modifiedsequences in both salts. As in vivo misincorporation ofA for a G in the telomeric sequence is possible andpotassium is a physiological salt, these findings may bebiologically important. In our next studies, we have com-pared the effect of the G to A substitutions in the humantelomere sequence with 8-oxoguanine substitutedsamples or samples containing guanine apurinic sites.Data obtained from our study show a noticeable trend: itis not the type of the lesion but the position of the modi-fication determines the effect on the conformation andstability of the quadruplex.

This research has been supported by Grant OP VK(CZ.1.07/2.3.00/30.0019), Grant P205/12/0466 from theGrant Agency of the Czech Republic and by the project‘CEITEC – Central European Institute of Technology’(CZ.1.05/1.1.00/02.0068) from the European RegionalDevelopment Fund.

ReferencesSagi, J., Renciuk, D., Tomasko, M., & Vorlickova, M. Quadru-

plexes of human telomere DNA analogs designed to con-tain G:A:G:A, G:G:A:A and A:A:A:A tetrads. Biopolymers,93, 880–886.

Skolakova, P., Bednarova, K., Vorlickova, M., & Sagi, J.Quadruplexes of human telomere dG3(TTAG3)3sequences containing guanine abasic sites. Biochemicaland Biophysical Research Communications, 399, 203–208.

Tomasko, M., Vorlickova, M., & Sagi, J. Substitution of ade-nine for guanine in the quadruplex-forming human telomereDNA sequence G(3)(T(2)AG(3))(3). Biochimie, 91, 171–179.

Vorlickova, M., Tomasko, M., Sagi, A.J., Bednarova, K., &Sagi, J. 8-oxoguanine in a quadruplex of the human telo-mere DNA sequence. FEBS Journal, 279, 29–39.


47 Incorporation of alternative nucleicacid chemistries intoG-quadruplex structure

Christopher J. Lech* and Anh Tuan Phan†

Department of Physics and Applied Physics, Nanyang Technol-ogy University, Singapore 637371, Singapore*Email: [email protected], †[email protected],Phone: (+65) 6513-7422, Fax: (+65) 6795-7981

Nucleic acids that form G-quadruplex (G4) structure havefound applications in a host of research and technologyregimes. Numerous G4 based aptamer drugs have beenidentified with pharmacological activity against cancer,HIV, prions, and blood coagulation (1). In the field of nano-technology, G4 based sensors and nano-machines have alsoreceived much attention. The ability to synthesize nucleicacid ex-vivo allows for the site-specific incorporation ofnon-natural chemistries into nucleic acids that can be usedto tune their physical and pharmacological properties. Wesummarize the results of a series of studies investigating theeffective incorporation of alternative nucleic acid chemis-tries into G4 DNA. These modified chemistries include C8-modified guanine bases, as well as 2′-F, 2′-F-ANA, andLocked nucleic acid (LNA) modifications to the ribosesugar. We report primarily on the effect of these modifica-tions on G-quadruplex folding topology, thermal stability,and structure. The substitution of LNA-guanosine into thecore guanine tetrads disrupts structure in specific structuralenvironments. On the other hand, 2′-F- and 2′-F-ANA gua-nosine can generally be incorporated without disrupting thestructure when substituted into guanine bases in certainstructural conformations. We find that 2′-F-ANA-guanosineand 2′-F-guanosine are powerful tools for controling theconformation of G4 structures (2). Functionalization at theC8 of the guanine base stabilizes in a manner dependent onthe glycosidic conformation of the base, with different mod-ification chemistries stabilizing to varying extents (3). Theresults of these studies provide useful insight on how toeffectively incorporate some useful chemical tools from thegrowing toolbox of modified nucleic acid chemistries intoG-quadruplex nucleic acid.

ReferencesGatto, B., Palumbo, M., & Sissi, C. (2009). Nucleic acid apta-

mers based on the G-quadruplex structure: therapeutic anddiagnostic potential. Current Medicinal Chemistry, 16,1248–1265.

Lech, C. J., Li, Z., Heddi, B., & Phan, A. T. (2012). 2′-F-ANA-guanosine and 2′-F-guanosine as powerful tools forstructural manipulation of G-quadruplexes. ChemicalCommunications, 48, 11425–11427.

Lech, C. J., Lim, J. K. C., Lim, J. M. W., Amrane, S., Heddi,B., & Phan, A. T. (2011). Effects of site-specific guanineC8-modifications on an tramolecular DNA G-quadruplex.Biophysical Journal, 101, 1987–1998.

48 NMR study of the interactionbetween G-quadruplexes and smallmolecules

Wan Jun Chung*, Brahim Heddi and Anh Tuân Phan†

School of Physical and Mathematical Sciences, NanyangTechnological University, Singapore 637371, Singapore*Email: [email protected], †[email protected],Phone: (+65) 6514 1915, Fax: (+65) 6795 7981

Guanine-rich oligonucleotides are able to adopt second-ary DNA structures, known as G-quadruplexes. SuchG-rich sequences are found in human telomeres,promoter regions of oncogenes, 5′ untranslated regions(UTRs) of mRNAs and human intronic sequences.Studies have shown that small molecules can induceanti-cancer effect through stabilizing or promotingG-quadruplex formation. In order to design and developa potent drug, structural details on the interactionbetween small molecules and G-quadruplexes areinvaluable. In this study, we seek to understand thestructural determinants involved in the interactionbetween G-quadruplexes and small molecules. NMRspectroscopy is employed to resolve the structures oftwo intramolecular G-quadruplexes bound to smallmolecules. These resolved complexes allow us to struc-turally design new potent drugs for their application inanti-cancer therapy.

49 Nucleic acids in anhydrous media:G-quadruplex folding governed byKramers rate theory

Irena Mamajanov*, Ford M. Lannan andNicholas V. Hud

School of Chemistry and Biochemistry, Georgia Institute ofTechnology, Atlanta, GA 30332, USA*Email: [email protected], Phone: (404) 385-1166

Structures formed by human telomere sequence (HTS)DNA are of interest due to G-quadruplex formingHTS DNA that has recently generated a tremendousinterest due to its involvement in aging process andcancer. The present study examines HTS in anhydrous,exceptionally viscous deep eutectic solvent (DES),comprised of choline, chloride, and urea. Under theseconditions, HTS adopts an extremely stable “parallel-propeller” form of G-quadruplex, consistent with thepreviously observed effects of diminished water activ-ity. Additionally, the high solvent friction of DESslows the dynamics of HTS folding on the order ofmonths, as opposed to milliseconds in aqueous solu-tion, and allows the entrapment of kinetic intermedi-


ates. Moreover, analogous transition studies of thequadruplex converting from the aqueous buffer struc-ture to the parallel form in 90% DES (w/v) and 40%PEG 200 (v/v) differ from hours to days scaling inver-sely with viscosity \tau � 1/n1.4. This diffusion con-trol over the HTS folding is consistent with Kramersrate theory and these findings highlight the necessityto consider the viscosity of intracellular conditionswhen exploring the structure dynamics of telomeresand drug binding interactions. Lastly, tuning solventviscosity could prove useful in the future study of G-quadruplex dynamics, and applied DNA nano-technol-ogy, where time dependent structural transitions aredesired.

This work was jointly supported by NSF and the NASAAstrobiology Program, under the NSF Center forChemical Evolution, CHE-1004570.

50 Single-molecule studies of humantelomeric G-quadruplexes and theeffect of oxidative damage

Aaron M. Fleming, Na An and Cynthia J. Burrows*

Department of Chemistry, University of Utah, 315 S. 1400 E,Salt Lake City, UT 84112-0350, USA*Email: [email protected], Phone: (801) 585-7290

In the human genome, telomeric DNA has tandemrepeats of the sequence 5′-TTAGGG terminating witha 3′ single-stranded overhang of 100–200 bases.These guanine-rich DNA sequences can fold into tet-rastranded structures, known as G-quadruplexes. Theprecise fold of the G-quadruplex structure is dictatedby the metal ions present which we studied throughthe use of the α-hemolysin ion channel. Being elec-trophoretically driven into the cis side of the α-hem-olysin, the hybrid fold (K+) entered the vestibulemouth leading to current blockages for the durationof the time the DNA resided in the vestibule. Due tothe polymorphic nature of the hybrid folds, therecorded current signatures could be correlated withthe major structural topologies that exist for this foldin solution (e.g. hybrid-1, hybrid-2, and triplex; Mash-imo et al., 2010). The hybrid folds were not capableof traversing to the trans side of the nanopore, whilethe triplex could achieve translocation. The basketfold (Na+) was also able to enter into the vestibulecausing current blockages that were indicative of theorientation in which they entered into the vestibule.When the basket fold entered tail first, slow transloca-tion events were observed. In contrast, the propeller


fold (�3.9 nm, 0.05MK+/5M Li+) exceeds the proteinchannel orifice (�3.0 nm) producing only swift andsmall disturbances to the open channel current. Sec-ondly, oxidative damage to the telomeric sequence isproposed to contribute to telomere shortening, dys-function, and cell aging (Epel et al., 2004). Locationsof the oxidative damages have different effects on theG-quadruplex folding that produced significant changesin their nanopore behavior. Placement of the guanineoxidation product, 8-oxoguanosine (OG), in a top orbottom tetrad results in destabilization of that layer,whereas the presence of OG in a middle tetrad leadsto complete unfolding of the G-quadruplex. Thesebehaviors were determined by their translocation timeswhich correlated with the folding’s free energy sup-ported by NIH GM093099.

ReferencesMashimo, T., Yagi, H., Sannohe, Y., Rajendran, A., &

Sugiyama, H. (2010). Folding pathways of humantelomeric type-1 and type-2 G-quadruplex structures.Journal of the American Chemical Society, 132,14910–14918.

Epel, E. S., Blackburn, E. H., Lin, J., et al. (2004). Acceleratedtelomere shortening in response to life stress. Proceedingsof the National Academy of Sciences of the USA, 101,17312–17315.

51 Platinum(II) phenanthroimidazoleswith “clicked” side chains as selectiveG-quadruplex DNA binders

Katherine J. Castor*, Johans Akhoury, Zhaomin Liu,Mark Hancock, Anthony Mittermaier, Nicolas Moitessierand Hanadi F. Sleiman

Department of Chemistry, McGill University, Montreal, H3A0B8 QC, Canada*Email: [email protected], Phone: (514) 398-6921, Fax: (514) 398-3797

G-quadruplexes (noncanonical secondary structures),have gained recognition as viable targets for chemo-therapeutic drug design based on their ability to inter-fere with cancer cell proliferation. These DNAstructures, held together by Hoogsteen hydrogen bonds,result from the folding of guanine (G)-rich DNAsequences in the presence of potassium or sodium cat-ions.(Lane et al., 2008) G-quadruplex (G4)-formingsequences have been identified throughout the humangenome, and depending on the sequence, differentmonomeric topologies prevail. These varying topolo-

gies present a means of specific targeting of one poly-morph rather than all polymorphs. Two regions thathave been highly investigated as G4 targets for smallmolecule drugs are the telomeres and the promoters ofoncogenes. To date, both organic and inorganic com-pounds with varying degrees of efficacy in their target-ing of G4 structures have been reported.(Georgiades et al., 2010; Monchaud & Teulade-Fichou,2008) We have previously shown that Pt(II) phenan-throimidazole binders are good stabilizers of bothintermolecular and intramolecular human-telomere-derived G4 motifs.(Castor et al., 2012; Kieltyka et al.,2008) Now, we focus on broadening our target toinclude those G4s derived from the c-myc and c-kitoncogene promoters due to their apparent ability incontrolling gene expression through regulating tran-scription and translation. We hypothesized that theaddition of side chains to our existing phenanthroimi-dazole core will enable our complexes to discriminatebetween different groove environments presented bytopologically distinct G4s. Thus, we have taken ourcore and have appended “clicked”-amine containingside chains from the phenyl moiety of the phenylphen-anthroimidazole. Through a high-throughput fluores-cence intercalator displacement assay, circulardichroism, surface plasmon resonance, and molecularmodeling, we have evaluated three complexes withG4s derived from human telomere, c-myc, and c-kitsequences and have shown enhanced binding of thecomplexes with the c-kit sequence and an unprece-dented kinetic profile from SPR. We hope that thiskinetic profile results in a long residence time whileinteracting with the c-kit promoter G-quadruplex,resulting in lower levels of c-kit mRNA in humanglioblastoma cells and subsequent down-regulation ofthe gene expression.

ReferencesCastor, K. J., Mancini, J., Fakhoury, J., Weill, N., Kieltyka, R.,

Englebienne, P., … Sleiman, H. F. (2012). Platinum(II)phenanthroimidazoles for targeting telomeric G-quadruplex-es. ChemMedChem, 7, 85–94.

Georgiades, S. N., Abd Karim, N. H., Suntharalingam, K., &Vilar, R. (2010). Interaction of metal complexes with G-quadruplex DNA. Angewandte Chemie InternationalEdition, 49, 4020–4034.

Kieltyka, R., Fakhoury, J., Moitessier, N., & Sleiman, H. F.(2008). Platinum phenanthroimidazole complexes as G-quadruplex DNA selective binders. Chemistry A EuropeanJournal, 14, 1145–1154.

Lane, A. N., Chaires, J. B., Gray, R. D., & Trent, J. O. (2008).Stability and kinetics of G-quadruplex structures. NucleicAcids Research, 36, 5482–5515.

Monchaud, D. & Teulade-Fichou, M. P. (2008). A hitchhiker’sguide to G-quadruplex ligands. Organic & BiomolecularChemistry, 6, 627–636.


52 Examining the effects of 2′-OHsubstitutions on the structure andstability of the S. cerevisiaetelomerase RNA pseudoknot andtertiary structure

Carla A. Theimer*, Fei Liu and Katelyn M. Jasper

Department of Chemistry, University at Albany, SUNY, Albany,NY 12222, USA*Email: [email protected], Phone: (518) 813-9560,Fax: (518) 442-3463

Structural and functional characterization of thepseudoknot in the Saccharomyces cerevisiae telomeraseRNA (TLC1) demonstrated that tertiary structural inter-actions occur between loop 1 uridines and stem 2 Wat-son–Crick A-U pairs, as previously observed for the K.lactis and human telomerase RNA pseudoknots. Thecontributions of backbone groups in the pseudoknot totelomerase catalysis were investigated using 2′-OH(ribose) to 2′-H (deoxyribose) substitutions and 2′-Omethylation at specific nucleotides within the stem 2pseudoknot region (Huang & Yu, 2010; Qiao & Cech,2008). Based on investigations of the structural andthermodynamic properties of the TLC1 RNA pseudo-knot region, which provided a more detailed descriptionof the secondary structure of the pseudoknot stem 2helical region (Liu et al., 2012), including an additionalupstream stem 2 base-paired sequence, we examinedthe structural and thermodynamic perturbations of the

2′-O methyl and 2′-H substituted pseudoknots usingUV-monitored thermal denaturation experiments, nativegel electrophoresis, CD spectroscopy, and nuclear mag-netic resonance spectroscopy (Liu & Theimer, 2012).These results show a correlation between A-form RNAgeometry, thermodynamic stability, and telomeraseactivity in the triple helix substitutions, and are consis-tent with the identification of the U809 2′-OH as a con-tributor to telomerase activity. We have since extendedthese observations to more completely characterize theeffects of additional substitution types and positions inthe pseudoknot and tertiary structure to obtain greaterinsight into thermodynamic, structural, and functionalconsequences of 2′-OH substitutions in this importantsecondary and tertiary structural element.

ReferencesHuang, C. & Yu, Y.-T. (2010). Targeted 2′-O methylation at a

nucleotide within the pseudoknot of telomerase RNAreduces telomerase activity in vivo. Molecular and CellBiology, 30, 4368–4378.

Liu, F., Kim, Y., Cruickshank, C., & Theimer, C. A. (2012).Thermodynamic characterization of the Saccharomycescerevisiae telomerase RNA pseudoknot domain in vitro.RNA, 18, 973–991.

Liu, F. & Theimer, C. A. (2012). Telomerase activity is sensi-tive to subtle perturbations of the TLC1 pseudoknot 3′ stemand tertiary structure. Journal of Molecular Biology, 423,719–735.

Qiao, F. & Cech, T. R. (2008). Triple-helix structure in telome-rase RNA contributes to catalysis. Nature Structural &Molecular Biology, 15, 634–640.


53 2D to 3D: interaction frequencymaps to chromatin higher-orderfolded conformations

Dario Meluzzi and Gaurav Arya*

Department of NanoEngineering, University of California,La Jolla, San Diego, CA 92093, USA*Email: [email protected], Phone: (858) 822-5542,Fax: (858) 534-9553

Chromatin is a fiber of histone proteins and DNApresent in the nuclei of eukaryotic cells. The higher-order folding of chromatin into chromosomes, whichaffects various genomic processes ranging from tran-scription to recombination, has so far remained elusive.Recently, sophisticated experimental techniques, knownas chromosome conformation capture, have been devel-oped to measure the frequencies of interaction betweendifferent DNA segments within and across chromo-somes (Dekker et al., 2002; Lieberman-Aiden et al.,2009). Such interaction frequencies (IFs) can in princi-ple be used to deduce higher-order folding of the chro-matin fiber, though this is a highly challengingcomputational problem. Here, we present novel compu-tational approach to recover the ensemble of chromatinconformations consistent with a set of given IF mea-surements (Meluzzi & Arya, 2012). Our approach com-prises three modules. A model builder builds arestrained bead-chain model of the examined chromo-somal domain, capturing both the physical properties ofthe chromatin fiber and the looping interactions. Anensemble generator performs a dynamical simulation ofthe restrained bead chain to determine its conforma-tional ensemble and to compute the IFs between allchromatin segments. An adaptive refiner uses a learningalgorithm to iteratively refine the strengths of theimposed restraints until a match between the computedand input IF map is achieved. Our approach thus offersmultiple advantages over existing alternatives: use ofphysical models of chromatin; avoidance ofpreconceived relationships between IFs and spatialdistances; prediction of ensembles rather than uniquestructures; and intrinsic validation of the computedensemble based on IF. The above approach has beenvalidated against multiple simulated test systems, andwe are currently refining the approach against knownexperimental IF and spatial distance measurements. Weexpect the final, refined approach to become a valuabletool for researchers examining the higher-order organiza-tion of chromatin.

This research has been supported by an AmericanCancer Society Instructional Research Grant 70-002provided to G.A. through the Moores Cancer Center,University of California, San Diego.

ReferencesDekker, J., Rippe, K., Dekker, M., & Kleckner, N. (2002).

Capturing chromosome conformation. Science, 295, 1306–1311.

Lieberman-Aiden, E., et al. (2009). Comprehensive mapping oflong-range interactions reveals folding principles of thehuman genome. Science, 326, 289–293.

Meluzzi, D. & Arya, G. (in press). Recovering ensembles ofchromatin conformations from contact frequencies. NucleicAcids Research. doi: 10.1093/nar/gks1029.

54 DNA conformation and energy innucleosome core: a theoreticalapproach

Davood Norouzi*

Department of Biological Sciences, Institute for AdvancedStudies in Basic Sciences, Zanjan, Iran*Email: [email protected], Phone: (+98) 241-415 3101

Present address: DNA and Nucleoproteins Section, Laboratoryof Cell Biology, National Cancer Institute, NIH, Bethesda, MD

In this work, we developed a mechanical model toaddress the problem of DNA structure and energy underdeformation. DNA in nucleosome core particle isdescribed as an example. The structure and energy ofnucleosomal DNA is calculated based on its sequenceand positioning state. Our theory is on the level of basepair step parameters. A quadratic elastic energy functiondescribes the deformational energy of the molecule inwhich sequence dependency appears in a matrix of cou-pling constants. This rigidity matrix has been evaluatedbased on molecular dynamics simulation data and X-ray


crystallography structures of DNA. The inferred structurehas been calculated through minimization of the energyand the results show remarkable similarity with X-raydata. Figure 1 compares the base pair step parameters ofa typical nucleosomal DNA (NCP147: PDB id 1kx5)with its inferred structure. Although there is nosequence-specific interaction of bases and the histonecore, we found considerable sequence dependency fornucleosomal DNA positioning. The wrapping affinity for5S rRNA, 601 positioning, TATA box, TGGA repeat,and NCP147 sequences are calculated and comparedwith the experimental data. The structural energy differ-ences, ΔΔG, are in good agreement with the data

obtained by calorimetric approaches for naturalsequences (Table 1). We argue that structural energydetermines the natural state of nucleosomal DNA and isthe main reason for affinity differences in vitro. This the-ory can be utilized for the DNA structure and energydetermination in protein–DNA complexes in general.

ReferenceNorouzi, D. & Mohammad-Rafiee, F. (2013). DNA conforma-

tion and energy in nucleosome core: A theoreticalapproach. Journal of Biomolecular Structure and dynamics,755134.

55 Dynamics of the nucleosome coreparticle revealed from a newdatabase of high resolution X-raycrystallographic and simulatedstructures

Gautam Singh*, Andrew V. Colasanti, Nicolas Clauvelinand Wilma K. Olson

BioMaPS Institute. Rutgers, The State University of NewJersey, Piscataway, NJ 08854, USA*Email: [email protected], Phone: (732) 445-4619

The nucleosome core particle is a highly conservedstructure which can play diverse roles depending on theorganism, cell, or part of chromatin in which it resides.The Protein Data Bank currently contains approximately70 nucleosome core particle structures, over half ofwhich were determined in the last three years. The recentemergence of the field of epigenetics, and the increase indata available from experiments, warrants a need todevelop new approaches to quantitatively compare

various features of interest across multiple structures. Asa first step, we have developed a database and newcomputational tools to allow researchers to quantitativelyanalyze and compare the nucleosome core particle struc-tures deposited in the Protein Data Bank. The features ofthe DNA-protein assembly can be examined in novelcoordinate frames placed on the structure, allowingresearchers to obtain a better understanding of the orga-nization and subtleties of the macomolecular complexes.This comparison allows one to examine the ‘motion’ ofany specific residue of interest, including sites of post-translational histone modification. The database alsoincludes DNA-histone contact points, DNA conforma-tional parameters, and information about protein features,such as the secondary structure in the globular histonecore and the ‘motion’ of the histone tails. Along withthese features, we also characterize the dynamics of theglobal structure of the nucleosome core particle, includ-ing the changes in superhelical path of the DNA and therearrangements of the histone tetramers. In addition todata obtained from crystallographically solved structures,


we are working to incorporate data from in silicoexperiments. The data and the results of the analysis areavailable to the public as an automatically updated,online-accessible web server. Cartoon diagrams of crystalstructures are shown with their reference frames aligned.The frames were computed by PCA of the globular coreof the histone octamer. The anionic atoms of asparticacid and glutamic acid are marked in light red. Thecationic atoms of the arginines and lysines are marked indark blue. Note the channels that the charges mayoccupy.

56 Large-scale alternation ofnucleosome positioning potential inpericentromeric chromosome regions

Viya B. Fedoseyeva* and Alexander A. Alexandrov

Institute of Molecular Genetics of RAS, Kurchatov Sq, Moscow123098, Russia*Email: [email protected], Phone: (732) 445-4619

It is difficult to obtain quantitative information on theprotein bindings in the biological systems in the large-scale variant. We used nucleosome positioning potential(NPP) calculations on the basis of method publishedearlier (Fedoseyeva & Alexandrov, 2007). This methodoften demonstrates cluster character of NPP. Calculationof Furrier coefficients allows to analyze regional prefer-ences of nucleosome binding. Often, the alternation ofstronger and weaker binding regions takes place. Specialinterest deals with large-scale alteration when this maybe interpreted in the terms of cellular biology. Wedetermined them in pericentromeric regions of 2 and 3chromosomes of Drosophila melanogaster. In one of thecases, sufficiently strong alternation of stronger andweaker NPP region was observed. The distance betweennearby stronger ones is approximately 70 kbp. Fortu-

nately, this observation may be interpreted by the compe-tition of nucleosome with cohesin-type proteins forbinding with sister chromatids. This binding maintainsthem side by side during appropriate stages in mitosis.The differences and likeness in NPP between left andright arms of chromosomes are discussed.

ReferenceFedoseyeva, V. B. & Alexandrov, A. A. (2007). Analysis

and development of the computer methods of thenucleosome localization on DNA fragments with differentAT-content. Journal Biomolecular Structure Dynamics,24, 481–488.

57 Nucleosome [mis]positioning,chromatin folding, and acomputational karyotype

Victoria Bamburg, William Johnston, James Liman,James Solow and Thomas C. Bishop*

College of Engineering and Science, Louisiana Tech University,Ruston, LA 71270, USA*Email: [email protected], Phone: (318) 257-5209,Fax: (318) 257-4000

Given a set of experimentally or theoretically deter-mined nucleosome positions, it is possible to rapidlyconstruct and interactively display 3D models of entirechromosomes. Our Interactive Chromatin ModelingWeb (ICM-Web) server can fold and display megabasesegments of chromatin in real time (Stolz & Bishop,2010). These models are first order approximations thatassume each nucleosome is a canonical octasome andthat the linker DNA assumes a sequence-specific con-formation similar to free DNA. Thermal fluctuations areincluded in the model that alters the nucleosome wrap-ping (i.e. entry/exit angle) and linker conformation. Themodels provide valuable insights, even if the chromatinfolds are not necessarily accurate, e.g. visualization ofDNA rotational phasing on individual nucleosomes andhow DNA defects alter global structure. The DNAdefects arise from known sequence-specific conforma-tions and thermal fluctuations of DNA. Nucleosomepositioning or mispositioning alters chromatin topologyby exposing or hiding DNA defects. We utilize theCHA1, MFA2, HIS3, PHO5, and MMTV promotercomplexes to illustrate these ideas. The six positionednucleosomes in the MMTV promoter complex yield amuch more extended structure is typically representedin literature. A compact chromatin structure for theMMTV requires more than six nucleosomes and thusmispositioning of some nucleosomes. Nucleosome [mis]positoning hides or exposes a DNA defect in theMMTV that may regulate chromatin looping associated


with this promoter. Using whole genome nucleosomepositioning data, we generate chromatin folds for eachchromosome of Saccharomyces cerevisiae to produce acomputational karyotype. Thermal motion associatedwith linker DNA plays a critical role in chromatin flex-ibility, reduces the spatial range of each fiber, andallows it to be compacted into the nucleus. ICM isavailable on the Chromatin Folding tab at http://www.latech.edu/~bishop.

This material is based upon work supported by theNational Science Foundation under the NSF EPSCoRCooperative Agreement No. EPS-1003897 with addi-tional support from the Louisiana Board of Regents

ReferenceStolz, R. & Bishop, T. C. (2010). ICM web: The interactive

chromatin modeling web server. Nucleic Acids Research,38, W254–261PMID: 20542915.

58 Packaging trinucleotide repeats: theeffect of CAG/CTG repeats onnucleosome formation

Vilma Medrano* and Sarah Delaney

Department of Molecular Biology, Cellular Biology, andBiochemistry, Brown University, Providence, RI 02906, USA*Email: [email protected],Phone: +1 401-863-2044, Fax: +1 401-863-1993

In neurological diseases such as fragile X syndrome,spinal and bulbar muscular atrophy, myotonic dystrophy,and Huntington’s disease, the molecular basis ofpathogenicity is the presence of an expanded trinucleotiderepeat (TNR) tract (Ashley & Warren, 1995). TNRsimplicated in many of these diseases are composed ofCAG/CTG repeats. For example, in healthy individuals5–35, CAG/CTG TNR repeats are present in thehuntingtin gene. However, individuals with 40 or greaterrepeats will develop Huntington’s disease (Andrew et al.,1993). We are particularly interested in how these TNRsequences are packaged in chromatin. Recent evaluationsof CAG/CTG TNR sequences in our laboratory havedemonstrated that the repeats increase the propensity forthe DNA sequences to incorporate into nucleosomes,where nucleosomes represent the minimal unit ofpackaging in chromatin (Volle & Delaney, 2012). In thiswork, we are interested in determining the minimumnumber of CAG/CTG repeats required to confer a signifi-cant increase in nucleosome incorporation relative tosequences that lack the TNR sequence. By defining thechanges imposed on these fundamental interactions bythe presence of a CAG/CTG repeat tract, we will gain

insight into the possible interactions that allow for theexpansion of these TNR tracts.

This work was supported by the National Institute ofEnvironmental Health Sciences (R01ES019296) and theBrown IMSD program, which is funded by NIGMSgrant R25GM083270.

ReferencesAshley, C. T., Jr, Stephen, T., & Warren, S. T. (1995). Trinucleo-

tide repeat expansion and human disease. Annual review ofgenetics, 29, 703–728.

Andrew, S.E., et al. (1993). The relationship between trinu-cleotide (CAG) repeat length and clinical features ofHuntington’s disease. Nature genetics, 4, 398–403.

Volle, C. B. & Delaney, S. (2012). CAG/CTG repeats alteraffinity for the histone core and positioning of DNA inthe nucleosome. Biochemistry, 51, 9814–9825.

59 The synergy between DNA andnucleosomes in chromatin

Nicolas Clauvelin* and Wilma K. Olson

BioMaPS Institute, Rutgers University, Piscataway, NJ 08854,USA*Email: [email protected], Phone: +1 (732) 445-4619, Fax: +1 (732) 445-5958

Although it is now common to look at the informationcarried by the genome as a linear sequence of nucleo-tides, such representation does not say much about theorganization of the genetic information within the cellnucleus. How the genome accommodates the tight pack-


ing needed to fit in the cell nucleus and at the same timemaintains the accessibility necessary for specific expres-sion is one of the open questions in modern biology. Ineukaryotic cells, DNA is wrapped and packaged intochromatin through the binding of histones assembled intonucleosomes. In addition to bundling DNA, the nucleo-somes also facilitate communication between distantgenomic sites, such as enhancers and promoters found atthe ends of protein-mediated loops. In order to under-stand the physical and chemical basis of such processes,we have begun to investigate chromatin organization andlooping. We have developed a mesoscale model of chro-matin at a resolution of a single base pair and usedMonte Carlo numerical strategies to understand how thepresence of nucleosomes on DNA can influence andpossibly control chromatin looping. We have validatedthis model by successfully reproducing experimentalmeasurements of gene expression on nucleosomal arrays(Kulaeva et al., 2012). Our results show a wide varietyof chromatin organization depending on the way nucleo-somes are positioned on DNA (i.e. on the spacingbetween successive nucleosomes as illustrated in the pic-tures below) and also on chemical details at the histone

level, such as modifications of the N-terminal tails. Thisdiversity in chromatin organization, which extendsbeyond the conventional solenoid and zigzag models,comes along with very different physical and mechanicalproperties and looping propensities. Furthermore, oursimulations reveal some surprising properties of chroma-tin: for example, we found that tightly packed chromatinfragments are highly flexible, a remarkable feature for amaterial that needs to fit inside a cell nucleus. In conclu-sion, our work uncovers parts of a rich and dynamic pic-ture of chromatin where the DNA sequence influencesthe positioning of nucleosomes, which in turn shape thegenomic material and have an impact on the accessibilityand expression of the genetic message.

This research has been supported by USPHS ResearchGrant GM 34809.

ReferenceKulaeva, O. I., et al. (2012). Internucleosomal interactions mediated

by histone tails allow distant communication in chromatin.Journal of Biological Chemistry, 287, 20248–20257.

60 Binding and transactivation by thetumor suppression protein p53 areencoded in the differential structuralproperties of its response elements

Tali E. Harana, Alon Senitzkia, Jenia Sharava,Itai Benoa*, Karin Rosenthala, Jennifer J. Jordanb,Daniel Menendezb and Michael A. Resnickb

aDepartment of Biology, Technion–Israel Institute of Technology,Technion City, Haifa 320003, Israel; bChromosome StabilitySection, Laboratory of Molecular Genetics, National Institute ofEnvironmental Health Sciences, National Institutes of Health,Research Triangle Park, NC 27709, USA*Email: [email protected], Phone: +972 4 8293767,Fax: +972 4 8225153

The tumor suppressor p53 is one of the most centralhubs in human cells, connected to a complex networkin the living cell. Mutations in the p53 tumor suppres-sor gene are the most frequent genetic alterations in

human cancer. In response to cellular stress, p53 actsas a transcription factor by binding to defined DNA tar-gets, thereby activating the expression of genes leadingto diverse cellular outcomes. How p53 decides betweenits various cellular functions and what determines thestrength of binding of p53 to its response elements(REs) at various levels of protein is currently unknown.We will discuss our recent studies and will show thatdifferential structural properties of p53 REs affect bothp53 binding mechanism as well as the transactivation(TA) level (Beno et al. 2011; Jordan et al. 2012;Menendez et al. 2012). Transactivation by p53 isconsidered to be dependent on its cellular level, butuntil recently how the binding strength of p53 to itsREs is related to transactivation level was unknown.We recently discovered that this relationship is protein-level dependent. At high protein level, we observed theexpected positive relationship between TA level andbinding affinity of p53 to its REs, but at low proteinlevel this relationship breaks down and instead we


observed a positive relationship between TA level andthe torsional flexibility of p53 REs. In addition, we dis-covered two sequences that supported high TA levelseven at basal p53 concentration (“super-transactivating”REs, or STA-REs), in both a highly controlled func-tional assay in yeast and when transfected into humancells. The transcriptional capacity of many p53 mutantsassociated with breast cancers could be discriminatedby transactivation from these REs. Our findings estab-lish that p53 transactivation is kinetically determined atlow levels and thermodynamically driven at high levels,and that the DNA sequence itself can strongly affectp53 transactivation even under conditions where thenumber of p53 molecules is small, suggesting thatDNA structural properties can be critical factors in p53-dependent gene regulation. The mechanistic implicationsof these findings for p53 binding on its targetsequences will be discussed.

ReferencesBeno, I., Rosenthal, K., Levitine, M., Shaulov, L., & Haran, T. E.

(2011). Sequence-dependent cooperative binding of p53 toDNA targets and its relationship to the structural propertiesof the DNA targets. Nucleic Acids Research, 39, 1919–1932.

Jordan, J. J., Menendez, D., Sharav, J., Beno, I., Rosenthal, K.,Resnick, M. A., & Haran, T. E. (2012). Low-level p53expression changes transactivation rules and reveals super-activating sequences. Proceedings of the National Academyof Sciences USA, 109, 14387–14392.

Menendez, D., Resnick, M. A., & Haran, T. E. (2012). Trans-activation by low and high levels of human p53 revealsnew physical rules of engagement and novel super-transac-tivation sequences. Cell Cycle, 11, 4287–4288.

61 High-resolution crystal structures ofp53 mutants and their interactionwith DNA

Haim Rozenberg*, Yael Diskin-Posner, Amir Eldar andZippora Shakked

Department of Structural Biology, Weizmann Institute ofScience, Rehovot 76100, Israel*Email: [email protected]

In response to cellular stress, the tumor suppressorprotein p53 acts as a transcription factor by binding toits DNA targets, leading to the expression of severalgenes that participate in a variety of biologicalprocesses including DNA repair, cell cycle arrest orapoptosis. Thus, p53 “protects” the integrity of thegenome. p53 binds as a tetramer to DNA response ele-ments made of two decameric half-sites of the consen-sus sequence RRRCWWGYYY (R=A/G, Y=T/C,W=A/T) separated by a variable number of base pairs(Kitayner et al., 2006; 2010). About 50% of all inva-

sive cancer cases show mutations in p53 and 97% ofthese mutations occur within the DNA-binding domain(DBD), among them, mutations in six “hot spot”codons account for more than 30% of cancer cases.These mutations lead to p53 loss of function. It wasshown that several oncogenic mutants can be rescuedby second-site suppressor mutations, resulting in wild-type-like activity in terms of DNA binding and tran-scriptional activation. To understand the structuraleffects caused by hot-spot mutations and the mecha-nisms of their rescue by suppressor mutations, wedetermined several crystal structures of human p53DBDincorporating these mutations as well as the rescuedproteins and their complexes with DNA. These includedDNA contact mutants, R273H and R273C, a structuralmutant G245S, and the rescued proteins incorporatingboth the oncogenic mutation and the corresponding sup-pressor mutation. The crystal structures elucidate thestructural basis of loss of function caused by the hot-spot mutations. The crystal structures of the rescuedproteins bound to DNA reveal different rescue mecha-nisms: formation of alternative p53-DNA interactionsfor the DNA-contact mutants, and intramolecular orintermolecular stabilizing interactions for the structuralmutant which compensate for the loss of p53 stabilitycaused by the oncogenic mutation. In contrast to can-cer-related mutations, specific mutations in other regionsof p53DBD such as its DNA-binding loop (L1), whichis part of the loop–sheet–helix motif at the p53-DNAinterface, were shown to modulate the protein’s DNA-binding affinity and transactivation (Zupnick & Prives,2006; Resnick & Inga, 2003). In particular, the replace-ment of T123 in the L1 loop by T123A or T123P lar-gely increases p53 transactivation capacity relative towild-type p53 (Resnick & Inga, 2003). The crystalstructures of these mutants and their complexes withDNA provide a structural basis for understanding theirbiological activity.

ReferencesKitayner, M., Rozenberg, H., Kessler, N., Rabinovich, D.,

Shaulov, L., Haran, T. E., & Shakked, Z. (2006). Structuralbasis of DNA recognition by p53 tetramers. Molecular cell,22, 741–753.

Kitayner, M., Rozenberg, H., Rohs, R., Suad, O., Rabinovich,D., Honig, B., & Shakked, Z. (2010). Diversity in DNA rec-ognition by p53 revealed by crystal structures with Hoogs-teen base pairs. Nature Structural and Molecular Biology,17, 423–429.

Resnick, M. A. & Inga, A. (2003). Functional mutants of thesequence-specific transcription factor p53 and implicationsfor master genes of diversity. Proceedings and NationalAcademy of Sciences USA, 100, 9934–9939.

Zupnick, A. & Prives, C. (2006). Mutational analysis of thep53 core domain L1 loop. Journal of Biological Chemistry,281, 20464–20473.


62 Distinct nucleosome organizationaround p53 response elementsassociated with cell cycle arrest andapoptosis

Feng Cuia* and Victor B. Zhurkinb†

aThomas H. Gosnell School of Life Sciences, Rochester Insti-tute of Technology, Rochester, NY 14623, USA; bLaboratory ofCell Biology, National Cancer Institute, NIH, Bethesda, MD20892, USA*Email: [email protected], †[email protected]

DNA is severely deformed in a tetrameric complex withthe tumor suppressor protein p53, both in solution and inco-crystal structures. The DNA deformations occur notonly at the CWWG motifs in the p53 half sites, but alsoin the central region of a full site, YYRR. As a result,the DNA fragment is bent in such a way that the p53tetramer is located on the external side of the DNA loop.The overall shape of the p53-bound DNA (bending andsliding) resembles nucleosomal DNA, suggesting that thep53 may recognize its cognate site embedded in thenucleosome. Recent studies established unambiguouslythat p53 is a nucleosome-binding protein. In particular,the p53 site in a nucleosome is accessible if it is bent inthe direction similar to that found in the p53-DNAco-crystals; the site becomes inaccessible if the orienta-

tion is changed by �180° (see Figure). This implies thatthe rotational setting of a p53 site in nucleosomes is crit-ical for its accessibility, which may have a direct impacton how p53 recognizes its target binding sites in thechromatin context. We illustrate the functional impor-tance of this idea by comparing the p53 sites associatedwith cell cycle arrest (CCA-sites) and sites associatedwith apoptosis (Apo-sites), the two extreme cellularoutcomes after the p53 activation. To elucidate the rota-tional setting of p53 sites in nucleosomal DNA, wedeveloped a computational approach (Cui & Zhurkin,2010) based on well-established DNA sequence patternsrelated to nucleosome positioning. Importantly, we foundthat the CCA-sites are oriented in such a way that theytend to be ‘open’ and ‘exposed’ on the nucleosomalsurface. This result is corroborated by human nucleo-somes mapped in high resolution (Gaffney et al., 2012).The nucleosome dyad positions are “out of phase” withthe CCA-sites, separated by �10n + 5 bp from the sitecenters. In other words, the CCA-sites embedded innucleosomes are accessible for direct p53 recognition,thereby facilitating p53 binding and subsequent geneinduction. By contrast, our computations suggest thatmost of the Apo-sites have a different rotational settingfrom the CCA-sites, consistent with the high-resolutionnucleosome mapping. That is, the Apo-sites in generalare likely to be ‘closed’ in the chromatin context. Thiswould hinder direct p53 binding to DNA, and require


additional factors such as chromatin remodeling com-plexes to expose the Apo-sites, which may account forthe ‘delayed’ kinetics of apoptotic genes in vivo.

ReferencesCui, F. & Zhurkin, V. B. (2010). Structure-based analysis of

DNA sequence patterns guiding nucleosome positioningin vitro. Journal of Biomolecular Structure and Dynamics,27, 821–841.

Gaffney, D. J., McVicker, G., Pai, A. A., Fondufe-Mittendorf,Y. N., Lewellen, N., Michelini, K., Widom, J., Gilad, Y., &Pritchard, J. K. (2012). Controls of nucleosome positioningin the human genome. PLoS Genetics, 8, e1003036.

63 EMSA detection of p53 sequencenon-specific binding in PCRproduced human DNA analogues

Jiri Chervena*, Pavla Bazantovaa, Petr Pecinkaa,c,Barbora Chvatalovaa and Marie Brazdovab

aDepartment of Biology and Ecology, Faculty of Nature Sci-ence, University of Ostrava, Chittussiho 10, 71000 Ostrava,Czech Republic; bInstitute of Biophysics, Academy of Sciencesof the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno,Czech Republic; cEnvironmental Center, Faculty of NatureScience, University of Ostrava, Chittussiho 10,71000 Ostrava, Czech Republic*Email: [email protected], Phone: +420 597092331,Fax: +420 597092382

p53 is a tumor suppressor that induces cell cycle arrestand apoptosis in response to DNA damage and cancero-genesis. Its ability to bind DNA, and thus play itsbiological role, is possible in two manners: sequence-spe-cific binding to its consensus sequence (p53CON) andsequence non-specific binding, which occurs preferably inhigher DNA structures. Recently, it has been proven thatDNA quadruplexes occur in regulation areas of most can-

cer genes. In our study, we have tested human DNAcloned into plasmid vectors. The DNAs were obtained bychromatin immunoprecipitation of regions which werebound by p53 with high affinity, although they do notcontain p53CON. The sequence studied in this work islocated in a noncoding region of human chromosome 7.We suggest that structure-specific binding is responsiblefor higher affinity of p53 binding in these areas. It hasbeen previously found that some single-stranded regionsappeared in these areas, suggesting the presence of higherDNA structures by S1 nuclease digestion (unpublishedresults). Because we were unable to detect the exact loca-tion of p53 binding with sufficient resolution by standardmethods, we have amplified different parts of immunopre-cipitated DNAs by PCR and found, using EMSA, to whatpart of the insert p53 binds with the highest affinity. Thisarea is represented by cca 150 nucleotides. The strongestpreference of p53 was found for the region whichcontained repeated short tracts of 3–4 Ts and a shortpolyPu.polyPy sequence. It is known that dAn:dTn blockscan cause DNA curvature, and the polyPu.polyPysequence is able to form an intramolecular triplex.

This work has been done in connection with projectInstitute of Environmental Technologies, reg. no.CZ.1.05/2.1.00/03.0100, supported by Research andDevelopment for Innovations Operational Programmefinanced by Structural Funds of Europe Union and bymeans of the state budget of the Czech Republic andCzech Science Foundation (13-36108S).

ReferencesBrooks, T. A., Kendrick, S., & Hurley, L. (2010). Making

sense of G-quadruplex and i-motif functions in oncogenepromoters. FEBS Journal, 277, 3459–3469.

Nejedly, K., Sykorova, E., et al. (1998). Analysis of a curvedDNA constructed from alternating dAn:dTn-tracts in linearand supercoiled form by high resolution chemical probing.Biophysical Chemistry, 73, 205–216.


64 Hoogsteen or not Hoogsteen?Iodine-125 radioprobing of thep53-induced DNA deformations

Igor G. Panyutina, Valery N. Karamycheva,Ronald D. Neumanna, Sharlyn Mazurb, Ettore Appellab,Difei Wangb and Victor B. Zhurkinb*aRadiology and Imaging Sciences, Clinical Center, NIH,Bethesda, MD 20892, USA; bLaboratory of Cell Biology,National Cancer Institute, NIH, Bethesda, MD 20892, USA*Email: [email protected], Phone: +1 (301) 496-8913,Fax: +1 (301) 402-4724

Radioprobing is suitable for tracing the DNA andRNA trajectories in nucleoprotein complexes in solu-tion. The method is based on the analysis of the sin-gle-strand breaks produced by decay of iodine-125incorporated in the C5 position of cytosine (Karamy-chev et al., 1999, 2012). Here, we used radioprobingto study the conformation of DNA in complex withthe DNA binding domain (DBD) of the tumor-sup-pressor protein p53. Two recently crystallized DNA-p53 DBD complexes have different conformations ofthe CATG motifs: one with the Hoogsteen A:T pairs(Kitayner et al., 2010) and the other with the Wat-son–Crick pairs (Chen et al., 2010). The two com-plexes differ in the sequence of the central YYY|RRR junction: the first one has the C|G step and thesecond has the T|A step. Thus, it is interesting toapply the radioprobing method to the two DNAsequences used in crystallography to see if the localchanges (T|A to C|G) in the center of the p53response element would produce significant distortionsin the CATG motifs. To this aim, the iodine-contain-ing cytosine was incorporated in the duplexes contain-ing p53-binding sites, in one of the two CATGmotifs and the frequencies of DNA breaks were ana-lyzed. Frequencies of breaks are negatively correlatedwith the iodine–sugar distances, thus, one can evalu-ate the changes in these distances upon DNA bindingto a protein. The radioprobing distances obtained forboth DNA sequences proved to be consistent with theWatson–Crick structure observed by Chen et al.(2010). We did not find any evidence of the Hoogs-teen A:T base pair formation in the DNA-p53 DBDcomplexes in solution using our radioprobing method.The most significant changes in the break frequencydistributions were detected in the central segment ofthe p53 binding site, YYY|RRR, which are consistentwith an increase in DNA twisting in this region andlocal DNA bending and sliding (Nagaich et al.,1999). We interpret these p53-induced DNA deforma-tions in the context of p53 binding to nucleosomalDNA (Sahu et al., 2010).

ReferencesChen, Y., Dey, R., & Chen, L. (2010). Crystal structure of the

p53 core domain bound to a full consensus site as a self-assembled tetramer. Structure, 18, 246–256.

Karamychev, V. N., Wang, D., Mazur, S. J., Appella, E.,Neumann, R. D., Zhurkin, V. B., & Panyutin, I. G. (2012).Radioprobing the conformation of DNA in a p53-DNA com-plex. International Journal of Radiation Biology, 88, 1039–1045.

Karamychev, V. N., Zhurkin, V. B., Garges, S., Neumann, R.D., & Panyutin, I. G. (1999). Detecting the DNA kinks ina DNA-CRP complex in solution with iodine-125 radiop-robing. Nature Structural Biology, 6, 747–750.

Kitayner, M., Rozenberg, H., Rohs, R., Suad, O., Rabinovich,D., Honig, B., & Shakked, Z. (2010). Diversity in DNArecognition by p53 revealed by crystal structures with Hoo-gsteen base pairs. Nature Structural and Molecular Biology,17, 423–429.

Nagaich, A. K., Zhurkin, V. B., Durell, S. R., Jernigan, R. L.,Appella, E., & Harrington, R. E. (1999). P53-inducedDNA bending and twisting: p53 tetramer binds on theouter side of a DNA loop and increases DNA twisting.Proceedings of the National Academy of Sciences USA,96, 1875–1880.

Sahu, G., Wang, D., Chen, C. B., Zhurkin, V. B., Harrington, R.E., Appella, E., Hager, G. L., & Nagaich, A. K. (2010). P53binding to nucleosomal DNA depends on the rotationalpositioning of DNA response element. Journal of BiologicalChemistry, 285, 1321–1332.

65 QM-MM simulations on p53-DNAcomplex: comparison of DNA-binding property between cancer andtheir rescue mutants

Shruti Koulgi*, Archana Achalere, Neeru Sharma,Uddhavesh Sonavane and Rajendra Joshi

Bioinformatics Group, Centre for Development of AdvancedComputing (C-DAC), Pune University Campus, Pune 411 007,India*Email: [email protected], Phone/Fax: +91 22-569-4084

p53 is a transcription factor involved in expression ofa number of downstream genes in response to geno-toxic stress. In normal cells, it is present in the latentor inactive state, and the in case of cancer cells it isactivated by various post translation modifications. It isfound to be mutated in 50% of the cancers. Thesemutations occur at a high frequency in the DNA bind-ing region of p53. All seven hot spot cancer mutationsR175H, Y220C, G245S, R248Q, R249S, R273C, andR282W have been studied here using quantum andmolecular mechanics hybrid simulations. The experi-mentally proven rescue mutations of the above men-tioned cancer mutants have also been included in thepresent work. Each of the p53 mutants has been simu-lated for 30 ns each. A comparative study of these


cancer mutations along with wild-type and their rescuemutations have been studied. The key residues whichcontribute to the binding of the p53 to the DNA byforming crucial hydrogen bonds have been studied indetail. Free energy changes, principal component analy-sis, hydrogen bonding, and various other structuralparameters have been calculated to quantify the lossand gain in DNA binding property and local structuralalterations of all the p53 mutants.

ReferencesJoerger, A. C. & Fersht, A. R. (2008). Structural biology of the

tumor suppressor p53. Annual Review of Biochemistry, 77,557–582.

Lu, Q., Tan, Y. -H., & Luo, R. (2007). Molecular dynamicssimulations of p53 DNA-binding domain. Journal of Physi-cal Chemistry B, 111(39), 11538–11545.

Nikolova, P. V., Wong, K. B., DeDecker, B., Henckel, J., &Fersht, A. R. (2000). Mechanism of rescue of common p53cancer mutations by second-site suppressor mutations.EMBO Journal, 19, 370–378.

66 Architectural role of HMO1 inbending, bridging, and compactingDNA

Divakaran Murugesapillaia*, Micah J. McCauleya

Ran Huoa, Molly H. Nelson Holteb,L. James Maher IIIb, Nathan E. Israeloff a andMark C. Williamsa

aDepartment of Physics, Northeastern University, Boston, MA02115, USA; bDepartment of Biochemistry and MolecularBiology, Mayo Clinic College of Medicine, Rochester, MN55905, USA*Email: [email protected]

HMO1 proteins are abundant Saccharomyces cerevisiae(yeast) High Mobility Group Box (HMGB) protein(Kamau, Bauerla & Grove, 2004). HMGB proteins arenuclear proteins which are known to be architecturalproteins (Travers, 2003). HMO1 possesses two HMGBbox domains. It has been reported that double boxHMGB proteins induce strong bends upon binding toDNA. It is also believed that they play an essential rolein reorganizing chromatin and, therefore, are likely to beinvolved in gene activation. To characterize DNA bind-ing we combine single molecule stretching experiments

and AFM imaging of HMO1 proteins bound to DNA.By stretching DNA bound to HMO1, we determine thedissociation constant, measure protein induced averageDNA bending angles, and determine the rate at whichtorsional constraint of the DNA is released by theprotein. To further investigate the local nature of thebinding, AFM images of HMO1-DNA complexes areimaged, and we probe the behavior of these complexesas a function of protein concentration. The results showthat at lower concentrations, HMO1 preferentially bindsto the ends of the double helix and links to the separateDNA strands. At higher concentrations HMO1 inducesformation of a complex network that reorganizes DNA.Although HMG nuclear proteins are under intense inves-tigation, little is known about HMO1. Our studiessuggest that HMO1 proteins may facilitate interactionsbetween multiple DNA molecules.

ReferencesKamau, E., Bauerle, K. T., & Gove, A. (2004). The Saccharo-

myces cerevisiae high mobility group box protein HMO1contains two functional DNA binding domains. Journal ofBiological Chemistry, 279, 55234–55240.

Travers, A. A. (2003). Priming the nucleosome: a role forHMGB proteins? EMBO Rep, 4, 131–6.


67 Covariation between homeodomainsand the DNA shape of their bindingsites provides new insights intoprotein–DNA recognition

Iris Drora,b, Tianyin Zhoua, Yael Mandel-Gutfreundb andRemo Rohsa

aMolecular and Computational Biology Program, University ofSouthern California, 1050 Childs Way, Los Angeles, CA 90089,USA; bFaculty of Biology, Technion – Israel Institute ofTechnology, Haifa, Israel

It is well established that transcription factors (TFs)identify their binding sites through direct contacts withunique chemical groups of the base pairs mainly in themajor groove. A second type of mechanism, which hasbeen relatively less studied, is the readout of the DNAshape, in which the protein recognizes the three-dimen-sional DNA structure (Rohs et al., 2010). Here, wefocused on the homeodomain family of TFs andanalyzed the DNA shape of thousands of sequences inorder to study the correlation between the amino acidsequence of homeodomains and the nucleotide sequenceand the shape of their DNA binding sites. We havefound regions in the homeodomains that are significantlycorrelated with the sequence or with the shape of theirpreferred binding sites, demonstrating the role of differ-ent homeodomain regions in attaining binding specificitythrough different modes of recognition. Next, we pre-dicted specific residues in homeodomains which likelyplay an important role in DNA recognition throughDNA shape attributes. Furthermore, we show that addingDNA shape information to the characterization of TFbinding sites can improve predictions of homeodomainbinding specificity. Finally, our work indicates that DNAshape information can provide new mechanistic insightsinto TF binding.

ReferenceRohs, R., Jin, X., West, S. M., Joshi, R., Honig, B., & Mann, R.

S. (2010). Origins of specificity in protein-DNA recognition.Annual Review of Biochemistry, 79, 233–269.

68 Deconvoluting the recognition ofDNA shape from DNA sequence

Namiko Abea, Matthew Slatterya, Iris Drorb,Remo Rohsb, Barry Honiga and Richard S. Manna*aDepartment of Biochemistry and Molecular Biophysics,Columbia University, New York, NY 10032, USA; bDepartmentof Biological Sciences, University of Southern California, LosAngeles, CA 90089, USA*Email: [email protected]

In previous work (Slattery et al., 2011; Joshi et al.,2007), we described the importance of the 3-D struc-ture of the DNA double helix – DNA shape – in therecognition of DNA binding sites by the Hox familyof transcription factors. In particular, we found thatlocal minima in the width of the DNA minor groovecreate electronegative pockets that are binding sitesfor amino acids with positively-charged side chainssuch as arginine. Moreover, our data argued thatDNA shape is a consequence of DNA sequence, andthus led to the idea that the recognition of specificDNA sequences by DNA binding proteins is mediatedby both base-readout, typically in the major groove,and shape-readout (Rohs et al., 2010). The relation-ship between DNA shape and DNA sequence leadsto a logical loop that is difficult to tease apart: ifshape is a consequence of sequence, then is the rec-ognition of a binding site by a transcription factormediated by the sequence of base pairs or by theresulting shape of the DNA molecule? One predictionof the shape recognition model is that if the shape-detecting amino acids are mutated, the shape of thepreferred binding sites would become less important.We tested this prediction by mutating the basic resi-dues of Hox proteins known to insert into narrowregions of the DNA minor groove and carrying outin vitro SELEX-seq experiments. Consistent with theimportance of DNA shape recognition by this familyof homeodomain proteins, we found that DNA mole-cules selected to bind these mutants had a muchsmaller propensity to have local minor groove widthminima compared with DNA molecules selected tobind the wild type proteins. Further, we were able totransfer the shape recognition properties of one Hoxprotein to another Hox protein by introducing residuesthat bind to narrow minor grooves. Together, thesefindings argue that the recognition of DNA shape isa key aspect of binding site selection by this familyof DNA binding proteins.

ReferencesJoshi, R., et al. (2007). Functional specificity of a Hox protein

mediated by the recognition of minor groove structure.Cell, 131, 530–543.

Rohs, R., et al. (2010). Origins of specificity in protein-DNA recognition. Annual Review of Biochemistry, 79,233–269.

Slattery, M., et al. (2011). Cofactor binding evokes latent differ-ences in DNA binding specificity between Hox proteins.Cell, 147, 1270–1282.


69 DNA-binding studies of largeantiviral polyamides

Elena Vasilieva, Gaofei He, Kevin J. Koeller,G. Davis Harris, Cynthia M. Dupureur* andJames K. Bashkin

Department of Chemistry & Biochemistry, Center forNanoscience, University of Missouri-St. Louis, One UniversityBlvd, St. Louis, MO, USA*Email: [email protected], Phone: +1 (314) 516-4392,Fax: +1 (314) 516-5342

Polyamides are minor groove DNA-binding agentsderived from the natural product distamycin A. PA1is a large 12 ring polyamide discovered by NanoVirLLC; it is bioactive against the HPV16 virus in celland tissue culture (Edwards et al., 2011). To betterunderstand the basis of this phenomenon, the interac-tions of PA1 with the regulatory sequence of theHPV16 genome (7662–122 bp) are being examined.Using affinity cleavage as detected by capillary elec-trophoresis, with PA1 attached to methyl propyl ethi-dium iron EDTA, 10 binding sites of PA1 were

identified in this part of the HPV genome. Polyamideperfect binding sites were as predicted by recognitionrules (Dervan & Edelson, 2003). Quantitative DNaseIfootprinting indicates that both perfect and single mis-match sites are bound with Kds in the low nm range.Interestingly, a wide range of Kds are observed fordouble mismatch sites (1–60 nm) and are under exam-ination. This work will permit us to build a map ofPA binding to HPV sequences, thus informing mecha-nisms of in vivo behavior.

This research has been supported by NIH (AI083803)and NanoVir, LLC under its NIH grant AI062182. Weare grateful for access to the NanoVir discoveries.

ReferencesDervan, P. B. & Edelson, B. S. (2003). Recognition of the

DNA minor groove by pyrrole–imidazole polyamides.Current Opinion in Structural Biology, 13, 284–299.

Edwards, T. G., Koeller, K. J., Slomczynska, U., Fok,K., Helmus, M., Bashkin, J. K., & Fisher, C.(2011). HPV episome levels are potently decreased bypyrrole-imidazole polyamides. Antiviral Research, 91,177–186.

70 Electrostatic properties of bacterialDNA and promoter predictions

Evgenia A. Temlyakova*, Timur R. Dzhelyadin,Svetlana G. Kamzolova and Anatoly A. Sorokin

Mechanism of Cell Functioning Group, Institute of CellBiophysics RAS, Institutskaya str. 3, Pushchino, MR,Russia*Email: [email protected], Phone: +7 (4967) 739-165

Electrostatic potential distribution (EPD) around a DNAmolecule seems to be the only physical property thatcould be recognized by other molecules from a dis-tance. More than 10 years ago, a method for simple

and fast calculation of EPD was proposed in our labo-ratory (Polozov et al., 1999). It is based on Coulombformula and allows us to estimate the main EPD pat-terns for DNA sequences of a size of a whole prokary-otic genome. We applied the projection on latentstructures discriminant analysis (PLS-DA) (Sarker &Rayens, 2003) to create three types of model to dis-criminate promoter and nonpromoter sequences of theEscherichia coli K-12 genome on the basis of its EPDprofiles. Randomized, coding, and promoter-like regionswere used to train models as the nonpromotersequences. The information about promoters and pro-moter-like regions was taken from the sources (Regulon6.0, S) Gama-Castro et al. (2008) and Shavkunov et al.


(2009), respectively. By our models, we evaluated theprobability of there being a possible transcription startsite (TSS) at the E. coli K-12 whole genome EPD witha 1 A step. It was shown that more than 2500 real pro-moters have TSS predicted in the regions [�50, +10]A around the annotated +1 position and so could beclassified as recognized TSS. No additional informationabout the nucleotide sequence, such as localization anddirection of nearby ORF or positions of annotated genestart codons, were taken into account for these predic-tions. This makes the methods of EPD analysis goodcandidates for the development of multistep promotersearching algorithms.

ReferencesPolozov, R. V., et al. (1999). Electrostatic potentials of DNA. Com-

parative analysis of promoter and nonpromoter nucleotidesequences. Journal of Biomolecular Structure & Dynamics,16, 1135–1143.

Sarker, M., & Rayens, W. (2003). Partial least squares fordiscrimination. Journal of Chemometrics, 17, 166.

Gama-Castro, S., et al. (2008). RegulonDB (version 6.0): Generegulation model of Escherichia coli K-12 beyond tran-scription, active (experimental) annotated promoters andTextpresso navigation. NAR, 36, D120–D124.

Shavkunov, K. S., et al. (2009). Gains and unexpectedlessons from genome-scale promoter mapping. NAR, 37,4919–4931.

71 Genomic regions flanking E-boxbinding sites influence DNA bindingspecificity of bHLH transcriptionfactors through DNA shape

Raluca Gordâna,b*, Ning Shenc,g, Iris Drord,g,Tianyin Zhoud, John Hortonb, Remo Rohsd

and Martha L. Bulyke,f

aDepartments of Biostatistics and Bioinformatics, ComputerScience, and Molecular Genetics and Microbiology, DukeUniversity, Durham, NC, USA; bInstitute for Genome Sciencesand Policy, Duke University, Durham, NC 27708, USA;cDepartment of Pharmacology and Cancer Biology, DukeUniversity, Durham, NC 27708, USA; dMolecular andComputational Biology Program, Departments of BiologicalSciences, Chemistry, and Physics and Astronomy, University ofSouthern California, 1050 Childs Way, Los Angeles, CA 90089,USA; eDivision of Genetics, Departments of Medicine and

Pathology, Brigham and Women’s Hospital and HarvardMedical School, Boston, MA 02115, USA; fHarvard-MITDivision of Health Sciences and Technology (HST), HarvardMedical School, Boston, MA 02115, USA*Email: [email protected], Phone: +1 (919) 667-8673,Fax: +1 (919) 668-0795 (shared)

Transcriptional regulation of gene expression isenacted mainly through binding of transcription factors(TFs) to specific, short DNA sites in cis-regulatoryregions of genes. Most TFs are members of proteinfamilies that share a common DNA-binding domainand thus recognize similar DNA-binding sequences. Itis not well understood why paralogous TFs often binddifferent genomic target sites in vivo to effect differentregulatory programs, despite apparently recognizing thesame sequence motifs. Here, we designed custom pro-tein- binding microarrays (PBMs) to analyze the


DNA-binding specificities of two Saccharomyces cere-visiae basic helix-loop-helix (bHLH) proteins, Tye7and Cbf1, as a model system. Our data reveal that E-box DNA-binding sequences (CAnnTG), when testedin the context of their native genomic flankingsequences, are bound differently by Cbf1 and Tye7.Computational models of the PBM data indicate thatDNA sequence features located in the genomicsequences outside the E-box contribute to DNA-bind-ing specificity in vitro. Our analyses suggest that theseflanking regions affect DNA-binding specificity indi-rectly by influencing the three-dimensional structure ofthe E-box binding sites. Finally, we show that thesesubtle differences in intrinsic sequence preferences ofCbf1 and Tye7 in vitro help to explain their differen-tial DNA-binding preferences in vivo. Our resultsprovide further evidence that the local shape of DNA-binding sites may be an important feature in distin-guishing the DNA-binding preferences among paralo-gous TFs and thus may play a widespread role indetermining how transcriptional regulatory specificitywithin TF families is achieved.

72 Get into shape: new insights into thebroadly observed mechanism of DNAshape readout and its relation togenetic variation

Ana C.D. Machado, Tianyin Zhou and Remo Rohs*

Molecular and Computational Biology Program, Department ofBiological Sciences, University of Southern California, LosAngeles, CA 90089, USA*Email: [email protected], Phone: +1 (213) 740 0552

Various biological processes are driven by interactionsbetween proteins and their DNA-binding sites. Toachieve DNA-binding specificity, an interplay betweenbase and shape readout modes by proteins is impor-tant (Rohs et al., 2010). Recently, we have demon-strated the important role of DNA shape readout fordifferent transcription factors and other protein fami-lies (Rohs et al., 2009). This mechanism includes therecognition of structural features of the DNA, andelectrostatic interactions between the narrow DNAminor groove and arginine residues. Here, we expandthis finding and demonstrate how histidine residuescan play a similar role in DNA shape readout, andillustrate that shape readout regulates many other pro-tein–DNA interactions beyond transcription. Whereaswe have previously demonstrated the importance ofshape readout for the homeodomain family of tran-scription factors (Slattery et al., 2011), we now showthe broader spectrum of this mechanism for protein

families involved in DNA looping, origin recognition,and transcription initiation. In a related approach, westudy the effect that single nucleotide variants canhave on DNA shape readout. These findings broadlyexpand our current understanding of the mechanismsby which proteins can recognize DNA, and raisemany interesting questions as to how genetic varia-tions in noncoding regions of the genome affect pro-tein–DNA binding.

ReferencesRohs, R., Jin, X., West, S. M., Joshi, R., Honig, B., &

Mann, R. S. (2010). Origins of specificity in protein-DNA recognition. Annual Review of Biochemistry, 79,233–269.

Rohs, R., West, S. M., Sosinsky, A., Liu, P., Mann, R. S., &Honig, B. (2009). The role of DNA shape in protein–DNArecognition. Nature, 461, 1248–1253.

Slattery, M., Riley, T., Liu, P., Abe, N., Gomez-Alcala, P., Dror,I., … Mann, R. S. (2011). Cofactor binding evokes latentdifferences in DNA binding specificity between Hoxproteins. Cell, 147, 1270–1282.

73 Molecular dynamics approach forDNA duplex thermal stabilityprediction

Alexander Lomzov*, Yury Vorobjev andDmitryi Pyshnyi

Institute of Chemical Biology and Fundamental Medicine SBRAS, Novosibirsk, Russia*Email: [email protected], Phone: +7 (383) 363-5134,Fax: +7 (383) 363-5134

Development of new derivatives and analogs of nucleicacids (NA) with precalculated physico-chemical proper-ties is important, both in practice and basic research.Unfortunately, these studies remain laborious, costly,and time-consuming. In silico research probably couldresolve this problem due to the significant progress indevelopment of computer software and hardware. Theaim of this work is to study a molecular dynamicsapproach for nucleic acid hybridization enthalpy calcu-lation. The enthalpies of DNA duplex formation werecalculated as a difference of the total internal energy ofdouble- and single-stranded states which were averagedfrom a 10 ns MD trajectory computed with Amber 11software (UCSF, USA). Computations were performedon NVIDIA GTX580/Intel i7-2600 hardware andresources of the Siberian Supercomputer Center(ICMMG SB RAS). The use of a GPU has speeded upthe modeling in implicit solvent up to 60 times and upto 30 times in explicit solvent in comparison with theone node of a CPU. To determine the optimal parame-


ter set of modeling, we have used Dickerson–Drewdodecamer (DDD) 5'-CGCGAA TTCGCG-3' with wellcharacterized secondary structure and thermal stability.We have varied force field, temperature, heating proto-col, and ion concentration in implicit and explicit sol-vent, solvent shell radius and compared averagedstructures with those experimentally obtained. Usingoptimal parameters of modeling, we have shown thathybridization enthalpy of DDD correlates well withexperimental and calculated ones of the nearest neigh-bor models’ enthalpies (Lomzov et.al., 2006). The dif-ferences were <15% whereas the experimental accuracyis about 10%. To verify the MD are predictive ability,we have collected a database of experimentally deter-mined thermodynamic parameters (enthalpy andentropy) of hybridization of 272 oligodeoxyribonucleo-tides. The length of oligonucleotides varies from 4 upto 16 base pairs (aver. 9 bp), GC-content 0-100% (aver.57%). The total energy of oligonucleotide or duplexwas averaged over 10 000 snapshots of 10 ns trajecto-ries simulated with the optimal parameter set. The cor-relation between the values of hybridization enthalpiesobtained experimentally and calculated using MD areshown in the figure. The RMSD and average error val-ues of calculated and experimental enthalpies were lessthan 12 and 15%, respectively. The results obtainedshow that MD modeling allows one to calculateenthalpy of matched DNA duplexes with surprisinglygood accuracy.

y = 0.84x - 1.88

R² = 0.86

-140

-120

-100

-80

-60

-40

-20

-140 -110 -80 -50 -20

HoExperimental,

kcal/mol

HoMD, kcal/mol

This research has been supported by RFBR (12-04-31776 mol_a), Contracts (8123, 8535), Integration grantSB RAS (86), and by MCB programs of RAS.

ReferenceLomzov, A. A., Pyshnaya, I. A., Ivanova, E. M., & Pysh-

nyi, D. V. (2006). Thermodynamic parameters for calcu-lating the stability of complexes of bridgedoligonucleotides. Doklady Biochemistry and Biophysics,409, 211–215.

74 Novel geometric approaches touniquely characterizeDNA-binding interfaces

Yael Mandel-Gutfreund*

Faculty of Biology Technion, 32000 Haifa, Israel*Email: [email protected], Phone: +972 4-8293958,Fax: +972 4-8225153

Protein–nucleic-acid interactions play a critical role inall steps of the gene expression pathway. DNA-bindingproteins interact with DNA via distinct regions on theirsurface that are characterized by an ensemble of chem-ical, physical, and geometrical properties. In previousstudies, we have developed unique approaches to char-acterize DNA-binding proteins combining geometricand/or electrostatic features (Stawiski et al., 2003;Shazman et al., 2007; Shazman & Mandel-Gutfreund,2010). Here, I will present a novel approach we haverecently developed to characterize protein structures bythe distribution of their overlapping local surfacepatches. In this approach, the protein surface is repre-sented by a bag of overlapping surface patches, whichare defined by a central surface residue and its nearestsurface neighbors. Using a similar approach to Frag-Bag, a method used to retrieve protein structure neigh-bors based on short contiguous backbone segments(Budowski-Tal et al., 2010), we characterize each pro-tein by a ‘bag-of-surface patches’ – a vector represent-ing the distribution of different patches which appearon the protein surface. The similarity between twoproteins is finally measured by the distance betweentheir corresponding vectors of surface patches. Ourresults, based on a large benchmark of proteindomains, show that the method has a great advantageover other methods for detecting remote function rela-tionships between proteins based solely on the proteinsurface. We further show the applicability of themethod to uniquely identify DNA-binding interfacesaccurately and efficiently.


ReferencesBudowski-Tal, I., Nov, Y., & Kolodny, R. (2010). FragBag, an

accurate representation of protein structure, retrieves struc-tural neighbors from the entire PDB quickly and accurately.PNAS, 107, 3481–3486.

Shazman, S., Celniker, G., Haber, O., Glaser, F., & Mandel-Gutfreund, Y. (2007). Patch Finder Plus (PFplus): a webserver for extracting and displaying positive electrostaticpatches on protein surfaces. Nucleic Acids Research, 35,W526–W530.

Shazman, S., Elber, G., & Mandel-Gutfreund, Y. (2011). Fromface to interface recognition: a differential geometricapproach to distinguish DNA from RNA binding surfaces.Nucleic Acids Research, 39, 7390.

Stawiski, E. W., Gregoret, L. M., & Mandel-Gutfreund, Y. (2003).Annotating nucleic acid-binding function based on proteinstructure. Journal of Molecular Biology, 326, 1065–1079.

75 OnTheFly database – structuralbasis to study TF’s DNA-bindingspecificity

Shula Shazmana,b,c*, Jie Chena,b,c, Hunjoong Leea,b,c,Peng Liua,b,c, Richard Manna and Barry Honiga,b,c

aDepartment of Biochemistry and Molecular Biophysics,Columbia University, New York, NY, USA; bCenter forComputational Biology and Bioinformatics, ColumbiaUniversity, New York, NY, USA; cHoward HughesMedical Institute*Email: [email protected]

We describe a systematic determination of Drosophilamelanogaster transcription factor DNA-binding specifici-ties. We annotated and classified all transcription factors(TFs) predicted in the Drosophila melanogaster genome(Pfreundt, U. et al. 2009) and collected the knownpreferred DNA binding sites of the TFs based on the

B1H (Zhu, L.J. et al. 2011), DNaseI (Bergman C.M.et al. 2005), and SELEX (Slattery, M. et al. 2011) meth-ods. Then, we identified the sequence and shape prefer-ences for all DNA-binding proteins (Kuziemko A et al.2011) and also characterized the shapes of their preferredDNA binding sites using structural models. The identifi-cation of the preferred DNA binding sites, and theirshapes, for all DNA binding proteins will provide anunprecedented and extremely valuable database for any-one attempting to decipher noncoding regulatory DNA.Specifically, we showed that using structural criteria suchas the width of the minor groove (Rohs R. et al. 2010),we distinguished DNA sequences bound by proteinswhich possess a Homeodomain from other proteins thatpossess a ZNF-C2H2 domain or ETS domains (picturedbellow from right to left). Furthermore, based on keyTF–DNA interactions from the template structure storedin PDB, we superpose query DNA structure onto thetemplate. Therefore, we obtain a homology model, whereTF is from the template and DNA is from the query.This model will provide potential structural basis tostudy TF’s DNA-binding specificity. The insights fromsuch a study could help in selecting the best DNA candi-dates to be bound by a certain TF for experimental test-ing. Finding such pairs will help to characterize theunique properties of protein–DNA interfaces and identifynew drug target sites.

ReferencesBergman, C. M., Carlson, J. W., & Celniker, S. E. (2005). Dro-

sophila DNase I footprint database: a systematic genomeannotation of transcription factor binding sites in the fruitfly.Drosophila melanogaster. Bioinformatics, 21, 1747–1749.

Kuziemko, A., Honig, B., & Petrey, D. (2011). Using structureto explore the sequence alignment space of remote homo-logs. PLoS Comput Biol. 2011 Oct; 7(10):e1002175.


Pfreundt, U., et al. (2009). FlyTF: improved annotation andenhanced functionality of the Drosophila transcription fac-tor database. Nucleic Acids Research, 38, D443–447.

Rohs, R., et al. (2010). Origins of specificity in protein-DNArecognition. Annual Reviews Biochemistry, 79, 233–269.

Slattery, M., et al. (2011). Cofactor binding evokes latent differ-ences in DNA binding specificity between Hox proteins.Cell, 147, 1270–1282.

Zhu, L. J., et al. (2011). FlyFactorSurvey: a database of Dro-sophila transcription factor binding specificities determinedusing the bacterial one-hybrid system. Nucleic AcidsResearch, 39, D111–117.

76 Protein–DNA binding and breathingdynamics of DNA

Boian S. Alexandrova,b*, Kim Ø. Rasmussena,Alan R. Bishopa and Anny Ushevab

aTheoretical Division, Los Alamos National Laboratory, LosAlamos, NM 87545, USA; bHarvard Medical School, BethIsrael Deaconess Medical Center, Boston, MA 02215, USA*Email: [email protected], Phone: +1 (505) 500-7831,Fax: +1 (505) 665-2659

In the living cell, the double-stranded DNA molecule expe-riences thermal motions that induce spontaneous openingsand re-closings of the double helix known as “DNA breath-ing,” or “DNA transient bubbles” (Englander et al., 1980).The propensity for breathing is interconnected with DNAlocal stability and flexibility (Vafabakhsh & Ha, 2012),which play a key role in DNA biological function (Bishopet al., 2012). Here, we investigate the relationship betweenthe DNA local propensity for breathing and binding of twotranscription factors (TFs): (i) the human TF YY1, (seee.g. Usheva & Shenk, 1996), and (ii) the nucleoid-associ-ated protein Fis in Escherichia coli (see e.g. Finkel & John-son, 1992). Using a mesoscopic nonlinear model ofdouble-stranded DNA (Peyrard & Bishop, 1989) that canbe augmented for rational design of DNA breathing(Alexandrov et al., 2010), we have simulated the dynamicsof known Fis- and YY1-binding sites, analyzed publishedin vitro and genomic data-sets, and conducted targetedexperimental tests of our predictions (Alexandrov et al.,2012; Nowak-Lovato et al., in press). We found a strongcorrelation between the propensity for breathing (at thebinding sites) and YY1/Fis binding. We identified a breath-ing profile that is characteristic for a strong Fis-binding sitethat is significantly enriched among the identified in vivoE. coli Fis-binding sites. To test our understanding of howFis binding is influenced by the breathing, we designedbase-pair substitutions, mismatch, and O6-Guanine methyl-ation modifications of nucleotides, in sequences that areknown to interact (or not interact) with Fis, seeking to makethe breathing either closer to or farther from the breathingprofile of a strong Fis-binding site. For the modified DNAsegments, we found that Fis-DNA binding, as assessed by

EMSA, changed in accordance with our expectations.Further, by using site-specific chromatin immunopecipita-tions, BIOBASE data, and simulations, we also found aspecific breathing profile at the binding cites of YY1in vivo. Our finding suggests that the genomic-flankingsequence variations and SNPs presence may exert long-range effects on DNA breathing and predetermine YY1binding in cells.

This research has been supported by a LANL, EarlyCareer Research Award 20110516ECR.

ReferencesAlexandrov, B. S., et al. (2012). DNA breathing dynamics dis-

tinguish binding from nonbinding consensus sites for tran-scription factor YY1 in cells. Nucleic Acids Research, 40,10116–10123.

Alexandrov, B. S., et al. (2010). DNA dynamics play a role asa basal transcription factor in the positioning and regulationof gene transcription initiation. Nucleic Acids Research, 38,1790.

Bishop, A. R., et al. (2012). Entropy–driven conformations con-trolling DNA functions. Verlag-Berlin-Heidelberg: Springer.

Finkel, S. E., & Johnson, R. C. (1992). The Fis protein: It’snot just for DNA inversion anymore. Molecular Microbiol-ogy, 6, 3257–3265.

Nowak-Lovato, K. et al. (in press). Binding of nucleoid-associ-ated protein fis to DNA is regulated by DNA breathingdynamics. PLOS Computational Biology.

Peyrard, M., & Bishop, A. R. (1989). Statistical mechanics of anonlinear model for DNA denaturation. Physical ReviewLetters, 62, 2755–2758.

Usheva, A., & Shenk, T. (1996). YY1 transcriptional initiator:Protein interactions and association with a DNA site con-taining unpaired strands. Proceedings of the National Acad-emy of Sciences USA, 93, 13571–13576.

Vafabakhsh, R., & Ha, T. (2012). Extreme bendability of DNAless than 100 base pairs long revealed by single-moleculecyclization. Science, 337, 1097–1101.

77 Sequence features and chromatinstructure around the genomic regionsbound by 119 human transcriptionfactors

Jie Wang, Jiali Zhuang, Sowmya Iyer, Xin Lin,Troy W. Whitfield, Melissa C. Greven, Brian G. Pierce,Xianjun Dong, Anshul Kundaje, Yong Cheng,Oliver J. Rando, Ewan Birney, Richard M. Myers,William S. Noble, Michael Snyder and Zhiping Weng*

Program in Bioinformatics and Integrative Biology, Departmentof Biochemistry and Molecular Pharmacology, University ofMassachusetts Medical School, Worcester, MA 01605, USA*Email: [email protected],Phone: +1 (508) 856-8866, Fax: +1 (508) 856-2392

Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) has become the dom-


inant technique for mapping the transcription factor(TF) binding regions genome-wide. We performed anintegrative analysis centered around 457 ChIP-seq datasets on 119 human TFs generated by the ENCODEConsortium. We identified highly enriched sequencemotifs in most data sets, revealing new motifs and val-idating known ones. The motif sites (TF binding sites)are evolutionarily highly conserved and show distinctfootprints upon DNase I digestion. We frequentlydetected secondary motifs in addition to the canonicalmotifs of the TFs, indicating tethered binding andcobinding between multiple TFs. We observed signifi-cant position and orientation preferences between manycobinding TFs. Genes specifically expressed in a cellline are often associated with a greater occurrence ofnearby TF binding in that cell line. We observed cell-line-specific secondary motifs that mediate the bindingof the histone deacetylase HDAC2 and the enhancer-binding protein EP300. TF binding sites are located inGC-rich, nucleosome-depleted, and DNase I sensitiveregions, flanked by well-positioned nucleosomes, andmany of these features show cell type specificity. TheGC-richness may be beneficial for regulating TF bind-ing, because, when unoccupied by a TF, these regionsare occupied by nucleosomes in vivo. We present theresults of our analysis in a TF-centric web repositoryFactorbook (http://factorbook.org) and will continuallyupdate this repository as more ENCODE data are gen-erated.

ReferenceWang, J., Zhuang, J., Iyer, S., Lin, X., Whitfield, T. W.,

Greven, M. C., … Weng, Z. (2012). Sequence features andchromatin structure around the genomic regions boundby 119 human transcription factors. Genome ResearchSeparation, 22, 1798–1812.

78 Structural studies of rolling circlereplication initiator proteins

Stephen B. Carra*, Lauren B. Meciab, Alice J. Stelfoxb,Simon E.V. Phillipsa,b and Christopher D. Thomasb

aResearch Complex at Harwell, Rutherford Appleton Laboratory,Harwell Science and Innovation Campus, Didcot, Oxon OX110FA, UK; bAstbury Centre for Structural Molecular Biology,University of Leeds, Leeds, LS2 9JT, UK*Email: [email protected], Phone: +44 1235567717, Fax: +44 1235 567799

Plasmids of the pT181 family replicate by a rolling-circle mechanism. The process is initiated by a plas-mid-encoded Rep initiator protein, which has sequence-specific DNA nicking and religation activity. The plas-

mid replication origin is nicked by Rep, which bindscovalently to one DNA strand via an active site tyro-sine, initiating rolling circle replication and religatingthe strand at the end of the cycle. Rep proteins alsoassociate with PcrA helicase to form a highly proces-sive complex. We have determined the structure of theRep protein from cryptic plasmid pSTK1 of Geobacil-lus stearothermophilus (Gst), and several variants ofRepD from Staphylococcus aureus (Sau), representingthe first structural information on this class of initia-tors. Cloning and expression of the designated 269 aaRep product from pSTK1 failed to yield soluble,active protein. However, expression from an arbitrarypoint upstream yielded an elongated product capableof relaxing plasmid substrates encoding an invertedrepeat sequence from pSTK1, which resembles the rep-lication origin of the pT181 family. Both this productand a 31 kDa proteolytically derived fragment omittingthe C-terminus additionally display activation of thecognate Gst PcrA helicase, but not that of S. aureus.The crystal structure of the 31 kDa fragment of GstRep has been solved at 2.3 Å resolution, showing anunusual ring-shaped dimer with a 20Å diameter pore.The inner surface of the ring is largely formed by an18-stranded β-sheet, while the outer surface is deco-rated with 18 α-helices. The protein represents a novelfold; however, the extended sheet does exhibit somesimilarities to those observed in both TATA-bindingprotein and transcription factor IID. The active siteTyr179 residues, one from each subunit, lie 26Å apartacross the pore, with a nearby catalytic magnesium ioncoordinated by three carboxylate side-chains. Crystalstructures for the Sau Rep variants termed RepDE,RepDN and RepDC have been solved by molecularrepacement using the Gst Rep as a model, and showsimilar structural features. The implications for themechanism of rolling circle replication will be dis-cussed in the light of extensive functional data avail-able for Sau RepD.

79 Thermodynamic and kinetic studiesof trinucleotide repeat (TNR)DNA

Ji Huang* and Sarah Delaney

Department of Chemistry, Brown University, Providence,RI 02912, USA*Email: [email protected], Phone: +1 (401) 863-2044,Fax: +1 (401) 863-1993

The expansion of trinucleotide repeat (TNR) DNA hasbeen linked to several neurodegenerative diseases(McMurray, 2010). The number of repeats is usually a


characteristic indication of the severity of TNR-relateddiseases, with longer repeats giving higher propensityto expand and earlier onset of symptoms (López,Cleary, & Pearson, 2010). It is generally accepted thatformation of noncanonical secondary structures, suchas stem-loop hairpins or slipouts, contributes to theexpansion mechanisms during aberrant DNA replicationor repair processes (Mirkin, 2007). The stability ofthese hairpins is considered an important factor (Paiva& Sheardy, 2005). In this work, we used differentialscanning calorimetry (DSC) and UV–Vis spectroscopyto study the thermodynamic and kinetic stability of aseries of (CTG)n and (CAG)n TNR stem-loop hairpinsand their corresponding (CTG)n/(CAG)n duplexes(n = 6–14). We found that hairpins with n = even andn = even + 1 (odd) repeats possess very similar thermo-dynamic stability. But, when converting to the canoni-cal duplex form, odd-repeat hairpins are morestabilized compared to those of their even-repeat coun-terparts. Within both even- and odd-repeat series, hair-pins with longer repeats are thermodynamically morestabilized compared to the shorter ones. Kineticexperiments of the stem-loop hairpin to duplex conver-sion revealed a longer lifetime for the even-repeat hair-pins, while the odd-repeat hairpins convert to duplexes10-fold faster. Also, hairpins with increased number ofrepeats are more resistant to the conversion when con-sidered within the even- or odd-repeat series individu-ally. Taken together, although it is thermodynamicallymore favored that hairpins containing longer repeatsconvert to canonical duplex form; On the contrary,these longer hairpins are kinetically trapped during theconversion and therefore can persist the noncanonicalstructures, which allows TNR expansion.

This research is supported by National Institute ofEnvironmental Health Science (ES019296).

ReferencesLópez, C. A., Cleary, J. D., & Pearson, C. E. (2010). Repeat

instability as the basis for human diseases and as apotential target for therapy. Nature Reviews Molecular CellBiology, 11, 165–170.

McMurray, C. T. (2010). Mechanisms of trinucleotide repeatinstability during human development. Nature ReviewsGenetics, 11, 786–799.

Mirkin, S. M. (2007). Expandable DNA repeats and humandisease. Nature, 447, 932–940.

Paiva, A. M., & Sheardy, R. D. (2005). The influence ofsequence context and length on the kinetics of DNA duplexformation from complementary hairpins possessing (CNG)repeats. Journal of the American Chemical Scoiety, 127,5581–5585.

80 A new approach in determining therigidity of nucleic acids and polymers

Anton V. Sergeeva*, Natalia V. Lekontsevab,Alexander A. Timchenkob and Igor N. Serdyukb

aInstitute of Mathematical Problems of Biology RussianAcademy of Sciences, Institutskaja str., 4, 142290 Pushchino,Moscow Region, Russia; bInstitute of Protein Research RussianAcademy of Sciences, Institutskaja str., 4, 142290 Pushchino,Moscow Region, Russia*Email: [email protected],Phone: +7 (4967) 318-504, Fx: +7 (4967) 318-500

In polymer physics, persistence length of chain mole-cules varying in degrees of stiffness is usually evalu-ated from hydrodynamic data for a set of polymers ofdifferent molecular masses М. It is known that fromthe extrapolation of translational diffusion coefficient toinfinite molecular mass, one can calculate persistencelength and hydrodynamic radius for rigid and semi-rigidmolecules. If the persistence length of the molecule ismuch greater than its contour length, then the hydrody-namic parameters of the chain are independent of itssize and such extrapolation is not applicable. In thiscase we proposed to plot the dependence M/s0

2 (s0 –sedimentation coefficient) or MD2 (D – translational dif-fusion coefficient) vs. M (in the absence of volumeeffects) or n (number of monomers). In these coordi-nates the appearance of a plateau will correspond to thecomplete impermeability of the molecule and from pla-teau height the persistence length can be calculated. Weanalyzed literature data for a few synthetic polymers byour approach and determined their persistence lengths,the values of which were very close to those in the lit-erature. Usually, the synthesis of polymer moleculeswith very high molecular mass is a very difficult task.But at the same time, natural polymers with highmolecular mass are common in biology. A typicalexample is a DNA molecule. With increasing contourlength of DNA, its conformation passes the next stages:(1) a rigid rod-like particle, (2) a semi-rigid permeablecoil, and (3) an impermeable Gaussian coil (Serdyuk,Zaccai, & Zaccai, 2007). We analyzed by our approachsedimentation data of DNA molecules from the litera-ture. It was shown that the plateau starts from 40,000base pairs of the DNA molecule and the estimatedvalue of persistence length was about 50 nm. This valueis close to that from literature data (Lu, Weers, & Stell-wagen, 2002). Thus, the proposed approach can beapplied for flexibility analysis of biological macromole-cules of different natures – from nucleic acids to pro-teins in strong denaturants.


ReferencesLu, Y., Weers, B., & Stellwagen, N. C. (2002). DNA persis-

tence length revisited. Biopolymers, 61, 261–275.Serdyuk, I. N., Zaccai, N., & Zaccai, J. (2007). Methods in

molecular biophysics: Structure, function, dynamicsCambridge University Press.

81 A new series of biologically activeDNA minor groove bindersbased on bisbenzimidazole andbenzimidazole-pyrrole motives

Alexander A. Ivanova*, Olga Yu. Susovaa,Victor I. Salyanovb, Kirill I. Kirsanova andAlexei L. Zhuzeb

aInstitute of Carcinogenesis Blokhin Cancer Research CenterRussian Academy of Medical Sciences, Kashirskoye Shosse 24,Moscow 115478, Russia; bEngelhardt Institute of MolecularBiology Russian Academy of Sciences, Vavilova st. 32, Moscow119991, Russia*Email: [email protected], Phone: 7495-324-1114,Fax: 7495-324-1464

As we reported earlier, dimeric bisbenzimidazoles DB(n) (Ivanov et al., 2011a) were able to noncovalentlybind in the DNA minor groove and displayed inhibi-tory activity at low concentrations toward three dif-ferent DNA-dependent enzymes (Susova et al., 2010;Ivanov et al., 2011b; Cherepanova et al., 2011; Tun-itskaya et al., 2011). However, these compounds hada substantial drawback: a low solubility in watersolutions, which prevent their potential use in vivoas biologically active preparations. Therefore, for thenew minor groove binder series, additional groupswere added into the molecule structures for the sakeof hydrophility and DNA affinity improvement. A

DBP(n) series contains piperazine cycle in the oli-gomethylene linker which connects bisbenzimidazolefragments of molecule together. A DBPy(n) serieshave a pyrrole and benzimidazole cycles combinationin their structure (Figure 1). DBP(n) (n= 1–4) com-pounds were studied as inhibitors of calf thymusDNA topoisomerase I (topo-I). All of them inhibitedtopo-I activity at 1–5 μM concentrations, besides, atconcentrations over 5 μM, the inhibitory activities ofDBP(n) were few times higher than that of DB(n).It was found that dimeric bisbenzoimidazoles DBP(1–3) were not mutagenic. They were inactive in thepoint mutagenesis assessment using the Ames test(with Salmonella Typhimurium bacteria as the testedobject) and did not display mutagenic or recombino-genic properties in the Drosophila wing somaticmutation and recombination test (SMART). All thecompounds of series were evaluated for cytotoxicityin the MTT test on breast cancer MCF-7, largebowel adenocarcinoma HCT-116, noncancer HEK-293cell lines and on primary murine fibroblasts. All thetested DBP(n) were not toxic up to concentrationsof 100 μM. Contrary, the one tested compound fromDBPy(n) series, DBPy(4) (n = 4), showed substantialcytotoxicity against cancer HCT-116 and human leu-kemic K-562 cell lines with an IC50 of 1–2 μM.Judging by the combination of such criterions aswater solubility, topo-I inhibition activity and mutage-nicity, it would be safe to assume that dimeric bis-benzoimidazoles of DBP(n) series are moreperspective minor groove binders than previouslysynthesized DB(n).

The work was supported by RFBR (Grants 11-04-00589and 12-04-31599) and by the program of the Presidiumof the Russian Academy of Sciences “Molecular andCellular Biology” and the “Protek” fund.

ReferencesCherepanova, N. A., Ivanov, A. A., Maltseva, D. V., Minero,

A. S., Gromyko, A. V., Streltsov, S. A., Zhuze, A. L., &Gromova, E. S. (2011). Dimeric bisbenzimidazoles inhibitthe DNA methylation catalyzed by the murine Dnmt3acatalytic domain. Journal of Enzyme Inhibition and Medici-nal Chemistry, 26, 295–300.

Ivanov, A. A., Salyanov, V. I., Streltsov, S. A., Cherepanova, N.A., Gromova, E. S., & Zhuze, A. L. (2011). Synthesis offluorescent biologicaly active dimeric bisbenzimidazoles –DB(3, 4, 5, 7, 11). Russ J. Bioorgan Chemistry, 37, 472–482.

Ivanov, A. A., Streltsov, S. A., Salyanov, V. I., Susova, O. Y.,Gromova, E. S., & Zhuze, A. L. (2011). Minor grooveligands based on dimeric bisbenzimidazoles as inhibitors ofDNA- dependent enzymes. Journal of Biomolecular Struc-ture & Dynamics, 28, 1131–1132.


Susova, O. Y., Ivanov, A. A., Morales Ruiz, S. S., Lesovaya,E. A., Gromyko, A. V., Streltsov, S. A., & Zhuze, A. L.(2010). Minor groove dimeric bisbenzimidazoles inhibitin vitro DNA binding to eukaryotic DNA topoisomerase I.Biochemistry (Moscow), 75, 695–701.

Tunitskaya, V. L., Mukovnya, A. V., Ivanov, A. A., Gromyko, A.V., Ivanov, A. V., Streltsov, S. A., Zhuze, A. L., & Kochetkov,S. N. (2011). Inhibition of the helicase activity of the HCVNS3 protein by symmetrical dimeric bis-benzimidazoles. Bio-organic & Medicinal Chemistry Letters, 21, 5331–5335.

N

NH

N

NH

N

NCH3

NH

O

N

NH

N

NH

N

NCH3

NH

O

(CH2)n

DB(n)

DBP(n)

DBPy(n)

(CH2)n NN

NH

N

CH3

NH

NNH

O

(CH2)nNN

NH

N

CH3

NH

NNH

O

NN

CH3

CH3 N

NH

N

NH

NN

N

NH

O

CH3

CH3N

NH

N

NH

NN

N

NH

O

(CH2)n

Figure 1. Structures of dimeric bisbenzimidazoles of DB(n), DBP(n) and DBPy(n) series.

82 Targeting intramolecular DNAstructures with complementarystrands

Calliste Reiling, Sarah E. Johnson, Hui-Ting Lee andLuis A. Marky*

Department of Pharmaceutical Sciences, University ofNebraska Medical Center, 986025 Nebraska Medical Center,Omaha, NE 68198-6025*Email: [email protected], Phone: 402-559-4628,Fax: 402-559-9543

Antisense, antigene, and siRNA strategies are currentlyused to control the expression of genes. To this end, ourlaboratory is mimicking the targeting of mRNA by react-ing DNA stem-loop motifs with their partially comple-mentary strands. Specifically, we used a combination ofisothermal titration (ITC), differential scanning calorime-try (DSC), and temperature-dependent UV spectroscopyto investigate: (1) the unfolding of a pseudoknot and acomplex containing joined triplex-duplex motifs (shownbelow); and (2) the reaction of these compact structureswith single strands that are complementary to the bases inthe loops and to a portion of their stem.

We used DSC to determine the temperature unfoldingthermodynamics for the reactants and products of eachreaction. The resulting unfolding data is then used to cre-ate thermodynamic (Hess) cycles that correspond to eachtargeting reaction. The resulting enthalpies are comparedwith the reaction enthalpies, obtained directly from iso-thermal titration (ITC) experiments. All reactions yieldedfavorable free energy contributions that were enthalpy-driven. These favorable heat contributions result fromthe formation of base pair stacks involving the unpairedbases of the loops, indicating that each complementarystrand was able to invade and disrupt the secondarystructure.

This research is supported by National Science FoundationGrant MCB-1122029 and GAANN grant P200A120231(C.R.) from the US Department of Education.


83 Application of differential scanningcalorimetry to measure thedifferential binding of ions, water,and protons in the unfolding of DNAmolecules

Chris M. Olson, Irine Khutsishvili, Sarah E. Johnson,Calliste Reiling and Luis A. Marky*

Department of Pharmaceutical Sciences, University ofNebraska Medical Center, 986025 Nebraska Medical Center,Omaha, NE 68198-6025, USA*Email: [email protected], Phone: 402-559-4628,Fax: 402-559-9543

The overall stability of DNA molecules globally dependson base-pair stacking, base pairing, polyelectrolyteeffect, and hydration contributions. In order to improveour understanding of the role of ions, water, and protonsin the stability and melting behavior of DNA structures,we report an experimental approach to determine the dif-ferential binding of ions (Δnion), water (ΔnW), and pro-tons (ΔnH+) in the helix-coil transition of DNAmolecules. A combination of differential scanning calo-rimetry (DSC) and temperature-dependent UV and CDspectroscopic techniques to investigate the unfolding ofa variety of DNA molecules: S.T. DNA, two dodeca-mers, one undecamer, nine short hairpins as a functionof the GC content of their stem, and two triplexes. Wedetermine complete thermodynamic profiles, includingall the three linking numbers, for the unfolding of eachmolecule. The CD spectra indicated that all moleculesadopted the B-conformation at low temperatures. Ther-modynamic profiles obtained from the DSC curves indi-cate that the favorable folding of each molecule resultsfrom the typical compensation of favorable enthalpy andunfavorable entropy contributions, and negligible heatcapacity effects. UV and DSC melting curves as a func-tion of salt, osmolyte, and proton concentrations yieldedreleases of ions, water, and protons (for the triplex withC+GC base triplets). Therefore, the favorable folding ofeach DNA molecule results from the formation of base-pair stacks and uptake of water and counterions. Thethermodynamic data will be discussed in terms of theeffects of DNA length, loop contributions and type ofwater molecules.

This research is supported by Grant MCB-1122029 fromthe National Science Foundation.

84 PPC measurements of unfoldingvolumes of DNA stem-loop motifs

Irine Khutshivilli, Iztok Prislan and Luis A. Marky*

Department of Pharmaceutical Sciences, University ofNebraska Medical Center, 986025 Nebraska Medical Center,Omaha, NE 68198-6025, USA*Email: [email protected], Phone: 402-559-4628,Fax: 402-559-9543

One focus of our research is to further our understandingof the physico-chemical properties of non-canonicalnucleic acid structures. In this work, DNA hairpins areused to mimic a common motif present in RNA, i.e. astem-loop motif with a bulge or internal loop in theirstem. Specifically, we used a combination oftemperature-dependent UV spectroscopy, differentialscanning (DSC), and pressure perturbation (PPC) calori-metric techniques to determine complete thermodynamicprofiles for the helix–coil transitions of two sets ofhairpins with 5′–3′ sequences: d(GCGCTnGTA-ACT5GTTACGCGC) and d(GCGCTnGTAACT5GTT-ACTnGCGC). “Tn” is a variable loop of thymines, n= 1,3 or 5; and “T5” is an end-loop of five thymines.Unfolding curves show monophasic transitions with TMsindependent of strand concentration, confirming theirintramolecular formation. DSC thermodynamic profilesindicate that the favorable folding of each hairpin resultsfrom the typical compensation of favorable enthalpy andunfavorable entropy contributions, while the DSC curvesas a function of salt concentration yielded an uptake ofcations and negative heat capacity effects. PPC meltingcurves yielded positive folding volumes ranging 12–31 cm3/mol, corresponding to releases of water mole-cules; in contrast, an uptake of water (ranging from 32to 63mol of H2O/mol) is observed from osmotic stressexperiments using ethylene glycol as the osmolyte. Over-all, the increase in the size of the variable bulge or inter-nal-loop yielded lower TMs and slightly more favorableenthalpies, corresponding to less favorable free energycontributions of �0.7 kcal/mol per thymine residue. Thevolume measurements will be correlated with the unfold-ing entropies and discussed in terms of the type of waterthat is hydrating these stem-loop motifs structures.

This research has been supported by Grants MCB-0616005 and MCB-1122029 from the National ScienceFoundation.


85 Bistable regime of adsorption ofligands on a macromolecule inducedby an external noise

Valeri B. Arakelyana*, Sos V. Harutyunyanb,Vardan K. Andriasyanc and Hakob V. Arakelyana

aDepartment of Molecular Physics, Yerevan State University,Aleq Manukyan 1, Yerevan 0025, Armenia; bDepartment ofMedical Physics, Yerevan State Medical University, MkhitarHeratsi, Koryun 2, Yerevan 0025, Armenia; cInstitute ofEvolutionary Biology and Environmental Studies, University ofZurich, Winterhurerstrasse, 190, CH -8085 Zurich, Switzerland*Email: [email protected], Phone: 37410-77689,Fax: 37410-554641

Practically in all experimental and theoretical worksdedicated to the interaction of ligands with the macromol-ecule (DNA, RNA), the investigations are carried out atthe fixed parameters of the external medium, that is at theabsence of an external noise, though it is obvious that theexternal noise is never exactly equal to zero. Note alsothat a number of experimental and theoretical studieshave shown that under the action of an external noise, thebehavior of a physicochemical system can greatly differfrom that of a system in a deterministic medium. Issuingfrom this fact, the influence of the external noise has beeninvestigated on the adsorption of ligands on a macromol-ecule. We consider the case of small fillings. The kineticsof adsorption of ligands on a macromolecule are consid-ered in complex with the change of the structure of theadsorption center, which is the profile deformation of thepotential energy of the adsorption center at entering of aligand into the adsorption center. The description of thedeformation of the potential energy of the adsorption cen-ter is carried out within the framework of the relaxationequation for the conformation variable. Thus, the kineticsof the adsorption of ligands on a macromolecule aredescribed by a set of two nonlinear differential equations,which describes the self-consistent process of adsorptionand variation of the conformation variable at each adsorp-tion center. Through the reduction of the system, only arelaxation equation is left allowing to calculate theconformation potential and determine the isotherm ofthe adsorption of ligands on the macromolecule at thepresence of the external noise. It is shown that thestochastic potential strongly depends on the intensity ofthe external noise. The analysis of the stochastic potentialreveals that in some region of changing of the intensityof the external noise, the stochastic potential has twominima separated by a maximum. This leads to a bistableregime of adsorption of ligands on the macromoleculeand the S-form of the isotherm of adsorption.

86 Characterization and differentiationof binding modes of water-solubleporphyrins at complexation withDNA

Vigen G. Barkhudaryan*, Gayane V. Ananyan andYeva B. Dalyan

Chair of Molecular Physics, Faculty of Physics,Yerevan State University, 1 Alex Manoogian str., 375025,Yerevan, Armenia*Email: [email protected]

The influence of water-soluble cationic meso-tetra-(4N-oxyethylpyridyl)porphyrin (H2TOEPyP4) and it’smetallocomplexes with Ni, Cu, Co, and Zn on hydro-dynamic and spectral behavior of DNA solutions hasbeen studied by UV/Vis absorption and viscosity mea-surement. It was shown that the presence of planarporphyrins such as H2TOEPyP4, NiTOEPyP4, andСuTOEPyP4 leads to an increase in viscosity at rela-tively small concentrations, and then decrease to sta-ble values. Such behavior is explained byintercalation of these porphyrins in DNA structurebecause the intercalation mode involves the insertionof a planar molecule between DNA base pairs whichresults in a decrease in the DNA helical twist andlengthening of the DNA. Further decrease of viscosityis explained by the saturation intercalation sites andoccurs outside the binding mode. But, in the case ofporphyrins with axial ligands such as CoTOEPyP4and ZnTOEPyP4, the hydrodynamic parametersdecrease, which is explained by self-stacking of theseporphyrins in DNA surface. This data are proved byspectral measurements. The results obtained from titra-tion experiments were used for calculation of bindingparameters: the binding constant Kb and the numberof binding sites per base pair n. Obtained data revealthat Kb varies between 3.4 and 5.4� 106M�1 for aplanar porphyrins, a range typical for intercalationmode interactions, and 5.6� 105M�1 and1.8� 106M�1 for axial porphyrins. In addition, theexclusion parameter n also testifies that at intercala-tion, (n�2) the adjacent base pairs are removed toplace the planar molecules, and for outside binders topack on the surface needs too few places (n�0.5–1).It is apparent that the binding is somewhat strongerat intercalation. The viscometric and spectrophotomet-ric measurements are in good agreement.


87 The melting of DNA in the presenceof meso-tetra-pyridyl porphyrinswith different peripheralsubstituents

L.R. Aloyana,b* and Y.B. Dalyana

aDepartment of Molecular Physics, Faculty of Physics,Yerevan State University, Al.Manoogian 1, 0025 Yerevan,Armenia; bThe Abdus Salam International Centerof Theoretical Physics, Strada Costiera 11, I-34151 Trieste,Italy*Email: [email protected], Phone: 374-10-554341

The influence of water-soluble cationic 3N- and4N-pyridyl porphyrins with different peripheral sub-stituents (oxyethyl, buthyl, allyl, and metallyl) onmelting parameters of DNA has been studied. Resultsindicate that the presence of porphyrin changes theshape and parameters of DNA melting curve. Theincrease of porphyrins concentration results in theincrease of the melting temperature (Tm) and themelting interval (ΔT) of DNA. At the porphyrin-DNA concentration ratio r= 0.01, changes in themelting temperature have not been observed. Themelting intervals almost do not change upon addingof the 4N-porphyrins, while the decrease of ΔT, inthe presence of 3N-porphyrins, is observed. Becausethe intercalation binding mechanism occurs in GC-rich regions of DNA, we assume that 3N-porphyrins,intercalated in GC-rich regions, reduce the thermalstability of these sites, bringing them closer to thethermal stability of the AT-sites, which is the reasonfor the decrease in the melting interval. While at therelative concentration r= 0.01 for 4-N porphyrins,already the external binding mechanism “turns on”and the destabilizing effect of porphyrins on GC-pairs compensates stabilizing effect on AT-pairs, as aresult of which change in the melting of DNA uponcomplexation with these porphyrins is not observed.The decrease of the hypochromic effect also indicatesthe intercalation of investigated porphyrins in theDNA structure, which weakens the staking interactionof base pairs of DNA. The increase of the hypo-chromic effect of DNA upon binding with porphyrindepends on the type of peripheral substituents of theporphyrin. The results show that porphyrins withbutyl and allyl substituents weaken staking interactionof base pairs less than porphyrins with other substit-uents. The largest change was observed for metallylporphyrins. It can be the result of bulky peripheralsubstituents, which make significant local changes inDNA structure.

88 Comparison of monovalent anddivalent ion distributions around aDNA duplex with molecular dynamicsimulation and Poisson–Boltzmannapproach

Timothy J. Robbins and Yongmei Wang*

Department of Chemistry, University of Memphis, Memphis, TN38152, USA*Email: [email protected], Phone: 901-678-2621,Fax: 901-678-3447

Ion interactions with nucleic acids (both DNA and RNA)are an important and evolving field of investigation.Positively charged cations may interact with highlynegatively charged nucleic acids via simple electrostaticinteractions to help screen the electrostatic repulsion alongthe nucleic acids and assist their folding and/or compac-tion. Cations may also bind at specific sites and becomeintegral parts of the structures, possibly playing importantenzymatic roles. Two popular methods for computation-ally exploring a nucleic acid’s ion atmosphere are atomis-tic molecular dynamics (MD) simulations and thePoisson–Boltzmann (PB) equation. In general, monovalention results obtained from MD simulations and the PBequation agree well with experiment. However, Bai et al.(2007) observed discrepancies between experiment andthe PB equation while examining the competitive bindingof monovalent and divalent ions, with more significant dis-crepancies for divalent ions. The goal of this project wasto thoroughly investigate monovalent (Na+) and divalent(Mg2+) ion distributions formed around a DNA duplexwith MD simulations and the PB equation. We simulatedthree different cation concentrations, and matched theequilibrated bulk ion concentration for our theoretical cal-culations with the PB equation. Based on previous work,our Mg2+ ions were fully solvated, the expected state ofMg2+ ions when interacting with a duplex, when the pro-duction simulations began and remained throughout thesimulations (Kirmizialtin, 2010; Robbins, 2012). Na+ iondistributions and number of Na+ ions within 10Å of theDNA obtained from our two methods agreed well. How-ever, results differed for Mg2+ ions, with a lower numberof ions within the cut-off distance obtained from the PBequation when compared to MD simulations. The Mg2+

ion distributions around the DNA obtained via the twomethods also differed. Based on our results, we concludethat the PB equation will systematically underestimateMg2+ ions bound to DNA, and much of this deviation isattributed to dielectric saturation associated with highvalency ions.


This research has been supported by ORAU/ORNL HighPerformance Computing Award and NSF TennesseeEPSCOR funding (grant EPS-1004083).

ReferencesBai, Y., & Greenfield, M. (2007). Quantitative and comprehen-

sive decomposition of the ion atmosphere around nucleicacids. Journal of the American Chemical Society, 129,14981–14988.

Kirmizialtin, S., & Elber, R. (2010). Computational explorationof mobile ion distributions around RNA duplex. Journal ofPhysical Chemistry B, 114, 8207–8220.

Robbins, T. J., & Wang, Y. (2012). Effect of initial ion posi-tions on the interactions of monovalent and divalent ionswith a DNA duplex as revealed with atomistic moleculardynamics simulations. Journal of Biomolecular Structureand Dynamics, iFirst, 1–13.

89 Computational mapping revealseffect of Hoogsteen breathing onduplex DNA reactivity withformaldehyde

T. Bohnuud, D. Beglov, C.H. Ngan, B. Zerbe, D.R. Hall,R. Brenke, S. Vajda, M.D. Frank-Kamenetskii andD. Kozakov

Department of Biomedical Engineering, Boston University,Boston MA, 02215, USAEmail: [email protected]

Formaldehyde has long been recognized as a hazard-ous environmental agent highly reactive with DNA.Recently, it has been realized that due to the activityof histone demethylation enzymes within the cellnucleus, formaldehyde is produced endogenously indirect vicinity of genomic DNA. Should it lead toextensive DNA damage? We address this question withthe aid of a computational mapping method, analogousto X-ray and nuclear magnetic resonance techniquesfor observing weakly specific interactions of smallorganic compounds with a macromolecule in order toestablish important functional sites. We concentrate onthe leading reaction of formaldehyde with free bases:hydroxymethylation of cytosine amino groups. Ourresults show that in B-DNA, cytosine amino groupsare totally inaccessible for the formaldehyde attack.Then, we explore the effect of recently discoveredtransient flipping of Watson–Crick (WC) pairs intoHoogsteen (HG) pairs (HG breathing). Our resultsshow that the HG base pair formation dramaticallyaffects the accessibility for formaldehyde of the cyto-sine-aminonitrogens within WC-base pairs adjacent toHG-base pairs. The extensive literature on DNA inter-

action with formaldehyde is analysed in light of thenew findings. The obtained data emphasize the signifi-cance of DNA HG breathing.

ReferencesBohnuud, T., Beglov, D., Ngan, C. H., Zerbe, B., Hall, D. R.,

Brenke, R., … Kozakov, D. (2012). Computational map-ping reveals dramatic effect of Hoogsteen breathing onduplex DNA reactivity with formaldehyde. Nucleic AcidsResearch, 40, 7644–7652.

Nikolova, E. N., Kim, E., Wise, A. A., O'Brien, P. J.,Andricioaei, I., & Al-Hashimi, H. M. (2011 Feb 24).Transient Hoogsteen base pairs in canonical duplex DNA.Nature, 470, 498–502.

90 Intra-accumbens injection of adopamine aptamer abates MK-801-induced cognitive dysfunction ina model of Schizophrenia

Erin M. McConnella*, Matthew R. Holahanb,Dan Madularub, Ryan Walsha and Maria C. DeRosaa

aDepartment of Chemistry, Carleton University, 1125 ColonelBy Drive, Ottawa, K1S 5B6, ON, Canada; bDepartment ofNeuroscience, Carleton University, 1125 Colonel By Drive,Ottawa, K1S 5B6, ON, Canada

Aptamers are short, single-stranded sequences of DNAor RNA, which exhibit conformationally uniquethree-dimensional structures. Aptamer binding to targetmolecular substrates (from small molecules to proteins)demonstrates a high degree of affinity and specificity, inan analogous fashion to antigen–antibody binding.Aptamers offer several advantages over antibodies,including highly reproducible synthesis and ease of mod-ification. Though the characteristics of aptamers and theirapplicability within the central nervous system showgreat potential, research in this area is limited. In thepresent work, the ability of a dopamine-binding DNAaptamer to regulate MK-801-induced cognitive deficitswhen injected into the nucleus accumbens was investi-gated. Systemic administration of the non-competitiveNMDA-receptor antagonist, MK-801, has been proposedto model cognitive deficits similar to those seen inpatients with schizophrenia. Rats were trained to barpress for chocolate pellet rewards then randomlyassigned to receive an intra-accumbens injection of aDNA aptamer (200 nM; n = 7), tris buffer (n= 6) or arandomized DNA oligonucleotide (n= 7). Animals werethen treated systemically with MK-801 (0.1mg/kg) andtested for their ability to extinguish their bar-pressingresponse. Injection of 200 nM dose of the dopamine apt-


amer reversed this MK-801-induced elevation in leverpressing to levels as seen in rats not treated with MK-801. Tests for activity showed that the aptamer did notimpair locomotor activity. Results demonstrate thein vivo utility of DNA aptamers as tools to investigateneurobiological processes in preclinical animal models ofmental health disease.

91 Kinetics of DNA overstretching:melting vs. B-to-S transition

Micah J. McCauleya, Ioulia Rouzinab* andMark C. Williamsa

aDepartment of Physics, Northeastern University,110 Forsyth Street, Boston, MA 02115, USA; bDepartmentof Biochemistry, Molecular Biology, and Biophysics,University of Minnesota, 321 Church Street, Minneapolis, MN55455, USA*Email: [email protected], Phone: 651-354-6006

Single B-form DNA molecules undergo an overstret-ching transition at force Fov to a �1.7-fold longerform when stretched. The nature of overstretchedDNA has been debated for over 10 years. Eitherpeeled (PL DNA), internally melted (M DNA), orunwound double-helical (S DNA) forms of over-stretched DNA have been suggested. Here, we charac-terize the kinetics of the overstretching transition inpolymeric torsionally unconstrained double-stranded(ds) DNA molecules. We pull �50Kbp λ–DNA mole-cules using optical tweezers with rates ν �10 nm/s to5� 104 nm/s, (overstretching time between 0.2 and103 s). The Fov(ν, [Na+]) dependence measured over abroad range of rates and solution ionic strength sug-gests the existence of all three forms of the over-stretched DNA. Thus, at [Na+] > 50mM and thestretching time >>1 s, internal melting dominatesoverstretching. This B-to-M transition is highly coop-erative (involves �100 bp), and slow (on/off time�1000 s). Faster overstretching during 61 s leads toB-to-S DNA transition, which is less cooperative(involves �10 bp) and faster (on/off time �1 s). Incontrast, in lower salt ([Na+] < 50mM), the overstret-ching during >1 s leads to DNA peeling. However,on the faster time scale of 0.2–1 s, even in low salt,the DNA overstretches into S DNA, as peelingbecomes kinetically prohibited. Our conclusions aresupported by several independent lines of evidence,including the salt and rate dependence of both theslope of the overstretched DNA force-extension curveand the value of the second transition force (from Mor PL DNA into S DNA).

92 Novel interpretation of the isothermsof multimodal ligands binding withDNA

Poghos O. Vardevanyana, Valeri B. Arakelyanb,Ara P. Antonyana, Zhanna H. Mukhaelyana,Lilit A. Hambardzumyana and Armen T. Karapetianc

aDepartment of Biophysics and Molecular Physics, YerevanState University, Aleq Manukyan 1, Yerevan 0025, Armenia;bDepartment of Molecular Physics, Yerevan State University,Aleq Manukyan 1, Yerevan 0025, Armenia; cDepartment ofPhysics and Electrotechnics, Yerevan State University ofArchitecture and Construction, Yerevan 0009, ArmeniaEmail: [email protected], Phone: 37410-571061,Fax: 37410-554641

The binding of ligands with DNA is a key moment in awhole range of cellular processes that provide not onlythe normal cell vital activity but also the development ofsome pathological processes. Depending on ligand type,structure of DNA adsorption centers, and physical–chemical conditions of the surrounding, the ligand maybind to DNA by several modes [1]. Particularly, adsorptionisotherm of multimodal ligands binding to DNA inScatchard’s coordinates has a concave shape with twobrightly expressed linear areas in the region of smallfillings. The analysis of such type of adsorption isothermfor determining of important binding parameters such asbinding constant and number of adsorption centers(the part of DNA polymer with which one ligand mole-cule binds) presents difficulties. Practically in all cases,the analysis of such adsorption isotherm is carried outby linear parts of curves. Such analysis mode of experi-mental points is approximate method, since all registeredof experimental points are roughly divided into twogroups and they are treated by linear binding isothermand therefore the binding parameters are determined. Inthe present work, the non-linear adsorption isotherm inScatchard‘s coordinates is obtained which allowed,provided, the more precise treatment of all experimentalpoints by unique curve which includes linear regions aswell. Such mode of treatment of experimental pointsmakes more precise the determination of not onlybinding constant and number of adsorption centers thatcorrespond to the one ligand molecule binding, but alsoadditional binding parameter – a proportion of adsorptioncenters of each binding to DNA type of multimodalligand.

ReferenceArakelyan, V. B., Babayan, Yu. S., & Potikyan, G. (2000).

Determination of constant rates of adsorption of ligand onDNA. Journal of Biomolecular Structure and Dynamics,25, 119–125.


93 Spectroscopic investigation ofmethylene blue binding toDNA

Poghos O. Vardevanyana, Ara P. Antonyana,Marine A. Parsadanyana, Mariam A. Shahinyana,Lilit A. Hambrdzumyana, Mikayel V. Minasyantsa andArmen T. Karapetianb

aDepartment of Biophysics, Yerevan State University, AleqManukyan 1, Yerevan 0025, Armenia; bDepartment of Physicsand Electrotechnics, Yerevan State University of Architectureand Construction, Yerevan 0009, ArmeniaEmail: [email protected], Phone: 37410-571061,Fax: 37410-554641

The interaction of methylene blue (MB) with DNAhas been investigated by UV absorption spectra,Fluorescence spectra and UV-melting method. Analysisof the results of the melting experiments shows thatmelting temperature (Tm) of the complexes increaseswith the [total ligand]: DNA ratio (r) at two concen-trations of Na+ (2mM Na+ and 20mM Na+) providingsupport for conclusion that MB is a stabilizer of DNAhelix structure. By contrast, the shapes of dependencesof width of transition (ΔT) on r at low and high[Na+] are different which points to the existence ofdifferent types of binding modes of MB with DNA.UV-spectroscopy experiments and fluorescence spectraindicated that the binding modes of MB with DNAdepended on r. At high r (r> 0.25), remarkable hypo-chromic effect with no shift of λmax in the absorptionspectra of MB was observed. The fluorescence of MBwas quenched which indicated that MB was bound tophosphate groups of DNA by electrostatic interaction.At low r ratios (r< 0.2), the absorption spectra of MBupon increasing the concentration of DNA showedgradually decrease in the peak intensities with a redshift. This phenomenon is usually associated withmolecular intercalation into the base stack of the ds-DNA. Using the Scatchard’s model, the complex for-mation constants for MB with DNA were determined:the binding constant K ≈ 6.5� 105 and binding site sizen ≈ 4. Obtained data are not typical for intercalationmodel of ligands to DNA. Moreover, comparisonbetween these data and our early experimental resultsof interaction of ethidium bromide with DNA made itpossible to suggest that this binding type of MB is,more probably, semi-intercalation mode (Vardevanyanet al., 2003). This conclusion is in accordance withthe analysis of the model structures of MB–DNA com-plexes which clearly shows the importance of solventcontributions in suggested structural form (Tong et al.,2010).

ReferencesTong, Ch., Hu, Zh., & Jianmin, W. (2010). Interaction between

Methylene blue and calf thymus deoxyribonucleic acid byspectroscopic technologies. Journal of Fluorescence, 20,261–267.

Vardevanyan, P. O., Antonyan, A. P., Parsadanyan, M. A.,Davtyan, H. G., & Karapetyan, A. T. (2003). The bindingof ethidium bromide with DNA: interaction withsingle- and double-stranded structures. EMM, 35, 527–533.

94 The investigation of Hoechst 33258interaction with DNA in aqueoussolutions of dimethylsulfoxide

Poghos O. Vardevanyana, Ara P. Antonyanb,Karen Yu. Amirbekyanb and Shiraz A. Markarianb

aDepartment of Biophysics, Yerevan State University, AleqManukyan 1, Yerevan 0025, Armenia; bDepartment of PhysicalChemistry, Yerevan State University, Aleq Manukyan 1, Yerevan0025, ArmeniaEmail: [email protected], Phone: 37410-571061,Fax: 37410-554641

The melting of DNA complexes with Hoechst 33258(H33258) has been carried out in both presence andabsence of dimethylsulfoxide (DMSO) to reveal theeffect of the mentioned compound on melting parame-ters of DNA-H33258 complexes at r = 0.1, where r=[H33258]/[DNA] and at 0.02M Na+ ion concentration.It is known that the structural–functional changes ofmacromolecules take place in aqueous solutions ofDMSO (Markarian et al., 2006). It has been shownearlier that DNA hydration degree increase results insharp changing of the binding mechanism of H33258to DNA: AT-specific mode of binding disappears andligand molecules start to intercalate into DNA atr6 0.1 (Vardevanyan et al., 2008). Moreover, theincrease of Tm of complexes is higher compared tothe case of relatively low hydration degree of DNA.This is conditioned by the fact that at DNA hydration,increasing the intercalation becomes more preferablesince in this case hydrophobic ligand molecules areshielded from water more effectively. The resultsobtained confirm that with the increasing of DMSOcontent in solution the melting temperature, Tm,decreases. Moreover, denaturation of complexes leadsto the decrease of the fluorescence intensity of ligandmolecule. We suggest that the observed phenomenonhappens due to the structural change of solution whichpromotes dissociation of ligand molecules bound toDNA, and hence a facilitated denaturation of DNA-H33258 complexes occurs.


ReferencesMarkarian, S. A., Asatryan, A. M., Grigoryan, K. R., &

Sargsyan, H. R. (2006). Effect of diethylsulfoxide on thethermal denaturation of DNA. Biopolymers, 81, 1–5.

Vardevanyan, P. O., Antonyan, A. P., Parsadanyan, M. A.,Pirumyan, K. V., Muradyan, A. M., & Karapetyan, A. T.(2008). Influence of ionic strength of hoechst 33258 bind-ing with DNA. Journal of Biomolecular Structure &Dynamics, 25, 641–646.

95 Orientational ordering of short DNAfragments in the polymer matrix

Zareh A. Grigoryana, Yevgeni Sh. Mamasakhlisovb* andArmen T. Karapetianc

aDepartment of Electronics, Goris State University, Avangard2, Goris 3201, Armenia; bDepartment of Biophysics, YerevanState University, Aleq Manukyan 1, Yerevan 0025, Armenia;cDepartment of Physics and Electrical Engineering, YerevanState University of Architecture and Construction, Yerevan,0009, Armenia*Email: [email protected], Phone 37410-554341,Fax: 37410-554641

As a vital part of modern nanotechnology, nanofabrica-tion aims to develop nanoscale components and nanoma-terials in large quantities at relatively low cost. Thepromising strategy is the bottom-up self-assembly tech-niques of chemical assembly and molecular recognitionto bring together individual atoms, molecules, or supra-molecular building blocks to form useful constructs. TheDNA-DNA self-assembly seems to be the key point reg-ulating the polymer composites formation. We addressthe mixture of a flexible polymer with short double-strand DNA fragments, where the persistence length is incomparable with the contour length of the molecule. Weinvestigate the conditions affecting the orientational orderformation of short double-strand DNA fragments,immersed in the flexible polymer. It is shown that shortdouble-strand DNA fragments exhibit the formation of aliquid crystalline ordered phase, in dependence on thevalue of the Flory–Huggins parameter, aspect ratio , andthe attraction energy (Mamasakhlisov et al., 2009; Toddet al., 2008) of the double strand DNA molecules andvolume fraction of polymer.

ReferencesMamasakhlisov, Y. Sh., Todd, B. A., Badasyan, A. V., Mkrtch-

yan, A. V., Morozov, V. F., & Parsegian, V. A. (2009).DNA stretching and multivalent-cation-induced condensa-tion. Physical Review E, 80, 1–9.

Todd, B. A., Parsegian, V. A., Shirahata, A., Thomas, T. J., &Rau, D. C. (2008). Attractive forces between cation con-densed DNA double helices. Biophysical Journal, 94,doi:10.1529/biophysj.107.127332

96 Study of the stacking of DNAhomoassociates by the extendedcluster approach

Victor I. Danilova* and Vladimir V. Dailidonisb

aInstitute of Molecular Biology and Genetics, NationalAcademy of Sciences of Ukraine, 150 Zabolotnoho St.,Kyiv-143, 03143 Ukraine; bBogolyubov Institute for TheoreticalPhysics, National Academy of Sciences of Ukraine,14b Metrologichaskaya St., Kyiv-143, 03143 Ukraine*Email: [email protected]

Metropolis Monte Carlo method based on the extendedcluster approach (Danilov, Dailidonis, van Mourik, &Fruchtl, 2011a, 2011b; Dailidonis, Danilov, Früchtl, &van Mourik, 2011) is used to investigate adenine–ade-nine (AA), guanine–guanine (GG), thymine–thymine(TT), and cytosine–cytosine (CC) homoassociates in acluster consisting of 400 water molecules. The startingstructures taken were AA N(7) amino symmetric, TT N(3) – O(4) symmetric, GG N(1) – O(6) symmetric, andCC N(3) amino symmetric base pairs. A water sphericalcluster with the density of water at room temperatureand a radius sphere equal 13.9 Å was used, which corre-sponds to the most difficult conditions for the formationof stacks (see Abraham, 1982). In spite of such initialconditions, it is shown that during the simulation, eachbase pair is transformed into a more favorable stackedconfiguration. The results obtained allow to observe thewhole process of convergence for the first time (for moreinformation, visit the Website http://biophys.in.ua/).

Table: Energetic characteristics of the transition from thehydrogen-bonded base pairs to the stacked associatesand base stacking reaction in water cluster (in kcal/mol).

Transformation ΔUtot ΔUww ΔUwb ΔUbb

AA base pair > A/A stackedassociate

�19.2 �4.7 �18.0 3.5

TT base pair > T/T stackedassociate

�12.1 �6.9 �11.1 5.9

GG base pair > G/G stackedassociate

�24.5 9.3 �45.2 11.4

CC base pair > C/C stackedassociate

�15.8 9.9 �45.4 19.7

(CC base pair > C/C stackedassociate)

�17.4 �6.4 �19.5 8.5

A + A > A/A stackedassociate

�21.6 �7.5 �8.9 �5.2

T + T > T/T stackedassociate

�23.6 �17.9 �1.1 �4.6

G + G > G/G stackedassociate

�16.7 �0.3 �8.9 �7.4

C + C > C/C stackedassociate

�38.2 �30.0 �11.2 3.0

(C + C > C/C stackedassociate)

�13.7 �10.9 5.3 �8.1


It follows from this Table that all stacked associates inthe water cluster are energetically more preferable to thecorresponding base pairs. The changes in the interactionenergies show that the water–base interaction (ΔUwb) isthe determining factor in favoring the stacked speciesover the base pair in an aqueous cluster. This may bedue to the smaller hydrophobic surface of the stacks.The data allows us to calculate the formation energy(ΔUtot) and its various contributions for the base stackingreaction of stacked associates investigated in the watercluster. These results are given in the Table. As can beseen, the formation of all stacked dimers was found tobe favorable, with the formation energies ranging from�16.7 to �38.4 kcal/mol. The preference for the forma-tion of these stacks results from the favorable change inthe water–water interaction (ΔUww) and partly from thewater–base (ΔUwb) and base–base interactions (ΔUbb)during the base association reaction. In contrast to theWatson–Crick base pairs, the formation of all stackedassociates is highly favorable. The water energy changeassociated with the structural rearrangement of the watermolecules around the bases during their association,contributes most to the stabilization of the stacks. Thestacked associates are significantly less stabilized by thebase–base interaction in comparison with the H-bondedbase pairs. It is especially necessary to underline that thebase–base interaction energy in C/C stacked associate ispositive, i.e. repulsion forces operate between bases, thatis conditioned by the competition between the water–water, water–base and base–base interactions. As a resultthe study of hydration of the base associates in anycomplexes with the dense packing or at the account ofvery small number of water molecules, as it is oftendone in a number of works, can result in quite erroneousresults. At the same time, the use of the spherical clus-ters with the radius of equal 24.3 and having a volumein five times greater than the volume of 400 watermolecules (see Abraham, 1982) brings base–base interac-tion energy to negative values (Table, values in brack-ets). Thus, the water–water interaction is one of the mainfactors promoting stacked dimer formation, and theobtained data are a direct confirmation of the crucial roleof the water–water interaction in base stacking reportedearlier in references.

ReferencesAbraham, F. F. (1982). Reports on Progress in Physics, 45,

1113–1161.Danilov, V. I., Dailidonis, V. V., van Mourik, T., & Fruchtl,

H. A. (2011a). Journal of Biomolecular Structure &Dynamics, 28, 1140–1141.

Danilov, V. I., Dailidonis, V. V., van Mourik, T., & Früchtl,H. A. (2011b). Central European Journal of Chemistry, 9,720–727.

Dailidonis, V. V., Danilov, V. I., Früchtl, H. A., & vanMourik, T. (2011). Theoretical Chemistry Accounts, 130,859–870.

97 Study on electron transfer throughRNA/DNA duplex using pyrene and5-bromouracil as an electron donorand acceptor pair

Tadao Takada, Kenji Maie, Yumiko Otsuka,Tomohiro Saeki, Yuta Takamatsu, Mitsunobu Nakamuraand Kazushige Yamana*

Department of Materials Science and Chemistry, GraduateSchool of Engineering, University of Hyogo, 2167 Shosha,Himeji 671-2201, Japan*Email: [email protected], Phone: 0081-79-267-4895

Because of potential applications in nanoscale devices,DNA-mediated charge transfer (CT) has attracted muchinterest. Through spectroscopic and chemical studies, ithas been shown that both positive and negativecharges injected into DNA bases can move throughDNA over significant distances. The factors affectingto DNA- mediated CT are the nature of the chargedonor and acceptor, the structural dynamics of DNA,and the intervening base sequences or the integrity ofthe base stacks. The last factor led to the electrochem-ical devices to detect the perturbations of DNA stackssuch as a mismatch base pair. The photo-inducedcharge migration in DNA possessing a donor–acceptorpair has resulted in the long-lived charge separatedstate. This charge separation offers important insightsfor the development of photo-energy conversiondevices such as solar cells. In contrast to the DNA-mediated CT, little attention has been paid for CT in aRNA duplex. Since RNA duplexes have the basestacking overlaps and dynamics that are significantlydifferent from those of DNA–DNA as well as ofDNA–RNA duplexes, they are another choice of attractivemedium for CT. We have conducted the research on CT inRNA duplexes consisting of a pendant donor (pyrene) andacceptor (5-bromouracil or nitrobenzene) pair. We havefound that long-range excess electron transfer occursthrough RNA π-stacks with double exponential distancedependence. This finding should contribute to uncoverthe mechanism of RNA-mediated electron transfer andopen a way for development of RNA-based devices thatcontrol electron migrations. By contrast, the pyrene-donor and 5-bromouracil-acceptor system indicates thatDNA may not act as an efficient medium for the excesselectron transfer.


ReferencesMaie, K., Miyagi, K., Takada, T., Nakamura, M., & Yamana,

K. (2009). RNA-mediated electron transfer: Double expo-nential distance dependence. Journal of the AmericanChemical Society, 131, 13188–13189.

Takada, T., Otsuka, Y., Nakamura, M., & Yamana, K. (2011).Electron transfer through RNA: Chemical probing of dualdistance dpendence. Bioorganic & Medicinal Chemistry,19, 6881–6884.

98 The impact of active metabolitesobtained from saffron onH1–Oligonucleotide interaction

Reyhane Hoshyar and S. Zahra Bathaie*

Department of Clinical Biochemistry, Faculty of MedicalSciences, Tarbiat Modares University, Tehran, Iran

In the recent years, the anti-tumor activity of saffroncomponents, monoterpene aldehydes (safranal and

picrocrocin), and carotenoids (crocins and crocetin) wasreported by us and other research groups around theworld. The molecular mechanisms of action of saffronand its constituents have been not known clearly, yet.The transcriptional activation of genes occurs due tothe histone H1 dissociation from linker DNA. There-fore, H1–DNA complex has been considered as amodel of chromatin. Our previous study on H1–DNAinteraction in the presence of SDS (which was boundto H1 electrostatically and covered all positive chargeson H1 surface) has shown the more folded structure ofH1 and thus complete dissociation from DNA. We havealso showed, previously, that some modification on his-tone H1 such as acetylation by aspirin, or somechanges in the environment (for example in the pres-ence of chemical chaperones like polyamines) can affectthis complex and dissociate it with various degrees. Inthe present study, the effect of saffron active metabo-lites, both carotenoids and monoterpene aldehydes, onthe histone H1–DNA interaction, using a specific oligo-nucleotide with higher affinity for histone H1 than thatof high molecular weight DNA, is evaluated. Differenttechniques including gel retardation electrophoresis andcircular dichroism (CD) were used. The results of gelretardation assay and CD spectra of Hl–Oligonucleotidecomplex showed the decrease in the complex formationin the following order crocin>picrocrocin>>safr-anal>crocetin. These observations in the in vitro condi-tion led to suggesting a mechanism in which the H1

depletion may affect transcription of some genes, forexample, tumor suppressing genes in vivo. In conclu-sion, saffron’s various applications as an anti-oxidant,anti-genotoxic, and anti-cancer agent are due to its sec-ondary metabolites (safranal, picrocrocin, crocins, andcrocetin), which can decrease interaction of histone H1with DNA and reduce the H1–DNA complex forma-tion.

99 The role of the linker and aromaticpendent in Zn(ii) complexes forrecognition to noncanonical thyminein DNA

Stephanie A. Sander* and Janet R. Morrow

Department of Chemistry, University at Buffalo SUNY, Amherst,NY, 14226 USA*Email: [email protected], Phone: 716-645-4192

It is challenging to design small molecules for the recog-nition of nucleic acid structural motifs such as bulges andloops. Ultimately, it is important to understand all of theenergetic factors involved in binding including electro-static interactions, hydrophobic transfer energetic, nucleic


acid structural rearrangements as well as interactionsbetween the nucleic acid and small molecule. We haverecently reported the selective binding of Zn(II) macrocy-clic complexes to DNA- and RNA-containing thymine oruracil bulges, respectively. The NMR structure of a DNAbulge alone and a DNA bulge with Zn(II) complex showsthat the Zn(II) complex binds to the deprotonated N3 ofthymine and the pendent aromatic stacks over the thy-mine in a pocket that abuts the major groove of the helix.Our current research focuses on understanding the role ofthe pendent aromatic group, and the linker between mac-rocycle and aromatic group. A single ring system was toosmall to stack on the thymine and led to weak binding,while a three ring system was more apt to intercalate intoDNA, leading to poor selectivity. Pendents with twofused aromatic rings are optimal for bulge recognition. Inaddition, the presence of a heteroatom in the pendent aro-matic group increases the polarity and electron deficiencyof the pi system which promotes favorable stacking withthe polar nucleobase. Studies are underway to better

understand the importance of the linker and how it canaffect recognition. It has been observed that a methylenelinker works well when there is a binding pocket, becauseit provides the flexibility for the aromatic pendent to slipinto the pocket and stack tightly on the top of the nucleo-base, as shown in the NMR structure below. (Fig. A)(Reprinted with permission from (1). Copyright (2012)American Chemical Society) In comparison, a less flexi-ble sulfone linker does not interact well with T-bulgeswhich could be in part due to its inability to fit into thepocket. To better elucidate the linker trends and impor-tance, direct attachment of a pendent to the macrocycle isbeing studied. The future application of this work istowards metal ion-based optical sensors and nucleic acidconformational switches.

Referencedel Mundo, I. M. A., et al. (2012). Inorganic Chemistry, 51,

5444–5457.

100 Bridging the gap: from 1D to 3Dgenomics

Lin Yang, Tianyin Zhou and Remo Rohs*

Molecular and Computational Biology Program, University ofSouthern California, Los Angeles, CA 90089*Email: [email protected], Phone: (213) 740-0552,Fax: (213) 821-4257

Combination of the nucleotides A, C, G, and T leadsto one-dimensional (1D) DNA sequence that furthergives rise to the three-dimensional (3D) structure ofDNA. In fact, proteins do not literally read the lettersA, C, G, and T. Instead, read out is based on spatialinteractions between 3D objects, in which the structuresof both DNA and proteins are important for the interac-

tions to occur (Rohs et al. 2009; Rohs et al. 2010). Onthe other hand, while the 3D structure of DNA isdependent on the primary sequence, there is degeneracyin mapping DNA sequence to shape. Moreover, whilehigh-throughput sequencing technologies continue toproduce large amounts of DNA sequence information,experimental data on 3D structures of DNA is limited.These facts all urge to develop new approaches thatprovide new insights into DNA structure. Given suchmotivation, we have developed a high-throughput toolfor predicting shape of “naked” DNA on a genomicscale (Slattery et al. 2011) and a web server for predict-ing DNA shape information. In parallel, we constructeda database of shape that features of transcription factorbinding sites using sequence-based databases as thesource for binding motifs. These tools provide a novel


approach for studying transcription factor binding sites,especially those of paralogous transcription factorswhich share highly similar core-binding motifs but binddifferent genome sites in vivo. Our approach can alsobe used to study less-specific protein–DNA interactions,for example, histone–DNA interactions in nucleosomes.Here, we present structural profiles of transcription fac-tor and nucleosome binding sites of various organismsand provide evidence that DNA shape generally playsan important role in protein–DNA recognition. Specifi-cally, we present insights in how paralogous transcrip-tion factors recognize DNA shape to achievedifferential binding specificity and suggest that mecha-nisms for nucleosome formation are somewhat differentin Plasmodium falciparum whose genome has an extre-mely high A/T content compared with Saccharomycescerevisiae and Drosophila melanogaster.

ReferencesRohs, R., Jin, X., West, S. M., Joshi, R., Honig, B., &

Mann, R. S. (2010). Origins of specificity in protein-DNA recognition. Annual Review of Biochemistry, 79,233–269.

Rohs, R., West, S. M., Sosinsky, A., Liu, P., Mann, R. S., &Honig, B. (2009). The role of DNA shape in protein-DNArecognition. Nature, 461, 1248–1253.

Slattery, M., Riley, T., Liu, P., Abe, N., Gomez-Alcala, P., Dror,I., Zhou, T., Rohs, R., Honig, B., Bussemaker, H. J., &Mann, R. S. (2011). Cofactor binding evokes latent differ-ences in DNA binding specificity between Hox proteins.Cell, 147, 1270–1282.

101 Exploring the 3D spatialorganization of the genome

Frank Alber*

Molecular and Computational Biology, University of SouthernCalifornia, 1050 Childs Way, RRI 413E, Los Angeles,CA 90089, USA*Email: [email protected], Phone: (213) 740-0778

Knowledge about the 3D organization of the genomewill offer great insights into how cells retrieve and pro-cess the genetic information. Knowing the spatial prob-ability distributions of individual genes will provideinsights into gene regulatory and replication processes,and fill in the missing links between epigenomics,functional genomics, and structural biology. We willdiscuss an approach to determine 3D genome structuresand structure–function maps of genomes by integratingdivers types of data. To address the challenge of mod-eling highly variable genome structures, we discuss apopulation-based modeling approach, where we con-struct a large population of 3D genome structures thattogether are entirely consistent with all available experi-mental data including data from genome-wide chromo-some conformation capture and imaging experiments.We interpret the result in terms of probabilities of asample drawn from a population of heterogeneousstructures. We will discuss results on the 3D spatialorganization of genomes in human lymphoblastoid cellsand budding yeast.

102 Genome engineering nucleasesderived from GIY-YIG homingendonucleases

David Edgella*, Benjamin Kleinstivera, Jason M. Wolfsa,Li Wangb, Tomasz Kolaczyka, Brendon McDowella andAdam Bogdanoveb

aDepartment of Biochemistry, Western University, London, ONN6A3B1; bDepartment of Plant Pathology and Plant-MicrobeBiology, Cornell University, Ithaca, NY 14853*Email: [email protected], Phone: (519)-661-3133,Fax: (519)-661-3175

Efficient targeted manipulation of complex genomesrequires highly specific endonucleases to generate dou-ble-strand breaks at defined locations (Bibikova et al.,


2003; Bogdanove and Voytas, 2011). The predomi-nantly engineered nucleases, zinc-finger nucleases(ZFNs), and TAL effector nucleases (TALENs) use thecatalytic domain of FokI as the nuclease portion. Thisdomain, however, functions as a dimer to nonspecifi-cally cleave DNA meaning that ZFNs and TALENsmust be designed in head-to-head pairs to target adesired sequence. To overcome this limitation andexpand the toolbox of genome editing reagents, weused the N-terminal catalytic domain and interdomainlinker of the monomeric GIY-YIG homing endonucleaseI-TevI to create I-TevI-zinc-fingers (Tev-ZFEs), andI-TevI-TAL effectors (Tev-TALs) (Kleinstiver et al.2012). We also made I-TevI fusions to LAGLIDADGshoming endonucleases (I-Tev-LHEs). All the threefusions showed activity on model substrates on parwith ZFNs and TALENs in yeast-based recombinationassays. These proof-of-concept experiments demonstratethat the catalytic domain of GIY-YIG homing endonu-cleases can be targeted to relevant loci by fusing thedomain to characterize DNA-binding platforms. Recentefforts have focused on improving the Tev-TAL plat-form by (1) understanding the spacing requirementsbetween the nuclease cleavage site and the DNA bind-ing site, (2) probing the DNA binding requirements ofthe I-TevI linker domain, and (3) demonstrating activityin mammalian systems.

This research has been supported by grants to D.E. fromthe Canadian Institutes for Health Research (MOP977800) and the Natural Sciences and EngineeringResearch Council of Canada (311610-2010), and a grantfrom the National Science Foundation to A.B. (0820831).

ReferencesBibikova, M., Beumer, K., Trautman, J. K., & Carroll,

D. (2003). Enhancing gene targeting with designed zincfinger nucleases. Science, 300, 764.

Bogdanove, A. J., & Voytas,, D. F. (2011). TAL effectors:Customizable proteins for DNA targeting. Science, 333,1843–1846.

Kleinstiver, B. P., Wolfs, J. M., Kolaczyk, T., Roberts, A. K.,Hu, S. X., & Edgell, D. R. (2012). Monomeric site-specificnucleases for genome editing. Proceedings of the nationalacademy of sciences, USA, 109(21), 8061–8066. doi:10.1073/pnas.1117984109. Epub 2012 May 7.

103 Probing DNA shape andmethylation state on a genomicscale with DNase I

Allan Lazarovicia,b, Tianyin Zhouc, Anthony Shaferd,Ana Carolina Dantas Machadoc, Richard Sandstromd,Peter J. Sabod, Yan Luc, Remo Rohsc,John A. Stamatoyannopoulosd andHarmen J. Bussemakerb,e,*aDepartment of Electrical Engineering, Columbia University,New York, NY 10027, USA; bDepartment of BiologicalSciences, Columbia University, New York, NY 10027, USA;cMolecular and Computational Biology Program,Departments of Biological Sciences, Chemistry, and Physicsand Astronomy, University of Southern California, Los Angeles,CA 90089, USA; dDepartments of Genome Sciences andMedicine, University of Washington, Seattle, WA 98195, USA;eCenter for Computational Biology and Bioinformatics,Columbia University, New York, NY 10032, USA*Email: [email protected], Phone: (212) 854-9932,Fax: (212) 854-1527

DNA binding proteins find their cognate sequenceswithin genomic DNA through recognition of specificchemical and structural features. Here, we demonstratethat high-resolution DNase I cleavage profiles canprovide detailed information about the shape andchemical modification status of genomic DNA. Ana-lyzing millions of DNA-backbone hydrolysis eventson naked genomic DNA, we show that the intrinsicrate of cleavage by DNase I closely tracks the widthof the minor groove. Integration of these DNase Icleavage data with bisulfite sequencing data for thesame cell type genome reveals that the cleavagedirectly adjacent to CpG dinucleotides is enhanced atleast eight-fold by cytosine methylation. This phenom-enon we show is attributable to methylation-inducednarrowing of the minor groove. Furthermore, we dem-onstrate that it enables simultaneous mapping ofDNase I hypersensitivity and regional DNA methyla-tion levels using dense in vivo cleavage data. Takentogether, our results suggest a general mechanismthrough which CpG methylation can modulate pro-tein–DNA interaction strength via the remodeling ofDNA shape.

ReferencesFox, K. R. (1986). The effect of HhaI methylation on DNA

local structure. Biochemistry Journal, 234, 213–216.Rohs, R., et al. (2009). The role of DNA shape in protein-

DNA recognition. Nature, 461, 1248–1253.Suck, D. (1994). DNA recognition by DNase I. Journal of

Molecular Recognition, 7, 65–70.


104 Probing DNA shape on molecularand genomic scales

Tianyin Zhou and Remo Rohs*

Molecular and Computational Biology Program,Departments of Biological Sciences, Chemistry, and Physicsand Astronomy, University of Southern California, 1050 ChildsWay, Los Angeles, CA 90089, USA*Email: [email protected], Phone: (213)740-0552

Linear DNA sequence encodes its three-dimensionalstructure. The resulting DNA shape gives DNA uniquephysical and chemical properties that are explored byDNA-binding proteins (Rohs et al., 2009). Probing DNAshape, therefore, helps to reveal the origin of specificityin DNA-protein recognition. We have developed aMonte Carlo (MC) method for all-atom DNA structureprediction (Joshi et al., 2007). With key componentssuch as parallelization and functionality to handle chemi-cal modifications added, the revamped MC method canefficiently recover the DNA shape of any DNAsequence. To embrace the genomic era, we have traineda high-throughput (HT) prediction method on 2121 inde-pendent MC simulations (Slattery et al., 2011). Applyingthis method on whole genome DNA sequences of 66Drosophila individuals, we found that among singlenucleotide polymorphisms (SNPs) within the immediatevicinity of in vivo transcription factor (TF) binding sites,low-frequency SNPs tend to change the DNA minorgroove width more than high-frequency SNPs. This indi-cates that shape-changing mutations are detrimental toTF binding and in vivo function. We also examined thedependency of DNase I cleavage events on DNA shapeusing our HT method. Our results show that the rate ofDNase I cleavage closely tracks the width of the minorgroove. MC predictions for chemically modified basesfurther demonstrate that cytosine methylation enhancescleavage directly adjacent to CpG dinucleotides throughnarrowing of the minor groove. In summary, with toolson hand to address biological questions on both molecu-lar and genomic scales, our work provides new insightsinto gene regulation and evolution.

ReferencesJoshi, R., Passner, J. M., Rohs, R., Jain, R., Sosinsky, A.,

Crickmore, M. A., … Mann, R. S. (2007). Functionalspecificity of a Hox protein mediated by the recognition ofminor groove structure. Cell, 131, 530–543.

Rohs, R., West, S. M., Sosinky, A., Liu, P., Mann, R. S., &Honig, B. (2009). The role of DNA shape in protein-DNArecognition. Nature, 461, 1248–1253.

Slattery, M., Riley, T., Liu, P., Abe, N., Gomez-Alcala, P., Dror,I., … Mann, R. S. (2011). Cofactor binding evokes latentdifferences in DNA binding specificity between Hoxproteins. Cell, 147, 1270–1282.

105 Activity of DNA ligase onsubstrates containingnon-canonical structures

Katharina Bilotti*, Kelly Schermerhorn andSarah Delaney

Department of Chemistry, Brown University, Providence,RI 02906, USA*Email: [email protected], Phone: (401) 863-2044,Fax: (401) 863-1993

The expansion of trinucleotide repeat tracts (e.g.(CAG)n tracts) has been shown to contribute to geno-mic instability and has been implicated in the patho-genesis of several neurodegenerative diseases,including Huntington’s Disease and Fragile X syn-drome (Kovtun et al., 2008). While the molecularmechanism of this expansion is unknown, the abilityof trinucleotide repeat sequences to form non-canoni-cal secondary structures, such as hairpins, has beenimplicated as a multifaceted source of error (Gacyet al., 1995). Non-canonical DNA secondary structureshave been shown to impact the action of enzymes inthe base excision repair (BER) pathway, by whichoxidatively damaged bases are removed. More specifi-cally, there is evidence that trinucleotide repeat-con-taining DNA mistakenly enters long-patch BER,which can potentially lead to the incorporation ofextra nucleobases by DNA polymerase (Jarem et al.,2011). The final enzyme in the BER pathway isDNA Ligase, which catalyses the formation of aphosphodiester bond to seal a nick site (Taylor et al.,2011). When extra nucleotides have been addedduring an erroneous long-patch BER process, theaction of DNA ligase may expand the repeat tract byincorporating these additional bases into duplex DNA.In this study, DNA constructs containing (CAG)n hair-pins at various distances from a nick site are used toinvestigate the ability of DNA Ligase to ligate sub-strates containing non-canonical secondary structureback into duplex DNA.

This research has been supported by National Institute ofEnvironmental Health Sciences (R01ES019296).

ReferencesGacy, A. M., Goellner, G., Juranic, N., Macura, S., & McMurray,

C. T. (1995). Trinucleotide repeats that expand in humandisease form hairpin structures in vitro. Cell, 81, 533–540.

Jarem, D., Wilson, N. R., Schermerhorn, K. M., & Delaney, S.(2011). Incidence and persistence of 8-oxo-7,8-dihydrogua-nine within a hairpin intermediate exacerbates a toxicoxidation cycle associated with trinucleotide repeatexpansion. DNA Repair, 10, 887–896.


Kovtun, I. V., & McMurray, C. M. (2008). Features oftrinucleotide repeat instability in vivo. Cell Research, 18,198–213.

Taylor, M. R., Conrad, J. A., Wahl, D., & O’Brien, P. J.(2011). Kinetic mechanism of human DNA ligase I revealsmagnesium-dependent changes in the rate-limiting step thatcompromise ligation efficiency. Journal of BiologicalChemistry, 286, 23054–23062.

106 Deriving force field dihedral angleparameters that account forconformation-dependent solvationeffects

Petr Jurečkaa*, Marie Zgarbováa**, F. Javier Luqueb,Jiří Šponera,c* and Michal OtyepkaaDepartment of Physical Chemistry, Palacky, UniversityOlomouc 17, listopadu 12, Olomouc, 771 46, Czech Republic;bFacultat de Farmàcia, Department de Fisicoquímica andInstitut de Biomedicina (IBUB), Universitat de Barcelona,Avgda Diagonal 643, Barcelona 08028, Spain; cInstitute ofBiophysics, Academy of Sciences of the Czech Republic,Kralovopolska 135, Brno 612 65, Czech Republic*Email: [email protected], **Email: [email protected],Phone: (420) 585 634 760, Fax: (420) 585 634 761

Dihedral angle parameters in force fields are oftenobtained with the help of quantum chemical calcula-tions. These are usually performed in vacuum and it isassumed that the solvent model used in the moleculardynamics simulation (explicit or implicit) satisfactorilyaccounts for the major part of the solvation energy.However, there are certain nonspecific solvation effectsthat are not properly accounted for, in this approach.They relate to conformation-dependent solute polariza-tion and solvation of conformation-dependent chargedistribution. On an example of the glycosidic torsion,we show that the contribution resulting from theseeffects is substantial and provides important correctionto the torsion potential. A parameterization procedureis suggested that incorporates the missing conforma-tion-dependent solvation effects into the torsion param-eters, based on the difference between the quantummechanical self-consistent reaction field and Poisson–Boltzmann continuum solvation models. The suggestedapproach avoids double counting of solvation effectsand provides parameters that may be used in combina-tion with any of the widely used nonpolarizable dis-crete solvent models or with the continuum solventmodels. Improvements are demonstrated for the latestAMBER RNA vOL3

parameters. The suggested proce-dure may help to provide consistently better parametersthan the conventional in vacuo parameterizationapproach.

107 Genomes to hit molecules InSilico: a country path today, ahighway tomorrow: a case studyof Chikungunya

Anjali Soni*, Priyanka Dhingra, Avinash Mishra,Tanya Singh, Goutam Mukherjee and B. Jayaram

Department of Chemistry & Kusuma School of BiologicalSciences & Supercomputing Facility for Bioinformatics &Computational Biology, Indian Institute of Technology Delhi,Hauz Khas, New Delhi 110016, India*Email: [email protected], Phone: (011) 2659-1505,Fax: (011) 2658-2037

The growing genomic and proteomic sequence/structuraldatabases trigger high expectations for a rapid and suc-cessful treatment of diseases and disorders. In the currentbiological information, rich and functional knowledge,poor scenario, the feasibility of creating an automated gen-omes to hits (G2H) assembly line in order to cut down thecost and time in drug discovery is discussed. The G2Hcomputational pathway involves several challengingresearch areas viz. functional annotation of genomes, iden-tification of druggable targets, prediction of three-dimen-sional structures of protein targets from their amino acidsequences, and hit molecule generation for these targetsfollowed by a transition from bench to bedside. Wedescribe the ‘G2H In Silico’ strategy (called Dhanvantari),and illustrate it on Chikungunya virus (CHIKV). G2H is anovel pathway incorporating, a series of steps such as geneprediction (Chemgenome), protein tertiary structure deter-mination (Bhageerath), automated active site identifica-tion, rapid hit molecule generation followed by atomiclevel docking and scoring of hits to arrive at lead com-pounds (Sanjeevini). The current state of the art for eachof the steps in the pathway will be highlighted and theresults will be presented and discussed.

ReferencesChemgenome: (a) Dutta, S., Singhal, P., Agrawal, P., Tomer, R.,

Khurana, K. E., & Jayaram, B. (2006). A physicochemicalmodel for analyzing DNA sequences. Journal of ChemicalInformation and Modeling, 46, 78–85; (b) Singhal, P., Jaya-ram B., Dixit S. B., & Beveridge, D. L. (2008). Prokaryoticgene finding based on physicochemical characteristics ofcodons calculated from molecular dynamics simulations.Biophysical Journal, 94, 4173–4183; (c) Khandelwal, G., &Jayaram, B. (2012). DNA-water interactions distinguish mes-senger RNA genes from transfer RNA genes. Journal of theAmerican Chemical Society, 134, 8814–8816; (d) Khandel-wal, G., Gupta, J., & Jayaram, B. (2012). DNA energeticsbased analyses suggest additional genes in prokaryotes. Jour-nal of Biological Sciences, 37, 433–444.

Bhageerath: (a) Jayaram, B., Bhushan, K., Shenoy, S. R.,Narang, P., Bose, S., Agrawal, P., Sahu, D., Pandey, V.(2006). Bhageerath: An energy based web enabled computer


software suite for limiting the search space of tertiary struc-tures of small globular proteins. Nucleic Acid Research, 34,6195–61204; (b) Shenoy, S. R., & Jayaram, B. (2010). Pro-teins: Sequence to structure and function-current status. Cur-rent Protein and Peptide Science, 11, 498–514; (c) Mittal, A.,Jayaram, B., Shenoy, S., & Bawa, T. S. (2010). A stoichiom-etry driven universal spatial organization of backbones offolded proteins: Are there Chargaff's rules for protein fold-ing? Journal of Biomolecular Structure and Dynamics, 28,133–142; (d) Jayaram, B., Dhingra, P., Lakhani, B., & She-khar, S. (2012). Bhageerath-Targeting the near impossible:Pushing the frontiers of atomic models for protein tertiarystructure prediction. Journal of Chemical Sciences, 124, 83–91.

Sanjeevini: (a) Jain, T., & Jayaram, B. (2005). An all atomenergy based computational protocol for predicting bindingaffinities of protein–ligand complexes. FEBS Letter, 579,6659–6666; (b) Shaikh, S. A., & Jayaram, B. (2007). Aswift all-atom energy-based computational protocol topredict DNA-ligand binding affinity and ΔTm. Journal ofMedicinal Chemistry, 50, 2240–2244; (c) Jayaram, B.,Singh, T., Mukherjee, G., Mathur A., Shekhar, S., & She-khar, V. (2012). Sanjeevini: A freely accessible web-serverfor target directed lead molecule discovery. BMC Bioinfor-matics, 13, S7; doi:10.1186/1471-2105-13-S17-S7; (d)Soni, A., Menaria, K., Ray, P., & Jayaram, B. (2013). Gen-omes to Hits in Silico – A country path today, a highwaytomorrow: A case study of Chikungunya. Current Pharma-ceutical Design, in press.

108 Individual-particle electrontomography: a method forstudying macromolecule dynamics

Lei Zhang and Gang Ren*

Molecular Foundry, Lawrence Berkeley National Laboratory,Berkeley, CA 94720, USA*Email: [email protected], Phone: (510) 495-2375, Fax: (510) 486-7268

In solution, macromolecules are naturally flexible anddynamic. Dynamic personalities and structural heterogene-ities of macromolecules are essential to understandingtheir proper function (Karplus & Kuriyan, 2005).However, structural determination of dynamic/heteroge-neous macromolecules is limited by current technologysuch as: X-ray crystallography, nuclear magnetic reso-nance spectrum, small angle scattering, and electronmicroscopy single-particle reconstruction. A commonweakness of all current techniques is requiring anaveraged signal from thousands to millions of differentmacromolecules. Using averaged “signal” must involve inan assumption that macromolecules remain in identicalstructures or few identical conformations. This assumptionis a good estimate for some macromolecule that have arigid body, but not for most macromolecules that have“soft”, flexible, and dynamic body, such as lipoproteinsand antibodies. An ideal approach for structure determina-tion regardless of macromolecular dynamics is to use non-

averaged signal, i.e. the signal from a single macromole-cule itself. We developed a ‘‘focused electron tomographyreconstruction’’ (FETR) algorithm to improve the resolu-tion by decreasing the reconstructing image size so that itcontains only a single-instance macromolecule (Zhang &Ren, 2012). FETR can tolerate certain levels of image dis-tortion and measuring tilt-errors, and can also preciselydetermine the translational parameters via an iterativerefinement process that contains a series of automaticallygenerated dynamic filters and masks. Since this approachcan obtain the structure of a single-instance macromole-cule, we named it individual-particle electron tomography(IPET) as a new robust strategy/approach that does notrequire a pre-given initial model, class averaging of multi-ple molecules or an extended ordered lattice, but can toler-ate small tilt-errors for high-resolution single ‘‘snapshot’’of molecule structure determination (Zhang & Ren, 2012).FETR/IPET provides a completely new opportunity for asingle-macromolecule structure determination, and couldbe used to study the dynamic character, equilibriumfluctuation, to reveal macromolecular mechanism, andeven to track the intermediate state of the reaction of mac-romolecules (Zhang et al., 2010; Zhang & Ren, 2010).

This research has been supported by the Office of Science,Office of Basic Energy Sciences of the United States Depart-ment of Energy (Contract No. DE-AC02-05CH11231), andthe National Heart, Lung, and Blood Institute of theNational Institutes of Health (No. R01HL115153).

ReferencesKarplus, M., & Kuriyan, J. (2005). Molecular dynamics and pro-

tein function. Proceedings of the National Academic of Sci-ences of the United States America, 102(19), 6679–6685.

Zhang, L., Cavigiolio, G., et al. (2010). Structure of 9.6nm dis-coidal high-density lipoprotein revealed by individual-parti-cle electron tomography. Biophysical Journal, 98, 440a.

Zhang, L., & Ren, G. (2010). Determining the dynamic proteinstructure by individual-particle electron tomography: Anindividual antibody structure at a nanometer resolution.Biophysical Journal, 98, 441a.

Zhang, L., & Ren, G. (2012). IPET and FETR: experimentalapproach for studying molecular structure dynamics bycryo-electron tomography of a single-molecule structure.PLoS ONE, 7(1), e30249.

109 Molecular dynamics done quick:efficient trajectory analysissoftware

Alexander V. Popov*, Yuri N. Vorobjev andDmitry O. Zharkov

SB RAS Institute of Chemical Biology and FundamentalMedicine, 8 Lavrentieva Ave, Novosibirsk, 630090, Russia*Email: [email protected], Phone: (383) 363-5174,Fax: (383) 363-5153


Molecular dynamics (MD) simulation nowadays is anessential part of biological, chemical, and physicalresearch. There is a vast variety of accurate and high-performance MD software facilitating the task. How-ever, simulations of biopolymers on meaningful timescales always produce large trajectories rarely amenableto manual analysis. Such analysis, and especially mean-ingful data search and extraction, often becomes a bot-tleneck of in silico experiment along with actual MDcomputations. Most of the existing software for analysisof MD simulation results is based on command-line,script-guided processes that require the researchers tohave an idea about programming language constructionsused, often applied to the one and only product, provid-ing an excessive set of analytic features, but sacrificingease of use, simplicity, and clarity. In this work, wepresent an open source, cross-platform, GUI-based pro-gram, Molecular Dynamics Trajectory Reader and Ana-lyzer (MDTRA), which may be helpful in addressingsuch issues. MDTRA is a versatile program and doesnot require scripting (yet supports it), is able to quicklyplot the analysis results, and minimizes RAM require-ments (Popov et al., 2012). A key MDTRA feature isthe logical organization of data handling and treatment.Our program introduces a convenient way to managedata based on a principle of a re-useable “conveyor”,which delivers results from “streams” (trajectories)through “data sources” to “result collectors.” Each stageis adjustable at any time, causing only the affected data

sources to be rebuilt. MDTRA allows users to plot andanalyze distances, angles, and forces in the molecule. Italso implements trajectory-related search and extractiontools, including determination of meaningful torsions ofprotein backbone, search for stable hydrogen bonds,building 2D-RMSD diagrams, and massive comparativeplotting of DNA parameters. MDTRA proved itself use-ful in a study of DNA repair enzymes OGG1, Fpg, andMutY. An example of analysis of the mobility of tryp-tophan residues contributing to fluorescence of E. coliFpg (observed experimentally by Kuznetsov et al.,2007) is shown in the figure.

This research has been supported by the Presidium ofthe Russian Academy of Sciences (6.14), SB RASIntegrative Grants (No. 26, No. 119), and by RussianFoundation for Basic Research (11-04-00807-a, 12-04-00135-a). MDTRA is available at http://bison.niboch.nsc.ru/en/mdtra.html.

ReferencesKuznetsov, N. A., Koval, V. V., Zharkov, D. O., et al. (2007).

Pre-steady-state kinetic study of substrate specificity ofEscherichia coli formamidopyrimidine DNA glycosylase.Biochemistry, 46, 424–435.

Popov, A. V., Vorobjev, Y. N., Zharkov, D.O., et al. (2012).MDTRA: A molecular dynamics trajectory analyzer with agraphical user interface. Journal of Computational Chemis-try, 46, 424–435 [Epub ahead of print].


110 Refinement of force field torsionparameters for nucleic acids basedon inclusion of conformation-dependent solvation effects

Marie Zgarbováa*, Michal Otyepkaa, Pavel Banáša,F. Javier Luqueb, Thomas E. Cheatham IIIc, Jiří Šponerd

and Petr Jurečkaa

aFaculty of Science, Department of Physical Chemistry,Regional Centre of Advanced Technologies and Materials,Palacky University, 17 listopadu 12, Olomouc 77146, CzechRepublic; bFacultat de Farmàcia, Department de Fisicoquímicaand Institut de Biomedicina (IBUB), Universitat de Barcelona,Avgda Diagonal 643, Barcelona 08028, Spain; cDepartment ofMedicinal Chemistry, College of Pharmacy, University of Utah,Salt Lake City, UT, USA; dInstitute of Biophysics, Academy ofSciences of the Czech Republic, Královopolská 135, Brno 61265, Czech Republic*Email: [email protected], Phone: (+420) 585 634 766,Fax: (+420) 585 634 761

Accurate representation of nucleic acids in moleculardynamics simulations depends critically on the quality ofthe applied empirical force field. Among force fieldterms, the torsion parameters are known to strongly influ-ence the conformational equilibria and molecular struc-tures. Unfortunately, past several years witnessed severeproblems in describing the torsion space in nucleic acidsby current force fields and more problems continueemerging. In an attempt for improvement, we suggested anovel parameterization procedure that incorporates somepreviously neglected solvation-related effects, whichproved to be essential for obtaining accurate torsion pro-files. The suggested approach avoids double counting ofsolvation effects and provides parameters that may beused in combination with any of the widely used nonpo-larizable discrete solvent models or with the continuumsolvent models. Improvements are demonstrated for thelatest AMBER force field for RNA simulations, ff10,which incorporates parameters for the glycosidic torsion(χOL3) developed by us using the above-described proce-dure (Banáš et al., 2010; Zgarbová et al., 2011). Result-ing parameters are verified by extensive moleculardynamics simulations of canonical RNA duplexes andRNA hairpin loops. We show that our modificationremoves overstabilization of the high-anti region found inthe ff99 force field and thus prevents formation of unde-sirable ‘ladder-like’ structural distortions in RNA simula-tions. In addition, we applied our parameterizationapproach to development of the glycosidic torsion inDNA (χOL4). This refinement focuses on adjustingdescription of the syn region and syn-anti balance of theχ potential. This modification exhibits a notable improve-ment of the description of the antiparallel G-DNA stem,which was not modeled correctly by the current ff99force field (Krepl et al., 2012).

The authors gratefully acknowledge the support by theOperational Program Research and Development forInnovations – European Regional Development Fund(project CZ.1.05/2.1.00/03.0058), the Operational Pro-gram Education for Competitiveness – European SocialFund (project CZ.1.07/2.3.00/20.0017), and Integrationof Regional Centre of Advanced Technologies andMaterials into International Networks of Nanotechno-logical and Optical Research (project CZ.1.07/2.3.00/20.0058).

ReferencesBanáš, P., et al. (2010). Performance of molecular mechanics

force fields for RNA simulations: Stability of UUCG andGNRA hairpins. Journal of Chemical Theory and Compu-tation, 6, 3836–3849.

Krepl, M., et al. (2012). Reference simulations of noncanoni-cal nucleic acids with different chi variants of theAMBER force field: Quadruplex DNA, quadruplex RNA,and Z-DNA. Journal of Chemical Theory and Computa-tion, 8, 2506–2520.

Zgarbová, M., et al. (2011). Refinement of the Cornellet al. nucleic acids force field based on reference quan-tum chemical calculations of glycosidic torsion profiles.Journal of Chemical Theory and Computation, 7,2886–2902.

111 Towards the manifoldrepresentations of biological object

Yu. N. Zhuravleva*, M.A. Guzevb and E.E. Skurichinb

aInstitute of Biology and Soil Science, Far Eastern Branch ofthe Russian Academy of Sciences, 159 Stoletiya Ave 690022,Vladivostok-22, Russia; bInstitute for Applied Mathematics,Far Eastern Branch of the Russian Academy of Sciences,159 Stoletiya Ave 690022, Vladivostok-22, Russia*Email: [email protected], Phone: +7(4232)-310-718,Fax: +7(4232)310-190

Most entities of biological world somebody considereda biological object are conditionally discrete, locallyisolated, and partly closed systems with capacity ofdevelopment. Concentrating on the last characteristics,we describe the individual development of object bymeans of expression O = (G,F,Ph), which is a kine-matic description of locally isolated object developingin accord with its internal laws. Mesoscopic organismsare well-known examples of such systems wherefgg 2 G, fphg 2 Ph, and ff g 2 F stand for signs andsets of genotype, phenotype, and for map, respectively,thus indicating that g entails ph causally. However,such superposition of genotype and phenotype can bebroken during the individual development in differentways, e.g. because some phenotype signs can bereceived from mother in their complete form before


fertilization (before appearing the new individual withnew genotype). The individual development per sestarts with a division of zygote. That division andsome follow-up ones giving globe embryos in the ves-sel plants or multicellular blastocyst in mammals canbe described as f0(g0)→ 2g0. This description is validfor meristem (in plants) and stem cells due to theirstatus of low level of differentiation. Diverging fromzygote as a central position in the imaginary diagramrepresentation, the trajectories of divisions describe thehistory of development whereas the real embryoincludes only the cells of current state of division anddifferentiation. The cell differentiations g0→ gd anddedifferentiations gd→ g0 assemble the main body ofevents of development in their relation to genotype.Besides, in the time intervals between divisions, themaps of f(g)→ ph type occur. Resulting from thesemaps the stable sets of phenotype signs correspond toontogeny stages. In the general case, the iterations of{g0↔ gd, gd→ phd} block can form a time series inspace of states. As a transition from one element oftime series to another one is directed by the partialorder set on the antecedent, it turned out that thebackbone of series is represented by succession ofgenotype states whereas there are no direct connectionsbetween phenotype states. During development, biolog-ical object becomes more and more open system.Thus, butterfly consumes nectar, transfers the blossomdust or becomes a pray of predator. In such represen-tations, the biological object can be defined as anoperator transforming one external object in the otherexternal one: O � U: uðO1Þ ! O2, O – O1;O2,u 2 U. It means that due to the neogenic phenotypicpart, an object receives an interaction potential whichwas absent in the former locally isolated entity consid-ered as a partly closed system. This potential can berealized in many ways. So, the dynamics of biologicalobject consists in the transitions of stable genotypestate in the other stable states, whereas the latter tran-sit into (final) states of phenotype. That demands todescribe the individual development as a process inthe complex space that changes with every small stepof ontogeny depending on the current states of itsgenotypic and phenotypic constituents and on theirinteraction. Such dynamics of object and its relatedspace provides the biological object with multifold rep-resentations on the wide spectrum of thermodynamicdescriptions.

The research was executed as a part of program of basicresearch of the Presidium of the Russian Academy ofSciences “Biosphere Origin and Evolution” (subprogramII). No 28.

ReferencesZhuravlev, Yu. N. (2012). Definition by means of indefiniteness

(Comment). Journal of Biomolecular Structure & Dynam-ics, 29(4), 643–644.

Zhuravlev Yu. N., & Avetisov V. A. (2011). Structure–functionanalysis of transformation events//genetic transformation(pp. 29–52). ISBN 978-953-307-364-4.

112 The importance of being modified:tautomeric forms of pyrimidinesprovide expanded use of thegenetic code

William A. Cantaraa,b,c,d,e,f, Franck A.P. Vendeixa,b,c,d,e,f,Franck V. Murphy IVa,b,c,d,e,f, Graźyna Leszcyńskaa,b,c,d,e,f,Kimberly Harrisa,b,c,d,e,f, Estella M. Gustiloa,b,c,d,e,f,Brian Sproata,b,c,d,e,f, Rob Kaisera,b,c,d,e,f,Andrzej Malkiewicza,b,c,d,e,f and Paul F. Agrisa,b,c,d,e,f *aDepartments of Biological Sciences and Chemistry, The RNAInstitute, University at Albany; bNortheastern CollaborativeAccess Team, Building 436, Argonne National Laboratory;cDepartment of Molecular Biology, Cell Biology andBiochemistry, Brown University; dTechnical University, Łodz,Poland; fIntegrated DNA Technologies, Belgium; Dharmacon,Thermo Fisher, Colorado*Email: [email protected], Phone: 518-437-4448,Fax: 518-437-4456

Tautomeric shifts of five-modified uridines have beenestablished in, Escherichia coli tRNAVal

cmo5UAC, as wellas in the human cytoplasmic tRNALys3

mcm5s2UUU. Theseshifts enable the recognition of multiple codons with thewobble base pair in Watson–Crick geometry, suggestingan energetic preference for perhaps an enhanced effi-ciency with this conformation. Thus, modifications at thefifth-position of wobble position uridines appear to be acommon mechanism for expanding tRNA’s codonrecognition. One deviation from the universal codewhich is particularly interesting is the reading of AUGand the universal isoleucine codon AUA as methioninein both the A- and P-sites of the mitochondrial ribosomeof most metazoans. We have discovered a mechanism bywhich five-formylcytidine (f5C) at the wobble position ofhuman mitochondrial tRNAMet (hmtRNAMet

f5CAU)expands the decoding ability of tRNA, enabling it toread AUA as methionine. We have also found thatanother modified cytidine can restrict wobble-base for-mation. This underscores the ability of cytidines, as wellas uridine, modifications within the anticodon loop tomodulate the decoding ability of the tRNA, providing aninsight into decoding mechanisms and evolution of thegenetic code.


This research is supported by: NIH 2RO1 GM23037-25;NSF MCB 0548602; NSF MCB 1101859.

ReferencesVendeix, F. A. P., Murphy, F. V.IV, Cantara, W. A., Les-

zczynska, G., Gustilo, E. M., Sproat, B., … Agris, P. F.(2012). Human tRNALys3

UUU is pre-structured by naturalmodifications for cognate and wobble codon bindingthrough keto-enol tautomerism. Journal of MolecularBiology, 416, 467–485.

Weixlbaumer, A., Murphy, F.IV, Vendeix, F. A. P., Dzier-gowska, A., Malkiewicz, A., Agris, P. F., & Rama-krishnan, V. (2007). Mechanism for expanding thedecoding capacity of transfer RNAs by modification ofuridines. Nature Structural & Molecular Biology, 14,498–502.

113 Conformational dynamics ofhuman 8-oxoguanine-DNAglycosylase

Nikita A. Kuznetsov and Olga S. Fedorova*

Institute of Chemical Biology and Fundamental Medicine,Novosibirsk, 630090, Russia*Email: [email protected], Phone: +7-383-363-5175,Fax: +7-383-363-5153

DNA continuously undergoes oxidation damage fromboth exogenous and endogenous sources, includingionizing radiation, ultraviolet light, and products ofmetabolism. Replication of damaged DNA sometimesgives rise to mutations which can contribute to diseaseand aging. One of the most mutagenic lesions causedby DNA oxidation is 7,8-dihydro-8-oxoguanine(oxoG), which, if not repaired, results in G→T trans-versions. In human cells, oxoG is repaired throughexcision by 8-oxoguanine-DNA glycosylase hOGG1.In addition to its glycosylase activity, hOGG1 pos-sesses an AP-lyase activity, which catalyzes the elimi-

nation of the 3’-phosphate (β-elimination) at thenascent, or preformed abasic (AP) site. The glycosidicbond breakage is initiated by a nucleophilic attack atC1’ by the Lys-249 residue resulting in a covalentenzyme–DNA-Schiff base intermediate, which thenrearranges, and undergoes elimination. The 3-D struc-ture of hOGG1shows that DNA binding is accompa-nied with drastic conformational changes, includingDNA kinking, eversion of oxoGua from the doublehelix, and insertion of few amino acid residues intoDNA. Previously (Kuznetsov et al., 2005, 2007), wehave studied the stopped-flow kinetics of oxoG andAP site lesions processing by hOGG1. The characterof tryptophan and 2-aminopurine fluorescence tracesrevealed that both the protein and the damaged DNAundergo extensive conformational changes in thecourse of DNA substrate binding- and -cleavage. Tounderstand better, the mechanism by which hOGG1recognizes DNA lesions, we have examined the influ-ence of amino acid substitutions on conformationaldynamics of hOGG1 and DNA during specific siterecognition and conversion. Fluorescence kinetics ofenzyme mutant forms F45W, F319W, Y203W,Y203A, H270W, K249Q demonstrated the multistepcharacter of catalytic process and made clear the roleof these amino acids for hOGG1 catalysis.

This research has been supported by The Ministry ofEducation and Science of Russian Federation, (projects8092, 8473 and 14.B37.21.0195) and RFBR (projects12-04-00013 and 12-04-31066).

ReferencesKuznetsov, N. A., Koval, V. V., Zharkov, D. O., Nevinsky,

G. A., Douglas, K. T., & Fedorova, O. S. (2005). Kineticsof substrate recognition and cleavage by human 8-oxogua-nine-DNA glycosylase. Nucleic Acids Research, 33, 3919–3931.

Kuznetsov, N. A., Koval, V. V., Nevinsky, G. A., Douglas,K. T., Zharkov, D. O., & Fedorova, O. S. (2007).Kinetic conformational analysis of human 8-oxoguanine-DNA glycosylase. Journal of Biological Chemistry, 282,1029–1038.

114 Kinetic and thermodynamic basisfor damaged bases excision by8-oxoguanine-DNA glycosylases

Anton Endutkin* and Dmitry Zharkov

SB RAS Institute of Chemical Biology and FundamentalMedicine, Novosibirsk, RU 630090*Email: [email protected], Phone: (383) 363-5128,Fax: (383) 363-5153


DNA glycosylases play the opening act in a highlyconserved process for excision of damaged bases fromDNA called the base excision repair pathway. DNAglycosylases attend to a wide variety of lesions arisingfrom both endogenous and exogenous factors. Thetypes of damage include alkylation, oxidation, andhydrolysis. A major DNA oxidation product is 8-oxo-guanine (8-oxoG), a base with a high mutagenicpotential. In bacteria, this lesion is repaired by for-mamidopyrimidine-DNA glycosylase (Fpg), while inthe case of humans this function belongs to 8-oxoG-DNA glycosylase (OGG1). We have attempted a com-prehensive characterization of 8-oxoG recognition byDNA glycosylases. First, we have obtained thermody-namic parameters for melting of DNA duplexes con-taining 8-oxoG in all possible nucleotide contexts. Theenergy of stacking interactions of 8-oxoG was in strictdependence on 8-oxoG nucleotide environment, whichmay affect the recognition of damage and the effi-ciency of eversion of 8-oxoG from DNA helix by gly-cosylases. Next, we established how the flexibility ofDNA context affects damage recognition by theseenzymes (Kirpota et al., 2011). Then, we have foundthat DNA containing 8-oxoG next to a single-strandbreak provides a good substrate for Fpg, as soon asall structural phosphate residues are maintained. Usingsite-directed mutagenesis, we have addressed the func-tions of many previously unstudied amino acid residu-ess that were predicted to be important for Fpgactivity by molecular dynamics simulation and phylo-genetic analysis. Of note, many substitutions abolishedthe excision of 8-oxoG, but did not affect the cleavageefficiency of abasic substrates. Finally, we investigatedthe contribution of separated structural domains of Fpgto specific enzyme-substrate interaction. Surprisingly,despite the absence of the catalytic domain, C-terminaldomain of Fpg possessed a low- residual ability torecognize and cleave abasic substrates. Our studysheds light on mechanism details of Fpg and OGG1activity, with the ultimate goal of understanding howbinding energy can be spent by these enzymes forcatalysis.

This work was supported by RAS Presidium (Molecularand Cellular Biology Program), RFBR (11-04-00807,12-04-33231), Russian Ministry of Science and Educa-tion.

ReferenceKirpota, O. O., Endutkin, A. V., Ponomarenko, M. P.,

Ponomarenko, P. M., Zharkov, D. O., & Nevinsky, G.A. (2011). Thermodynamic and kinetic basis for recogni-tion and repair of 8-oxoguanine in DNA by human 8-oxoguanine-DNA glycosylase. Nucleic Acids Res, 39,4836–4850.

115 The rate of hOGG1-mediated8-oxoG removal from nucleosomalDNA

Eric D. Olmon* and Sarah Delaney

Department of Chemistry, Brown University, Providence02912, England*Email: [email protected], Phone: (401) 863-2044,Fax: (401) 863-1993

When left unrepaired, DNA damage can lead to muta-genesis and carcinogenesis. Organisms have evolved torepair DNA damage through several pathways, includ-ing the removal and replacement of damaged DNAbases via base excision repair (BER). This pathwayinvolves an enzymatic cascade: following removal ofthe damaged base by a glycosylase, the damage site istreated by an apurinic/apyrimidinic lyase, and thenpatched by a polymerase. Much is known about theBER pathway and the enzymes involved in it (Brookset al., 2013); however, studies of BER in the contextof eukaryotic DNA have only recently begun (Odell,Wallace, & Pederson, 2013). In eukaryotic cells, shortstretches (146 base pairs) of genomic DNA arewrapped around octameric clusters of histone proteins,forming macromolecular structures called nucleosomes(Eickbush & Moudrianakis, 1978). The nucleosomepresents a particular challenge to BER enzymes: therate and efficiency of repair at a particular lesion siteare expected to depend on the relative rotational andtranslational position of the lesion site on the histonecore. Here, we examine the rate and efficiency of theremoval of 8-oxo-7,8-dihydroguanine (8-oxoG), thepredominant oxidative lesion in DNA (Michaels &Miller, 1992), by the glycosylase hOGG1 as a functionof position within the nucleosome. These results arecompared with the rate and efficiency of the hOGG1reaction on free DNA substrates, which have previ-ously been observed in our laboratory (Jarem, Wilson,& Delaney 2009; Jarem et al., 2011). These experi-ments will provide a basis for further explorations intothe factors affecting the efficiency of BER in eukary-otic DNA.

This research has been supported by National Institute ofEnvironmental Health Sciences award R01ES019296.

ReferencesBrooks, S., Adhikary, S., Rubinson, E., & Eichman, B. (2013).

Recent advances in the structural mechanisms of DNAglycosylases. Biochimica et Biophysica Acta, 1834, 247–271.

Eickbush, T. H., & Moudrianakis, E. N. (1978). The histone corecomplex: An octamer assembled by two sets of protein-pro-tein interactions. Biochemistry, 17, 4955–4964.


Jarem, D., Wilson, N., & Delaney, S. (2009). Structure-depen-dent DNA damage and repair in a trinucleotide repeatsequence. Biochemistry, 48, 6655–6663.

Jarem, D., Wilson, N., Schermerhorn, K., & Delaney, S. (2011).Incidence and persistence of 8-oxo-7,8-dihydroguaninewithin a hairpin intermediate exacerbates a toxic oxidationcycle associated with trinucleotide repeat expansion. DNARepair, 10, 887–896.

Michaels, M., & Miller, J. (1992). The GO system protectsorganisms from the mutagenic effect of the spontaneouslesion 8-hydroxyguanine (7,8-dihydro-8-oxoguanine).Journal of Bacteriology, 174, 6321–6325.

Odell, I., Wallace, S., & Pederson, D. (2013). Rules of engage-ment for base excision repair in chromatin. Journal ofCellular Physiology, 228, 258–266.

116 Deamination of both methyl- andnormal-cytosine by the foreignDNA restriction enzymeAPOBEC3A

Michael A. Carpentera*, Ming Lia, Anurag Rathorea,Lela Lackeya, Emily K. Lawa, Allison M. Landa,Brandon Leonarda, Shivender M.D. Shandilyab,Markus-Frederik Bohnb, Celia A. Schifferb,William L Browna and Reuben S. Harrisa

aDepartment of Biochemistry, Molecular Biology & Biophysics,University of Minnesota, Minneapolis, MN 55455, USA;bDepartment of Biochemistry & Molecular Pharmacology,University of Massachusetts Medical School, Worcester,MA 01605, USA*Email: [email protected], Phone: (612) 624-0459,Fax: (612) 624-0426

Multiple studies have indicated that the TET oxidasesand, more controversially, the AID/APOBEC deaminaseshave the capacity to convert genomic DNA 5-methyl-cytosine (MeC) into altered nucleobases that provokeexcision repair and culminate in the replacement of theoriginal MeC with a normal cytosine (C). We show thathuman APOBEC3A (A3A) efficiently deaminates bothMeC to thymine (T) and normal C to uracil (U) in sin-gle-stranded DNA substrates. In comparison, the relatedenzyme APOBEC3G (A3G) has undetectable MeC-to-Tactivity and 10-fold less C-to-U activity. Upon 100-foldinduction of endogenous A3A by interferon, the MeCstatus of bulk chromosomal DNA is unaltered whereasboth MeC and C nucleobases in transfected plasmidDNA substrates are highly susceptible to editing. Knock-down experiments show that endogenous A3A is thesource of both of these cellular DNA deaminase activi-ties. This is the first evidence for non-chromosomalDNA MeC-to-T editing in human cells. These biochemi-cal and cellular data combine to suggest a model inwhich the expanded substrate versatility of A3A may bean evolutionary adaptation that occurred to fortify its

innate immune function in foreign DNA clearance bymyeloid lineage cell types.

This research has been supported by the NIGMS F32GM095219 and by NIDCR T32 DE07288.

ReferenceCarpenter, M. A., et al. (2012). Methyl- and normal-cytosine deam-

ination by the foreign DNA restriction enzyme APOBEC3A.Journal of Biological Chemistry, 287, 34801–34808.

117 Non-B DNA structure-inducedgenetic instability in mammaliancells

Guliang Wang*, Laura A. Christensen, Scott A. Spitserand Karen M. Vasquez*

Division of Pharmacology &Toxiology, The University of Texasat Austin, Austin, TX, 78723 USA*Email: [email protected], [email protected], Phone: (512)495 3040, Fax: (512)496 4946

Repetitive regions of genomic DNA can adopt a num-ber of different structures, e.g. Z-DNA and they are atrisk for increased genetic instability and disease devel-opment [As reviewed in (Zhao, Bacolla, Wang, & Vas-quez, 2010)]. We found Z-DNA structure isintrinsically mutagenic in cultured mammalian cellsand in chromosomes of transgenic mice (Wang Carba-jal, Vijg, DiGiovanni, & Vasquez, 2008). Our recentdata showed CG14 repeats, a model Z-DNA-formingsequence, could stall DNA polymerase progress inmammalian COS-7 cells, while AT14 repeats that doesnot form Z-DNA conformation, did not affect replica-tion. These data suggested a replication-dependentmodel of Z-DNA-induced mutagenesis in mammaliancells. In addition, we also found that Z-DNA causedmutation and DNA breakage in the absence of DNAreplication, and the spectrum of mutation from unrepli-cated system tended to have more large deletions than


replicated DNA, suggested a replicated-independentpathway of Z-DNA-induced genetic instability (Wang,Christensen, & Vasquez, 2004). Several DNA repairproteins were found to be involved in Z-DNA inducedmutagenesis, but in different ways: adding XPA pro-tein back in, otherwise, deficient XP12RO cellsincreased Z-DNA-induced mutation frequency by�50%, while expressing MSH2 protein in Hec59 cellsreduced Z-DNA-induced mutation frequency by �25%.Interestingly, cytosine methylation in CpG repeatsinduced higher frequencies of deletions and rearrange-ments with a broader mutant size distribution than thatfound with unmethylated CpG repeats. We discoveredthat unusual structural features of the methylated CpGsequences precluded local nucleosome assembly lead-ing to genetic instability. Thus, non-B DNA-inducedmutation appears to be a very complex process whereDNA replication, repair and epigenetic modification areall involved.

This work was supported by an NIH/NCI grant to K.M.V. (CA093729).

ReferencesWang, G., Carbajal, S., Vijg, J., DiGiovanni, J., & Vasquez, K. M.

(2008). DNA structure-induced genomic instability in vivo.Journal of the National Cancer Institute, 100, 1815–1817.

Wang, G., Christensen, L. A., & Vasquez, K. M. (2006). Z-DNA-forming sequences generate large-scale deletions inmammalian cells. Proceedings of the National academy ofSciences of the United States of America, 103, 2677–2682.

Zhao, J., Bacolla, A., Wang, G., & Vasquez, K. M. (2010). Non-B DNA structure-induced genetic instability and evolution.Cellular and Molecular Life Sciences, 67, 43–62.

118 DNA structure-induced geneticinstability in mammals

Guliang Wang, Laura A. Christensen andKaren M. Vasquez*

Division of Pharmacology and Toxicology, The University ofTexas at Austin, 1400 Barbara Jordan Blvd., Dell PediatricResearch Institute, Austin, TX 78723*Email: [email protected], Phone: (512) 495-3040, Fax: (512) 495-4945

Naturally occurring repetitive DNA sequences can adoptalternative (i.e. non-B) DNA secondary structures, andoften co-localize with chromosomal breakpoint “hot-spots,” implicating non-B DNA in translocation-relatedcancer etiology. We have found that sequences capableof adopting H-DNA and Z-DNA structures are intrinsi-cally mutagenic in mammals. For example, an endoge-nous H-DNA-forming sequence from the human c-MYCpromoter and a model Z-DNA-forming CpG repeat

induced genetic instability in mammalian cells, largelyin the form of deletions resulting from DNA double-strand breaks (Wang & Vasquez, 2004; Wang et al.,2006). Characterization of the mutants revealed micro-homologies at the breakpoints, consistent with a micro-homology-mediated end-joining repair of the double-strand breaks (Kha et al., 2010). We have constructedtransgenic mutation-reporter mice containing thesehuman H-DNA- and Z-DNA-forming sequences todetermine their effects on genomic instability in a chro-mosomal context in a living organism (Wang et al.,2008). Initial results suggest that both H-DNA- and Z-DNA-forming sequences induced genetic instability inmice, suggesting that these non-B DNA structures repre-sent endogenous sources of genetic instability and maycontribute to disease etiology and evolution. Our currentstudies are designed to determine the mechanisms ofDNA structure-induced genetic instability in mammals;the roles of helicases, polymerases, and repair enzymesin H-DNA and Z-DNA-induced genetic instability willbe discussed.

This work was supported by grants from the NationalInstitutes of Health (CA097175 and CA093729) to K.M.V.

ReferencesKha, D. T., Wang, G., Natrajan, N., Harrison, L., & Vasquez,

K. M. (2010). Pathways for double-strand break repair ingenetically unstable Z-DNA-forming sequences. Journal ofMolecular Biology, 398, 471–480.

Wang, G., Carbajal, S., Vijg, J., DiGiovanni, J., & Vasquez, K.M. (2008). DNA structure-induced genomic instabilityin vivo. Journal of the National Cancer Institute, 100, 1815–1817.

Wang, G., Christensen, L. A., & Vasquez, K. M. (2006).Z-DNA-forming sequences induce large-scale deletions inmammalian cells. Proceedings of the National Academyof Sciences of the USA, 103, 2677–2682.

Wang, G., & Vasquez, K. M. (2004). Naturally occurringH-DNA-forming sequences are mutagenic in mammaliancells. Proceedings of the National Academy of Sciences ofthe USA, 101, 13448–13453.

119 Fork stalling at AT-rich sequencesand failure of origin activation leadto chromosomal instability atfragile sites

Efrat Ozeri-Galai, Michal Irony-Tur Sinai andBatsheva Kerem*

Department of Genetics, The Hebrew University of Jerusalem,Jerusalem, Israel 91904*Email: [email protected], Phone: (972)-2-6585689,Fax: (972) 2-2 6584810


Perturbed DNA replication in early stages of cancerdevelopment induces chromosomal instability preferen-tially at fragile sites. However, the molecular basis forthis instability is unknown. Using DNA combing, westudied the replication dynamics along two commonfragile sites on chromosome 16, FRA16C and FRA16D.We found in FRA16C that under normal growth condi-tions, the replication along the fragile region showsstress-like dynamics. Replication forks along the fragilesite progress significantly slower than in the entiregenome, and frequently stall at AT-rich sequences. Inter-estingly, under these conditions, most of the stalled forksare concentrated near the largest 3.3Kbs AT-richsequence. Furthermore, the distance between originsalong FRA16C are significantly shorter than in the entiregenome. Together, these results indicate that even undernormal growth conditions, replication along the fragilesite is continuously perturbed leading to activation ofadditional origins to enable replication of the region.Under mild replication stress induced by aphidicolin, thereplication rate at the FRA16C region is further sloweddown and the frequency of fork stalling at AT-richsequences is further increased. Strikingly, unlike theentire genome, additional origins are not activatedsuggesting that all potential origins in the FRA16Cregion are already activated under normal conditions.These results demonstrate the inability of FRA16C tocompensate for replication stress leading to failure ofnormal replication completion (Ozeri-Galai et al., 2011).To analyse directly, the role of AT-rich sequences infragile site expression we targeted the 3.3 Kbs long AT-rich sequence derived from FRA16C into a non-fragilegenomic region using a homologous recombination-based system. The results of these experiments will bediscussed. Replication dynamic analysis of another frag-ile site, FRA16D, which is enriched with AT-richsequences, reveals for the first time that a combinationof DNA sequences and abnormal origin activation under-lies the molecular basis of replication stress sensitivity ofa fragile site. Moreover, the results provide a novelmechanism underlying the replication delay and replica-tion stress sensitivity of fragile sites and shed light onthe basis of genomic instability during the early stagesof cancer development.

This research has been supported by The Israel CancerAssociation, Israel Science Foundation, Legacy funds ofHanna Polani and the Israel Ministry of Health.

120 Genome-wide mapping of DNAstrand breaks in Saccharomycescerevisiae

Hari K.K Subramaniana, Sarah E. Bernarda,Cheryl Chianga, Stephen C. J. Parkerb andThomas D. Tulliusa,c*aDepartment of Chemistry, Boston University, Boston,MA 02215, USA; bNHGRI, NIH, Bethesda, MD 20892,USA; cProgram in Bioinformatics, Boston University,Boston,MA 02215, USA*Email: [email protected], Phone: 617-353-2482,Fax: 617-353-6466

DNA damage due to reactive oxygen species (ROS)has severe consequences to cellular metabolism andits accumulation can lead to many diseases. One typeof ROS-induced DNA damage is produced primarilyby hydroxyl radicals which generate both single- anddouble-strand breaks in DNA. Our aim is to map thelandscape of such damage on genome-wide scale athigh resolution. A genome-wide map of ROS-inducedDNA strand breaks is likely to show a greateramount of damage in open chromatin due to higheraccessibility of genomic DNA in these regions. Inorder to map oxidative damage throughout a genomeat single-nucleotide resolution, we have developed anew method (OH-Seq) to biochemically process oxida-tively-damaged genomic DNA to make it suitable forhigh-throughput sequencing. We use gamma radiationto generate hydroxyl radicals in vivo and then processthe damaged genomic DNA to tag the breaks with asequencing adapter. By using Illumina high-throughputsequencing to locate such tagged sites in the genome,it is possible to obtain a high resolution genome-widemap of DNA strand breaks. This map yields adetailed picture of functional regions and transcriptionfactor binding sites in the genome, and has thepotential to provide a new level of detail in the studyof DNA repair and oxidatively-induced aging.

This research has been supported by the Ellison MedicalFoundation.


121 Influence of changes in DNAconformation on thermodynamiccontribution during interaction ofhuman genomic DNA and itsmutant with anticancer drugs

Debjani Ghosh, Subrata Kumar Dey and Chabita Saha*

School of Biotechnology and Biological Sciences, West BengalUniversity of Technology, BF-142, Sector-I, Salt Lake, Kolkata,700 064, West Bengal, India*Email: [email protected], Phone: +9133 2321-0731/2334-1021 ext 113, Fax: +9133 23341030

Isothermal calorimetry (ITC) is efficient in characteriz-ing and recognizing both high affinity and low affin-ity intermolecular interactions quickly and accurately.Adriamycin (ADR) and daunomycin (DNM) are thetwo anticancer drugs whose activity is achievedmainly by intercalation with DNA. During chemother-apy, normal human genomic DNA and mutated DNAfrom K562 leukemic cells show different thermody-namic properties and binding affinities on interactionwith ADR and DNM when followed by ITC. NormalDNA shows more than one step in kinetic analysis,which could be attributed to outside binding, interca-lation and reshuffling as suggested by Chaires et al.(1985); whereas K562 DNA fits a different bindingpattern with higher binding affinities (by one order or

more) compared to normal DNA. Structural propertiesof the interaction were followed by laser Raman spec-troscopy, where difference in structure was apparentfrom the shifts in marker B DNA Raman bands(Ling et al., 2005). A correlation of thermodynamiccontribution and structural data reveals step wisechanges in normal genomic DNA conformation ondrug binding. The overall structural change is higherin normal DNA–DNM interaction suggesting a partialB to A transition on drug binding. Such largechanges were not observed for K562 DNA–DNMinteraction which showed B to A transition propertiesin native from itself corroborating with our earlierfindings (Ghosh et al., 2012).

ReferencesChaires, J. B., Duttagupta, N., & Chrothers, D. M. (1985).

Kinetics of daunomycin–DNA interaction. Biochemistry,24, 260–267.

Ghosh, D., Saha, C., Hossain, M., Dey, S. K., & Kumar,G. S. (in press). Biophysical studies of mutated K562DNA (erythroleukemic cells) binding to adriamycin anddaunomycin reveal that mutations induce structuralchanges influencing binding behaviour. Journal ofBiomolecular Structure & Dynamics. DOI: 10.1080/07391102.2012.698190.

Ling, J. Y., Yang, Q. Z., Luo, S. S., Li, Y., & Zhang, C. K.(2005). Preliminary Study on Cordycepin–DNA Interactionby Raman Spectroscopy. Chinese Chemical Letters, 16, 71–74.


122 Kinetics of apurinic/apyrimidinicendonuclease 1 (APE1) on anauthentic AP site or an AP siteanalog

Kelly M. Schermerhorn* and Sarah Delaney

Department of Chemistry, Brown University, Providence,RI 02912, USA*Email: [email protected],Phone: (401) 863- 3590, Fax: (401) 863-1993

Our genomic DNA is endlessly exposed to a widevariety of exogenous and endogenous DNA-damagingagents. One of the most abundant DNA lesions is anapurinic/apyrimidinic (AP) site, which in vivo, can formspontaneously or through various cellular pathways,including the repair activity of DNA glycosylaseenzymes (Wilson & Barsky, 2001). Persistence of theseAP sites is both highly mutagenic and cytotoxic to thecell (Loeb & Preston, 1986). AP endonuclease 1(APE1), an Mg2+ dependent enzyme, is the majorhuman endonuclease responsible for incising the DNAbackbone at AP sites. Repair to canonical duplex DNAis then completed by DNA polymerase and DNAligase. Recently, APE1, in conjunction with delivery ofDNA-damaging agents, has become a target for chemo-therapeutic research with the aim to inhibit APE1 activ-ity (Fishel & Kelley, 2007). Therefore, anunderstanding of APE1 activity and its molecular mech-anism is essential. In vitro, the authentic AP site ishighly unstable and can undergo β-elimination, leadingto a strand break (Strauss, Beard, Patterson & Wilson,1997). Due to the fragility of the AP site, stable APsite analogs, such as the reduced AP site or tetrahydro-furan (THF) site, are typically used to study APE1(Maher & Bloom, 2007; Strauss, Beard, Patterson &Wilson, 1997). In this work, we have performed thefirst comprehensive kinetic study of APE1 acting onthe authentic AP site as well the reduced AP site andTHF AP site analog. Transient-state kinetic experimentsreveal that the strand incision chemistry step is fast,upwards of �700 s�1 for all substrates, making APE1one of the fastest DNA repair enzymes. Steady-statekinetic experiments reveal for each substrate, a slow,post chemistry step limits the steady-state rate. Thesteady-state rate for APE1 acting on authentic AP andAP-Red substrates is highly dependent on Mg2+ con-centration, while the steady-state rate for THF site wasnot dependent on Mg2+ concentration. This comprehen-sive kinetic analysis reveal differences and similaritiesin the way APE1 processes the authentic AP site com-pared to AP site analogs. Furthermore, these differencesrequire consideration when choosing AP site analogs tostudy APE1.

This research has been supported by NIEHS: ES019296.

ReferencesFishel, M. L., & Kelley, M. R. (2007). The DNA base

excision repair protein Ape1/Ref-1 as a therapeutic andchemopreventive target. Molecular Aspects of Medicine, 28,375–395.

Loeb, L. A., & Preston, B. D. (1986). Mutagenesis by apurinic/apyrimidinic sites. Annual Review of Genetics, 20, 201–230.

Maher, R. L., & Bloom, L. B. (2007). Pre-steady-state kineticcharacterization of the AP endonuclease activity of humanAP endonuclease 1. Journal of Biological Chemistry, 282,30577–30585.

Strauss, P. R., Beard, W. A., Patterson, T. A., & Wilson, S. H.(1997). Substrate binding by human apurinic/apyrimidinicendonuclease indicates a Briggs-Haldane mechanism.Journal of Biological Chemistry, 272, 1302–1307.

Wilson, D. M.III, & Barsky, D. (2001). The majorhuman abasic endonuclease: Formation, consequencesand repair of abasic lesions in DNA. DNA Repair,485, 283–307.

123 Mini-chromosome maintenancecomplexes form a filament toremodel DNA structure andtopology

Ian M. Slaymakera,b, Yang Fua, Daniel B. Tosoc,Nimna Ranatungaa, Aaron Brewsterc,Susan L. Forsburga, Z. Hong Zhouc andXiaojiang S. Chena*

University of Southern California, Los Angeles, CA 90089,USA; University of California, Los Angeles, CA 90095, USA;University of California, Berkeley, CA 94720-3220, USA*Email: [email protected], Phone: (213) 740-2409,Fax: (213) 740-2437

Deregulation of mini-chromosome maintenance (MCM)proteins are associated with genomic instability andcellular abnormality. MCM complexes are recruited toreplication origins for S phase genome duplication.Paradoxically, MCM proteins are expressed in largenumber of origins and are associated with unreplicat-ed chromatin regions away from the origins duringG1 and S phases. We report an unusually wide left-handed filament structure for an archeal MCM, asdetermined by X-ray and electron microscopy. Thecrystal structure reveals that an α-helix bundle formedbetween two neighboring subunits plays a critical rolein filament formation which we show through muta-tion and electron microscopy. The filament interiorhas a remarkably strong electro-positive surface spiral-ing along the inner filament channel which we estab-lish as the binding site for double-stranded DNA. Wefind that MCM filament binding to DNA causes dra-


matic topological changes which negatively supercoilcircular DNA through inducing loosening of the dou-ble helix. This newly identified biochemical activityof MCM may imply a wider functional role forMCM in DNA metabolism beyond helicase function,

for the non-origin bound MCMs. Finally, using yeastgenetics, we show that the inter–subunit interactionsimportant for MCM filament formation play a rolefor cell growth and survival.

124 Evaluation of different DNApolymerases for their effectivenessin using a cytosine analogueduring real-time PCRamplification

Cyntia R. Flores-Juáreza, Eva González-Jassoa

Anaid Antaramianb and Reynaldo C. Plessa*aInstituto Politécnico Nacional, Centro de Investigación enCiencia Aplicada y Tecnología Avanzada, Cerro Blanco 141,Colonia Colinas del Cimatario, Querétaro, QRO 76090,Mexico; bInstituto de Neurobiología, Universidad NacionalAutónoma de México, Juriquilla, QRO 76230, Mexico*Email: [email protected], Phone: +52 (442) 126-8166

GC-rich regions of the DNA are of special interest, asthey generally occur within transcribed or controlregions of the genome. However, the high GC-contentmakes these sequences prone to the formation of hair-pin structures, which may persist even at the relativelyhigh temperature of the extension step in PCR proto-cols, making their amplification difficult in manyinstances. Conversion to a different nucleotidic alpha-bet, resulting in thermodynamically weaker versions of

the G:C base pair, may provide a resolution of thisproblem. The 7-deaza analogue of the guanine base hasbeen tried in this context; however, it appears that thec7G:C base pair, while less stable than the canonical G:C pair, is still too strong, so that problems due to hair-pin formation persist. N4-alkylated cytosines form spe-cific, but much weaker base pairs with guanines, andmay represent a more effective solution to the problem,provided not only that they are stably inserted into thenewly formed DNA during the extension step, but alsocorrectly read in subsequent extensions. We have exam-ined the dCTP analogue, N4methyl-dCTP, for its abilityto sustain a PCR, both with HotStart Taq DNA poly-merase and with Pfu exo� DNA polymerase, amplify-ing a 200-bp amplicon within the pUC18 sequence.The Taq enzyme produced the expected product withthe nucleotide complement dATP/dGTP/dTTP/N4methyl-dCTP, albeit in reduced yield, compared to the yieldobtained with the use of dCTP or of dCTP/N4methyl-dCTP mixtures. A slowdown thermal protocol (Freyet al., 2008, Nature Protocols, 3, 1312–1318), usingsuccessively lower hybridization temperatures and slowtemperature ramps, proved beneficial in giving a highyield of the desired amplicon, with the expected size


and the correct nucleotide sequence. The all-N4-meth-ylC amplicon showed a Tm reduced by 11 °C, comparedto the amplicon obtained with the canonical set ofnucleotides, while amplifications performed with mix-tures of dCTP and N4methyl-dCTP gave productsof the expected size, showing intermediate meltingtemperatures (see Figure), all of which attests to thelower thermal stability of the N4-methylC:G pair,compared to the G:C base pair. In contrast, the ampli-con synthesized with the dATP/c7dGTP/dTTP/dCTPnucleotide set showed a Tm reduction of only 5°C. ThePfu exo�enzyme was less capable of sustaining PCRwith the N4-methyl nucleotide, providing a small yieldof 200-bp amplicon only in the presence of higher con-centrations of N4-methyl-dCTP.

This research was supported by CONACYT CienciaBásica grant No. 61322.

125 Put a stop to it: termination ofmitochondrial transcription

E. Yakubovskaya, E. Mejia, J. Byrnes, E. Hambardjievaand M. Garcia-Diaz

Department of Pharmacological Sciences, Stony Brook University,Stony Brook, NY 11794, USA*Email: [email protected], Phone: (631) 444-4169,Fax: (631) 444-3218

Deficiencies in mitochondrial protein production areassociated with human disease and aging. Given thecentral role of transcription in gene expression, recentyears have seen a renewed interest in understandingthe molecular mechanisms controlling this process.Mterf proteins have been implicated in modulatingtranscription, replication, and protein synthesis. Wehave solved the structure of a member in this family,the human mitochondrial transcriptional terminatorMTERF1, bound to dsDNA containing the terminationsequence. The structure indicates that upon sequencerecognition, MTERF1 unwinds the DNA molecule,promoting eversion of three nucleotides. Base flippingis critical for stable binding and transcriptionaltermination. Additional structural and biochemicalresults provide insight into the DNA binding mecha-nism and explain how MTERF1 recognizes its targetsequence. Furthermore, the identification of terminationdefects resulting from a number of mtDNA mutationshas led to the suggestion that this could be a commonmechanism influencing pathogenesis in a number ofmitochondrial diseases, highlighting the importance ofunderstanding the processes that regulate transcriptionin human mitochondria. Our results provide insightinto the role of mterf proteins and suggest a linkbetween mitochondrial disease and the regulation ofmitochondrial transcription.

This research has been supported by NIH R00 CS015421.


ReferencesYakubovskaya, E., Mejia, E., Byrnes, J., Hambardjieva, E., &

Garcia-Diaz, M. (2010). Helix unwinding and base flippingenable human MTERF1 to terminate mitochondrial tran-scription. Cell, 141, 982–993.

Jiménez-Menéndez, N., Fernández-Millán, P., Rubio-Cosials,A., Arnan, C., Montoya, J., Jacobs, H. T., Bernadó, P.,Coll, M., Usón, I., & Solà, M. (2010). Human mitochon-drial mTERF wraps around DNA through a left-handedsuperhelical tandem repeat. Nature Structural & MolecularBiology, 17, 891–893.

126 Replication impairment as asource of transcription- andR-loop-associated recombination

Maikel Castellano-Pozo, Irene Felipe-Abrio,María L. García-Rubio, Juan F. Lafuente-Barquero,Jose María Santos-Pereira, Tatiana García-Muse andAndrés Aguilera*

Department of Molecular Biology, CABIMER, Universidad deSevilla, Seville, 41092, Spain*Email: [email protected], Phone: +34 954 468372,Fax: +34 954 461 664

Coordination of DNA replication with DNA-damagesensing, repair, and cell cycle progression ensures withhigh probability genome integrity during cell divisions,thus preventing mutations and DNA rearrangements.Such events are usually associated with pathologicaldisorders, including premature aging, various cancerpredispositions, and inherited diseases (Aguilera &Gómez-González, 2008). One important type of genomeinstability is that associated with transcription. Tran-scription of a DNA sequence increases its frequency ofrecombination, a phenomenon referred to as transcrip-tion-associated recombination (TAR). We will providenew data on the analysis of protein factors involved intranscription and RNA export that provide evidence thatTAR is mediated by replication impairment and that itcan be further enhanced by dysfunction of replicationand repair factors in yeast and human cells via R-loops.Notably, our study provides a first connection betweenRNA processing, R-loops, and chromatin structure thatwill be discussed. In addition, we have analyzed anumber of mutants of the yeast RNA polymerase II thatare dependent on double-strand repair functions fortheir viability. We show that in these mutants, replica-tion fork progression and DNA-damage response areimpaired by the presence of strong transcription-depen-dent obstacles. Our data provide evidence that specificalterations of mRNP biogenesis/export machinery leadto different ways by which chromatin structure and pro-gression of replication can be compromised leading togenome instability.

This research has been supported by the Spanish Ministryof Economy and Innovation, Junta de Andalucía and theEuropean Union (FEDER).

ReferenceAguilera, A., & Gómez-González, B. (2008). Genome instabil-

ity: A mechanistic view of its causes and consequences.Nature Reviews Genetics, 9, 204–217.

127 Template-switching during DNAreplication

Susan T. Lovett

MS029 Rosenstiel Center Brandeis University, Waltham, MA02454-9110, USAEmail: [email protected], Phone: (781) 735-2497,Fax: (781) 736-2405

Short repetitive DNA sequences are frequent sites ofgenomic rearrangements. Using the bacterium Esche-richia coli, we have studied the mechanisms of thesefrequent events and have proposed that they arise by atemplate-switch mechanism during DNA replication. Wehypothesize that the template-switch mechanism is areplication-gap filling repair mechanism, which, liketranslesion DNA synthesis, can overcome replication-blocking lesions on the DNA template strand. Both tran-slesion synthesis (TLS) and template-switching are com-ponents of what has been termed “post-replicationrepair.” Tranlesion synthesis has been considered “error-prone” because of its propensity to induce point muta-tions because of the low fidelity of the TLS polymer-ases. Template-switching, we propose, comprises whathas been considered the “error-free” pathway of post-replication repair. We note that the pathway is not trulyerror-free, as it is “rearrangement-prone.” Using geneticassays for deletions or expansions at short repeats, wehave investigated the properties of the template-switchpathway. Our analysis shows that template-switchingfrequently occurs, concomitant with sister-chromosomeexchange. These crossovers occur independently of theknown homologous recombination machinery includingRecA. Whereas homologous recombination requires athreshold homology of about 250 bp (below whichrecombination is inefficient), the template-switchingcrossovers can occur between microhomologies as lowas 25 bp. Blocking replication with the chain-terminatorazidothymidine (AZT) stimulates template-switching asdo mutations in the DNA polymerase III replisome, con-sistent with the idea that template-switching occurs as aresponse to replication gaps. We have identified three


proteins whose functions are required for template-switching. Mutants in these functions exhibit enhancedsensitivity to DNA replication inhibitors AZT andhydroxyurea and lower rates of genetic rearrangements.The first template-switch factor is chaperone DnaKJ,identified through a genetic screen. Mutations in dnaKalso block the “error-prone” repair pathway, as theyexhibit lower rates of DNA polymerase V-dependentmutagenesis induced by UV irradiation. Mutations inthe processivity clamp for replication (dnaN) and in onesubunit of the clamp loader complex (dnaX) also dimin-ish template-switching. By a programmed translationalframeshift, the dnaX gene produces two variants, a lar-ger form required for replication and a shorter form ofunknown function. Our recent experiments suggest thatthe shorter form is required for template-switching. Wepropose that this short form is used in an alternativeclamp loader complex employed during DNA repair.

This research has been supported by NIH R01 AwardGM51753.

128 Some derivatives of1,3-diazaadamantane stronglystimulate strand exchangereaction between shortoligonucleotides

Anna Gabrieliana*, Tatiana N. Bocharovab,Elena A. Smirnova Alexanderb, A. Volodinb,Amalya Harutjunyana, Knarik Gevorkyana andGayane Harutjunyana

aResearch-technological Center of Organic & PharmacologicalChemistry, Armenian National Academy of Sciences), 26 AzatutianAvenue, 0014 Yerevan, Armenia; bInstitute of Molecular Genetics(Russian Academy of Sciences), 2 Kurchatov, Square, 123182Moscow, Russia*Email: [email protected],Phone: (37410) 524501

Earlier, we characterized the behavior of HG122(compound III) in the DNA Strand Exchange Reaction(SER) and found that 1,3-diazaadamantane derivative

facilitates SER in the system of short oligonucleotides(Gabrielian et al., 2011). In the present study, a series ofnew derivatives of 1,3-diazaadamantane have beensynthesized with the purpose to discern how small varia-tions in the compound structure can influence its activityin SER and try to get more effective substances for stim-ulation of SER. We revealed that most of the small vari-ations in the structure significantly influenced thecompounds’ efficacy in accelerating SER. For example,an increase in the compounds’ aliphatic chain lengthsconsiderably enhanced its efficiency in SER stimulationand in the series of compounds presented in theFigure HG188 (compound IV) was eminently the mostpotent agent in the stimulation of SER. Small modifica-tions in other parts of the 1,3-diazaadamantane moleculealso influenced the SER stimulation and several deriva-tives more efficient in facilitating SER than HG122 wererevealed. Some of the compounds exhibited virtuallynegligible capability to stimulate SER but, interestingly,out of 12 derivatives characterized, agents that retardspontaneous SER were not found. Earlier, the stimulationof the DNA strand exchange was documented for differ-ent ligands of the policationic nature such as CationicComb Copolymers (Kim et al., 2003), linker histones(Bocharova et al., 2012), cobalt hexamine (unpublishedobservation) etc. The present results provide us with anovel class of SER facilitating compounds – the cationicamphiphiles that can serve as interesting objects forfurther understanding of different aspects of DNA SER.Biomedical implications and prospects in biomedicalapplications of these and similar compounds’ activity inSER remain to be investigated.

ReferencesGabrielian, A., Bocharova, T. N., Smirnova, E. A., Volodin, A.

A., & Harutjunyan, G. (2011). Strand exchange reactionbetween short oligonucleotides promoted by a derivative of1,3-diazaadamantane. Journal of Biomolecular Structure &Dynamics, 27, 1124–1125.

Kim, W. J., Cato, Y., Akaike, T., & Maruyama, A. (2003).Cationic comb-type copolymers for DNA analysis. NatureMaterials, 2, 815–820.

Bocharova, T. N., Smirnova, E. A., & Volodin, A. A. (2012).Linker histone H1 stimulates DNA strand exchangebetween short oligonucleotides retaining high sensitivity toheterology. Biopolymers, 97, 229–239.


129 The molecular mechanism ofbreakage at fragile site FRA16D

Simran Kaushal*, Soo-Mi Alison Lee, Nealia House,Keerthana Gnanapradeepan, Adam Snider,Xiaofeng Allen Su and Catherine H. Freudenreich

Department of Biology, Tufts University, 200 Boston Ave Suite,4700, Medford, MA 02155, USA*Email: [email protected], Phone: (617) 627-5682,Fax: (617) 627-0309

Replication stress induces physical breakage at discreteloci in chromosomes, which can be visualized on a meta-phase chromosome spread. These common fragile sites(CFS) are conserved across species and are hotspots forsister chromatid recombination, viral integration, rear-rangements, translocations, and deletions (Glover et al2005). Despite multiple theories, the molecular mecha-nisms of CFS expression and genomic instability are stillnot well understood. The fragile site FRA16D is of specialinterest because it is the second most highly expressedfragile site and is located within the WWOX tumor sup-pressor gene. Previous data identified a polymorphic ATrepeat within a FRA16D subregion called F1 that causeschromosome fragility and replication fork stalling in ayeast model (Zhang and Freudenreich 2007). Recently, wehave found that breakage increases in an AT repeat length-dependent manner. Our results suggest that the AT repeatin the context of F1 forms a secondary structure, makingthe region more vulnerable to breakage.

This research has been supported by the Tufts UniversityBiology Department and the Tufts University Dean’s Fund.

ReferencesGlover, T. W., Arlt, M. F., & Casper, A. (2005). Mechanisms

of common fragile site instability. Human MolecularGenetics, 2, R197–R205.

Zhang, H., & Freudenreich, C. H. (2007). An AT-Rich sequencein human common fragile site fra16d causes fork stallingand chromosome breakage in S. cerevisiae. Molecular andCell. Biology, 27, 367–379.

130 Transcription blockage by single-strand breaks in varioussequences and the general modelfor transcription blockage by R-loop formation

Boris P. Belotserkovskiia*, Alexander J. Neila,Syed Shayon Saleha, Jane Hae Soo Shina,Sergei M. Mirkinb and Philip C. Hanawalta

aDepartment of Biology, Stanford University, Stanford, CA94305, USA; bDepartment of Biology, Tufts University,Medford, MA 02155, USA*Email: [email protected], Phone: (650) 723-2425,Fax: (650)725-1848

Transcription blockage can strongly affect gene expres-sion and trigger other important biological phenomenalike transcription-coupled repair (Hanawalt & Spivak,2008). Thus, it is of interest to study the various factorsthat can cause transcription blockage and to elucidatemechanisms of their action. We studied T7 RNApolymerase (T7 RNAP) transcription blockage caused bysingle-stranded breaks localize either in the template orthe nontemplate DNA strand (Belotserkovskii et al.,2013; Neil, Belotserkovskii, & Hanawalt, 2012). PartialT7 RNAP blockage was observed in both cases, but thepatterns of blockage signals differed dramatically forthese two types of lesions. A break in the templatestrand produces a sharp predominant blockage signalcorresponding to the position of the break, as expectedfor an interruption in the DNA strand that is continu-ously tracked by RNAP during transcription. In contrast,a break in the nontemplate strand produces an irregularladder of weak blockage signals that begins approxi-mately at the position of the break and then extends fardownstream from the break position, without either apredominant signal at the break position, or apronounced downstream “end” of the ladder. The block-ages produced by the break in the nontemplate strandincrease dramatically when they are closely adjacent toG-rich homopurine sequences. These sequences causepartial transcription blockage, as we have previouslyestablished (Belotserkovskii et al., 2010); and in thepresence of the nearby nontemplate strand break, theresulting blockage is greatly enhanced (Belotserkovskiiet al., 2013). Based upon these and other observations,we suggest that transcription blockage by breaks in thenontemplate strand is due to their propensity to induceR-loop formation which destabilizes the transcriptioncomplex and renders it prone to spontaneous prematureblockage/termination.

This research was supported by NIH grants; CA077712from the National Cancer Institute to P.C.H., andGM60987 from the National Institute of General MedicalSciences to S.M.M., and Undergraduate Research Grantsat Stanford to A.J.N., S.S., and J.H.S.S.

ReferencesBelotserkovskii, B. P., Liu, R., Tornaletti, S., Krasilnikova, M.

M., Mirkin, S. M., & Hanawalt, P. C. (2010). Mechanismsand implications of transcription blockage by guanine-richDNA sequences. Proceedings of the National Academy ofSciences USA, 107, 12816–12821.


Belotserkovskii, B. P., Neil, A. J., Saleh, S. S., Shin, J. H.,Mirkin, S. M., & Hanawalt, P. C. (2013). Transcriptionblockage by homopurine DNA sequences: Role ofsequence composition and single-strand breaks. NucleicAcids Research, 41, 1817–1828.

Hanawalt, P. C., & Spivak, G. (2008). Transcription-coupledDNA repair: Two decades of progress and surprises. NatureReviews Molecular Cell Biology, 9, 958–970.

Neil, A. J., Belotserkovskii, B. P., & Hanawalt, P. C. (2012).Transcription blockage by bulky end termini at single-strand breaks in the DNA template: Differential effects of5′ and 3′ adducts. Biochemistry, 51, 8964–8970.

131 Using whole-genome sequencingto search for trans-modifiers ofrepeat expansions

Ryan McGintya*, Anna Aksenovaa, Eric Wangb,David Hausmanb and Sergei Mirkina

aDepartment of Biology, Tufts University, Medford, MA 02155,USA; bCenter for Cancer Research, Massachusetts Institute ofTechnology, Cambridge, MA 02139, USA*E-mail: [email protected], Phone: (617) 966-7728

Expansions of simple repetitive DNA sequences areresponsible for a large number of human hereditarydiseases. The severity and age of onset of each disor-der can be estimated from the length of the inheritedrepeat tract; however, the subsequent expansion rate isitself a heritable trait. This points to a role for trans-acting modifier genes, whose actions may be alteredby single nucleotide polymorphisms. Previously, ourlab has used a yeast system to study large-scaleexpansions of GAA trinucleotide repeats, and a num-ber of gene knockouts have been shown to induceexpansions. While this has been highly informative,gene knockouts are a rather blunt tool, which are notsuitable to approximate the spectrum of SNPs thatmight affect repeat expansions, as has been predictedin human populations. To address this problem, wehave developed a novel, alternative screening methodwhich employs UV mutagenesis followed by thewhole-genome sequencing. This approach allowed usto detect SNPs that modified the likelihood of repeatexpansions in yeast. Unexpectedly, several such SNPsappeared in a yeast gene whose product is involved inthe cleavage and polyadenylation of RNA transcripts.We discuss possible mechanisms linking transcriptprocessing with repeat instability.

ReferencesMorales, F., et al. (2012). Somatic instability of the expanded

CTG triplet repeat in myotonic dystrophy type 1 is a herita-ble quantitative trait and modifier of disease severity.Human Molecular Genetics, 21, 3558–3567.

Shishkin, A., et al. (2009). Large-scale expansions of Friedreich’sataxia GAA repeats in Yeast. Molecular Cell, 35, 82–92.

Zhang, Y., et al. (2012). Genome-wide screen identifies pathwaysthat Govern GAA/TTC repeat fragility and expansions individing and nondividing yeast cells.Molecular Cell, 48, 1–12.

132 Impact of sticky end length on thediffraction of self-assembled DNAcrystals

Yoel P. Ohayona, Arun Richard Chandrasekarana,Esra Demirela, Sabrine I. Obbada, Rutu C. Shaha,Victoria T. Adesobaa, Matthew Lehmanna,Jens J. Birktofta, Ruojie Shaa, Paul M. Chaikinb andNadrian C. Seemana*aDepartment of Chemistry, New York University, New York, NY10003, USA; bDepartment of Physics, New York University,New York, NY 10003, USA*Email: [email protected], Phone: (212) 998-8395,Fax: (212) 995-4475

Our laboratory has reported a self-assembled 3-D crystalbased on a DNA tensegrity triangle. The tensegritytriangle is a rigid DNA motif with three-fold rotationalsymmetry consisting of three helices whose axes aredirected along three linearly independent directions (1).The triangles form a crystalline lattice stabilized viasticky ends (2). The length of the sticky ends reportedpreviously was two nucleotides (nt) GA:TC. Althoughdiffracting to 4 Å resolution at the APS-ID19 beam line,they diffract only to 4.9 Å at the NSLS-X25 beam line.In the current study, we have analysed the effect ofsticky end length and sequence on crystal formation andthe resolution of the X-ray diffraction pattern on NSLS-X25. Tensegrity triangle motifs having 1-, 2-, and 3-ntsticky ends have all formed crystals. X-ray diffractiondata from the same beam line revealed that the crystalresolution was somewhat better for the 2-nt sticky endhaving an AA:TT base pair (4.75 Å) than GA:CT andCC:GG (8.0 Å). Moreover, the 1-nt sticky end (C:G)yielded a diffraction pattern whose resolution (3.5 Å)compared favorably with all the three 2-nt sticky endsystems. However, the triangle motif having a 1-nt stickyend with an A:T base pair did not yield any crystals. Formotifs with 3-nt sticky ends, the sequence GAG:CTCproduced small crystals (10–20 μm), while larger crystals(150 μm) were obtained with the sequences TAG:ATCand TAT:ATA. Our results indicate that not only do thelengths and sequences of the sticky ends define the inter-actions between motifs, but they also have an impact onthe resulting resolution. We expect redesigned assembliesto form 3-D crystals with better resolution that can aidin the scaffolding of biological macromolecules forcrystallographic structure determination. Applications in


many areas of DNA nanotechnology are expected tobenefit from a complete analysis of the effects of stickyend length, sequence, and free energy.

We acknowledge support of the following grants to NCS:grant GM-29,554 from NIGMS, grants CTS-0,608,889 andCCF-0,726,378 from the NSF, grant W911FF-08-C-0057from ARO, grants N000140910181 and N000140911118from ONR and DE-SC0007991 from DOE.

ReferencesLiu, D., Wang, W., Deng, Z., Walulu, R., & Mao, C. (2004).

Tensegrity: Construction of rigid DNA triangles with flexiblefour-arm junctions. Journal of American Chemical Society,126, 2324–2325.

Zheng, J., Birktoft, J. J., Chen, Y., Wang, T., Sha, R., Constanti-nou, P. E., Ginell, S. L., Mao, C., & Seeman, N. C. (2009).From molecular to macroscopic via the rational design of aself-assembled 3D DNA crystal. Nature, 461, 74–77.

133 Designed DNA crystals with atriple-helix veneer

Arun Richard Chandrasekarana, David A. Ruslingb,Yoel P. Ohayona, Ruojie Shaa and Nadrian C. Seemana*aDepartment of Chemistry, New York University, New York, NewYork 10003, USA; bCentre for Biological Sciences, University ofSouthampton, Highfield, Southampton SO17 1BJ, UK*Email: [email protected], Phone: (212) 998-8395,Fax: (212) 995-4475

DNA has been used as a tool for the self-assembly ofnano-sized objects and arrays in two and three-dimen-sions. Triplex-forming oligonucleotides (TFOs) can beexploited to recognize and introduce functionality atprecise duplex regions within these DNA nanostructures(Rusling et al., 2012). Here we have examined the feasi-bility of using TFOs to bind to specific locations within a3-turn DNA tensegrity triangle motif. The tensegrity tri-angle is a rigid DNA motif with three-fold rotationalsymmetry, consisting of three helices directed along threelinearly independent directions (Liu et al., 2004). Thetriangles form a three-dimensional crystalline latticestabilized via sticky-end cohesion (Zheng et al., 2009).The TFO 5′-TTCTTTCTTCTCT was used to target thetensegrity motif containing an appropriately embeddedoligopurine–oligopyrimidine binding site. Formation ofDNA triplex in the motif was characterized by an electro-phoretic mobility shift assay (EMSA), UV melting stud-ies and FRET analysis. Non-denaturing gel analysis ofannealed DNA motifs showed a band with slower mobil-ity only in the presence of TFO and only when the DNAmotif contained the triplex binding site. Experimentswere undertaken at pH 5.0, since the formation of a tri-plex with cytidine-containing TFOs requires slightly

acidic conditions (pH< 6.0). TFOs with modified C-ana-logs and T-analogs having a higher pKa worked at a moreneutral pH, also evidenced by EMSA. UV melting stud-ies revealed that the melting point of the 3-turn trianglewas 64 °C and the TFO binding increased the meltingpoint to 80 °C. FRET analysis was done by labeling thetriangle with fluorescein and the TFO with a cyanine dye(Cy5). The FRET melting curve revealed that a signalwas observed only when the TFO was bound to the DNAmotif and the results were consistent with UV meltingstudies. These results indicate that a TFO can be specifi-cally targeted to the tensegrity triangle motif.

We acknowledge support of the following grants to NCS:grant GM-29554 from NIGMS, grants CTS-0608889 andCCF-0726378 from the NSF, grant W911FF-08-C-0057from ARO, grants N000140910181 and N000140911118from ONR and DE-SC0007991 from DOE.

ReferencesLiu, D., Wang, M., Deng, Z., Walulu, R., & Mao, C. (2004).

Tensegrity: Construction of rigid DNA triangles with flexi-ble four-arm DNA junctions. Journal of the AmericanChemical Society, 126, 2324–2325.

Rusling, D. A., Nandhakumar, I. S., Brown, T., & Fox, K. R.(2012). Triplex-directed recognition of a DNA nanostructureassembled by crossover strand exchange. ACS Nano, 6,3604–3613.

Zheng, J., Birktoft, J. J., Chen, Y., Wang, T., Sha, R., Constan-tinou, P. E., … Seeman, N. C. (2009). From molecular tomacroscopic via the rational design of a self-assembled 3DDNA crystal. Nature, 461, 74–77.

134 Encapsulation of antitumor drugstamoxifen, 4-hydroxytamoxifenand endoxifen by chitosannanoparticles

H.A. Tajmir-Riahi*, D. Agudelo and S. Sanyakamdhorn

Departement de Chemistry-Biology, University of Québec inTrois-Rivières, C. P. 500, Trois-Rivières (Québec), G9A 5H7,Canada*Email: [email protected]

Synthetic polymers are often used as drug deliverysystems in vitro and in vivo (1). Here, we used biodegrad-able chitosan of different sizes to encapsulate antitumordrug tamoxifen (tam) and its metabolites 4-hydroxytamox-ifen (hydroxytam) and endoxifen (end). The interactionsof tamoxifen and its metabolites with chitosan 15, 100,and 200KD were investigated in aqueous solution, usingFTIR, fluorescence spectroscopic methods, and molecularmodeling. The structural analysis showed that tamoxifenand its metabolites bind chitosan via both hydrophilic andhydrophobic contacts with overall binding constants of


Ktam-ch-15 = 8.7 (±0.5)� 103 M�1 , Ktam-ch-100 = 5.9(±0.4)� 105 M�1, and Ktam-ch-200 = 2.4 (±0.4)� 105 M�1;Khydroxytam-ch-15 = 2.6 (±0.3)� 104 M�1, Khydroxytam-ch-100

= 5.2 (±0.7)� 106 M�1, and Khydroxytam-ch-200 = 5.1(±0.5)� 105 M�1; and Kend-ch-15 = 4.1 (±0.4)� 103 M�1 ,Kend-ch-100 = 1.2 (±0.3)� 106 M�1, and Kend-ch-200 = 4.7(±0.5)� 105 M�1 with the number of drug moleculesbound per chitosan (n) 2.8–0.5. The order of bindingis ch-100 > 200 > 15KD with stronger complexesformed with 4-hydroxytamoxifen than with tamoxifenand endoxifen. The molecular modeling showed theparticipation of polymer charged NH2 residues withdrug OH and NH2 groups in the drug–polymeradducts. The free binding energies of -3.46 kcal/molfor tamoxifen, �3.54 kcal/mol for 4-hydroxytamoxifen,and �3.47 kcal/mol for endoxifen were estimated forthese drug–polymer complexes. The results show thatchitosan 100KD is a stronger carrier for drug deliverythan for chitosan-15 and chitosan-200KD, which isconsistent with our recent report on doxorubicin–chito-san complexes (2).

ReferencesShi, M., Ho, K., Keating, A., & Shoichet, M. S. (2009).

Doxorubicin-conjugated immuno-nanoparticles forintracellular anticancer drug delivery. Advanced FunctionalMaterials, 19, 1689–1696.

Sanyakamdhorn, S., Agudelo, D., & Tajmir-Riahi, H. A. (inpress). Encapsulation of antitumor drug doxorubicin and itsanalogue by chitosan nanoparticles. Biomacromolecules.

135 Arranging enzymes and receptorswith nanometer-scale precision onmolecular switchboards

Ngo Anh Tien, Eiji Nakata and Takashi Morii*

Institute of Advanced Energy, Kyoto University, Uji,Kyoto 611-0011, Japan*Email: [email protected], Phone: +81-774-38-3585,Fax: +81-38-3516

DNA is a useful material for constructing nanoscale struc-tures in nearly any three-dimensional (3D) shape desired.The DNA nanostructure can also be equipped with specificdocking sites for proteins. Cellular processes and chemicaltransformations take place in several reaction steps. Multi-ple enzymes cooperate in specific fashion to catalyze thesequential chemical transformation steps. Such naturalsystems are effectively reconstructed in vitro if the individ-ual enzymes locate in the correct relative orientations.DNA-origami structures can be used as “molecular switch-boards” to arrange enzymes and other proteins with nano-meter-scale precision. A new method was developed forlocating the proteins by means of special “adapters”known as zinc-finger proteins based only on proteins. Zincfingers are suitable site-selective adapters for targetingspecific locations within DNA-origami structures. Severaldifferent adapters carrying different proteins can indepen-dently bind at defined locations on this type of nanostruc-ture. A basic leucine zipper (bZIP) protein is also acandidate for the site-selective adaptor. A well-character-ized bZIP protein GCN4 was chosen as an adaptor for spe-cific addresses. Analyses by atomic force microscopy andgel electrophoreses demonstrate specific binding of GCN4adaptor to the addresses containing the GCN4 bindingsites on DNA origami. The adaptor derived from GCN4and that form a zinc-finger protein zif268, for which wehave reported previously, acted as orthogonal adaptors tothe respective addresses on DNA origami. Therefore, theseorthogonal adaptors would be useful to place multipleengineered proteins at different addresses on DNA ori-gami. Especially, the homodimeric nature of GCN4 adap-tor is indispensable for constructing the assembly of thenaturally abundant dimeric proteins and/or enzymes toefficiently carry out chemical reactions and signal trans-ductions in vitro on DNA origami.

ReferenceNakata, E., Liew, F. F., Uwatoko, C., Kiyonaka, S., Mori, Y.,

Katsuda, Y., … Morii, T. (2012). Zinc finger proteins forsite-specific protein positioning on DNA origami.Angewandte Chemie International Edition, 51, 2421–2424.


136 Dendritic Alkyl Chains on DNACages: A Geometry-DependentInter- or Intramolecular“Handshake”

Thomas G.W. Edwardson, Karina M.M. Carneiro,Christopher K. McLaughlin, Christopher J. Serpell andHanadi F. Sleiman*

Department of Chemistry, McGill University, Montreal, QC,Canada*Email: [email protected], Phone: (1)514-398-6921

The selective association of hydrophobic sidechains isa strong determinant of protein organization. Wehave observed a parallel mode of assembly in DNAnanotechnology. Firstly, dendritic DNA amphiphiles(D-DNA) were synthesized (Carneiro, Aldaye, &Sleiman, 2009) comprising an addressable oligonu-cleotide portion and a hydrophobic alkyl dendron atthe 5’ terminus. DNA amphiphiles have gatheredinterest recently as they can self-assemble in aqueousmedia to form well defined micelles while alsoretaining the ability to hybridize to their complement(Kwak & Herrmann, 2011; Patwa, et al. 2011) Twovariations of alkyl D-DNA were hybridized to thesingle-stranded edges of a DNA cube (McLaughlin,et al., 2012). It was found that anisotropic organiza-tion of these hydrophobic domains on the 3D scaf-fold results in a new set of assembly rules,dependent on spatial orientation, number, and chemi-cal identity of the D-DNA on the cubic structure(Edwardson. et al. 2012). When four amphiphiles areorganized on one cube face, the hydrophobic residues

engage in an intermolecular “handshake” betweentwo cubes, resulting in a dimer. When eight amphi-philes are organized on the top and bottom faces ofthe cube, they engage in a “handshake” inside thecube. Combining the highly specific recognition ofthe oligonucleotide sequence with the orthogonalassociation of hydrophobic moieties can lead to avariety of structures with such diverse applications asmembrane anchoring, cell uptake, directed hydropho-bic assembly, and encapsulation and release of smallmolecules.

This research has been supported by NSERC, CFI,CSACS, CIHR, and CIFAR.

ReferencesCarneiro, K. M. M., Aldaye, F. A., & Sleiman, H. F.

(2009). Long-range assembly of DNA into nanofibersand highly ordered networks using a block copolymerapproach. Journal of the American Chemical Society,132, 679–685.

Edwardson, T. G. W. et al. (under review). Dendritic alkylchains on DNA cages: A geometry-dependent inter- orintramolecular “handshake”. Nature Chemistry.

Kwak, M., & Herrmann, A. (2011). Nucleic acid amphiphiles:Synthesis and self-assembled nanostructures. ChemicalSociety Reviews, 40, 5745–5755.

McLaughlin, C. K., et al. (2012). Three-dimensional organi-zation of block copolymers on “DNA- minimal” scaf-folds. Journal of the American Chemical Society, 134,4280–4286.

Patwa, A., et al. (2011). Hybrid lipid oligonucleotideconjugates: Synthesis, self-assemblies and biomedicalapplications. Chemical Society Reviews, 40, 5844–5854.


137 DNA nanostructure serumstability: greater than the sum oftheir parts

Justin W. Conway, Christopher K. McLaughlin,Katherine J. Castor and Hanadi Sleiman*

Department of Chemistry, McGill University, 801 SherbrookeSt. West, Montreal, QC, Canada, H3A 0B8*E-mail:[email protected], Phone: 514-398-2633,Fax: 514-398-3797

DNA cages hold tremendous potential to encapsulateand selectively release therapeutic drugs, and can pro-vide useful tools to probe the size and shape depen-dence of nucleic acid delivery (McLaughlin & Sleiman,H. F., 2011). These structures have been shown to site-specifically present ligands, small molecule drugs, orantisense/siRNA motifs, in order to increase their thera-peutic efficiency (Li & Fan, C. 2012). One of themajor barriers towards their in vivo applications is thesusceptibility of their strands towards nuclease degrada-tion. A number of chemical strategies have been usedto block nuclease digestion of oligonucleotides andimprove potency, such as the use of a phosphorothioatebackbone, 2´-O-methyl, locked nucleic acids, and shorthybrid gapmers. However, the synthesis of these oligo-nucleotides is often complicated and expensive, drivingthe need for simple modifications to enhance serum sta-

bility and address in vivo biodistribution. We show herea simple method to significantly enhance the nucleasestability of DNA strands, through introduction of com-mercially available, single-endmodifications (Conway &Sleiman 2013). We use these oligonucleotides to con-struct DNA cages in a single step and in quantitativeyields. Even in single-stranded form, these cages stabi-lize their component strands towards nucleases, withmean lifetimes as long as 62 h in 10 % (v/v) fetalbovine serum (FBS). We examine the effect of otherDNA-end modifications on nuclease susceptibility.Finally, we show the ligation of these single-strandedcages into topologically interesting catenane ‘necklaces,’with mean lifetimes in serum of �200 h.

ReferencesConway, J. W., McLaughlin, C. K., Castor, K. J., & Sleiman,

H. F. (2013). DNA Nanostructures Serum Stability: Greaterthan the Sum of its Parts. Chemical Communication, 49,1172–1174.

Li, J., Pei, H., Zhu, B., Liang, L., Wei, M., He, Y., Chen, N.,Li, D., Huang, Q., & Fan, C. (2012). Self-Assembled Mul-tivalent DNA Nanostructures for Noninvasive IntracellularDelivery of Immunostimulatory CpG Oligonucleotides.ACS Nano, 11, 8783–8789.

McLaughlin, C. K., Hamblin, G. D., & Sleiman, H. F. (2011).Supramolecular DNA Assembly. Chemical Society Reviews,40, 5647–5656

138 Optimizing DNA nanotube designfor future applications

Graham D. Hamblin* and Hanadi F. Sleiman

Department of Chemistry, McGill University, Montreal, QCH3A0B8*Email: [email protected],Phone: (514) 398-6921, Fax: (514) 398-3797

DNA nanotubes hold promise as scaffolds for proteinorganization, as templates of nanowires and photonicsystems, and as drug delivery vehicles (Cao, 2004). Wepresent a new DNA economic strategy for the construc-

tion of DNA nanotubes with a backbone produced byrolling circle amplification (RCA) which results inincreased stability and templated length (Hamblin et al.,2012). These nanotubes are more resistant to nucleasedegradation, capable of entering human cervical cancercells with significantly increased uptake over double-stranded DNA, and are amenable to encapsulation andrelease behavior. As such, they represent a potentiallyunique platform for the development of cell probes, drugdelivery, and imaging tools. In a second iteration, wealso present a modified design that has been simplifiedeven further (Hamblin et al., 2013). This allows rapidroom temperature assembly of high aspect ratio nano-


tubes from just five unmodified DNA strands and theRCA backbone.

This research has been supported by NSERC & CSACS.

ReferencesCao, G. (2004). Nanostructures and nanomaterials – Synthesis,

properties and applications. London: Imperial College.

Hamblin, G. D., Carneiro, K. M., Fakhoury, J. F., Bujold, K.E., & Sleiman, H. F. (2012). Rolling circle amplification-templated DNA nanotubes show increased stability and cellpenetration ability. Journal of the American Chemical Soci-ety, 134, 2888–2891.

Hamblin, G. D., Hariri, A., Carneiro, K. M., Lau, K. L., Cosa,G., & Sleiman, H. (submitted for publication). A simpledesign for DNA nanotubes from a minimal set of unmodi-fied strands: rapid, room temperature assembly and readilytunable structure. ACS Nano.

139 Development and application ofcrop exudate specific aptamers

Emily Mastronardia, Maureen Mckeagueb,Carlos Monrealc and Maria DeRosaa

aDepartment of Chemistry, Carleton University, 1125 ColonelBy Drive, Ottawa, ON Canada, K1S 5B6; bDepartment ofBioengineering, Stanford University, 473 via, Ortega, Stanford,CA 94305; cEastern Cereal and Oil Seed Research Centre,Agriculture and AgriFood Canada, 960 Carling Avenue,Ottawa, Ontario, Canada, K1A 0C6

The fertilizer industry is lucrative, though it facesenvironmental challenges. The amount of nitrogenapplied to crops far exceeds the nitrogen utilized bycrops leading to excess nitrogen in the form ofnitrates, gaseous ammonia, and nitrogen oxides(DeRossa et al., 2010). This excess nitrogen canspread into groundwater contaminating drinking waterand causing excess algal growth. Developments innanotechnology may alleviate some of these environ-mental challenges. Although there are examples ofnanotechnology being utilized for fertilizer products,none of these methods are able to respond to thespecific nutrient needs of the plant. This project aimsto produce a nanofertilizer product that cansynchronize the release of its nutrients with the

uptake of nutrients by the plant (DeRossa et al.,2010). Aptamers are synthetic molecules of DNA orRNA that can form 3-D shapes, which are capable ofstrongly and selectively binding a target of interest.Aptamers have been found to have binding affinitiessimilar to, if not surpassing, those of monoclonalantibodies (Sultan et al., 2009). The goal of this pro-ject is to use polyelectrolyte microcapsules containingaptamers in their walls that are specific for key plantsignals. This will allow the delivery of nutrient mole-cules from inside the microcapsules only whenrequired by the plants. Root exudate specific aptamersare being developed using SELEX (Systematic Evolu-tion of Ligands through Exponential enrichment) froma random DNA pool, as well as from an existingaptamer pool. These aptamers will act as molecularrecognition probes in the development of an intelli-gent fertilizer system. Progress from these selectionswill be presented.

ReferencesDeRosa, M. C., Monreal, C., Schnitzer, M., Walsh, R., & Sultan,

Y. (2010). Nanotechnology in fertilizers. Nature Nanotech-nology, 5, 91.

Sultan, Y., Walsh, R., Monreal, C., DeRosa, M. C. (2009).Preparation of functional aptamer films by layer-by-layerself-assembly. Biomacromolecules, 10, 1149–1154.


140 Development of nanoscaleaptamer films for controlledrelease of encapsulated payloads

Amanda G. Fostera, Yasir Sultana, Carlos Monrealb andMaria C. DeRosaa*aDepartment of Chemistry, Carleton University, 1125 ColonelBy Drive, Ottawa, K1S 5B6, Canada; bEastern Cereal and OilSeed Research Centre, Agriculture and AgriFood Canada, 960Carling Avenue, Ottawa, K1A 0C6, Canada*Email: [email protected], Phone: (613) 520-2600ext 3844, Fax: (613) 520-3749

Aptamers are short, single-stranded nucleic acids thatfold into well-defined 3D structures which bind to asingle target molecule (from small molecules to cells)with affinities and specificities that can rival those ofantibodies (Jeong et al., 2009). Unlike antibodies, apta-mers can be chemically synthesized eliminating the needfor animals or cell culture, which also allows for selec-tion under non-physiological conditions and broadenspotential targets to include toxic molecules (Banka &Stockley, 2006). The compatibility of aptamers withnanomaterials, in combination with their affinity,selectivity, and conformational changes upon target inter-action, have allowed for the development of a largenumber of therapeutic and targeted delivery systems inrecent years exploiting these properties. Despite this,many challenges still exist as unprotected DNA is readilydegraded by nucleases prevalent in biological and envi-ronmental systems (Bouchard et al., 2010). Embeddingaptamers within multilayer polyelectrolyte films couldprovide a biodegradable shelter, while allowing the detec-tion of diffusible small molecules. An understanding ofthese materials will allow for the eventual encapsulationof relevant payloads into aptamer–polyelectrolyte micro-capsules towards the development of a controlled releasesystem. In this work, films composed of natural poly-electrolytes chitosan and hyaluronan are employed dueto their biocompatibility, strong presence in currentliterature, and amiability to layer-by-layer film construc-

tion. Initial progress towards the development of anaptamer-embedded polyelectrolyte film system will bepresented.

ReferencesBouchard, P. R., Hutabarat, R. M., & Thompson, K. M. (2010).

Discovery and development of therapeutic aptamers. AnnualReview of Pharmacology and Toxicology, 50, 237–257.

Bunka, D. H. J., & Stockley, P. G. (2006). Aptamers come ofage – at last. Nature, 4, 588–596.

Jeong, E., Lee, J. W., & Ellington, A. D. (2009). Applicationsof aptamers as sensors. Annual Review of AnalyticalChemistry, 2, 241–264.

141 DNA origami as a platform forassembly of nanophotonicelements

Yan Liu*

Department of Chemistry and Biochemistry and the BiodesignInstitute, Arizona State University, Tempe, AZ 85287, USA*Email: [email protected], Phone: (480) 727-0397, Fax: (480)727-2378

Achieving DNA-functionalized semiconductor quantumdots (QDs) that are robust enough to be compatiblewith the DNA nanotechnology that withstand precipita-tion at high temperature and ionic strength is a chal-lenge. Here we report a method that facilitates thesynthesis of stable core/shell (1–20 monolayers) QD-DNA conjugates in which the end part (5–10 nucleo-tides) of the phosphorothiolated oligonucleotides isembedded within the shell of the QD. These reliableQD-DNA conjugates exhibit excellent chemical,colloidal and photonic stability over a wide pH range(4–12) and at high salt concentrations (>100mM Na+

or Mg2+), bright fluorescence emission with quantumyields of upto 70%, and broad spectral tunability withemission ranging from UV to NIR (360–800 nm). Theassembly of these different QDs into DNA origami in a


well-controlled pattern was demonstrated (Deng,Samanta, Nangreave, Yan, & Liu, 2012). We also usedDNA origami as a platform to co-assemble a goldnanoparticle with 20 nm diameter (AuNP) andan organic fluorophore (TAMRA) and studied thedistance dependent plasmonic interactions between theparticle and the dye using steady state fluorescence andlifetime measurements. Greater fluorescence quenchingwas found at smaller inter-particle distances, which wasaccompanied by an enhancement of the decay rate. Wefurther fabricated 20 nm and 30 nm AuNP homodimerswith different inter-particle distances using DNA ori-gami scaffolds and positioned a Cy3 fluorophorebetween the AuNPs in both the assemblies. Up to 50%enhancement of the Cy3 fluorescence quantum effi-ciency was observed for the dye between the 30 nmAuNPs. These results are in good agreement with thetheoretical simulations (Pal et al., 2013).

This research has been supported by NSF and ONR.

ReferencesDeng, Z., Samanta, A., Nangreave, J., Yan, H., & Liu, Y.

(2012). Robust DNA functionalized core/shell quantumdots with fluorescent emission spanning from UV–vis tonear IR and compatible with DNA directed self-assembly.Journal of the American Chemical Society, 134, 17424–17427.

Pal. S., Dutta, P., Wang, H., Deng, Z., ZOu, S., Yan, H., &Liu, Y. (2013). Quantum efficiency modification of organicfluorophores using gold nanoparticles on DNA origamiscaffold. Journal of Physical Chemistry C, In revision.

142 Non-canonical base pairing motifsin DNA crystal design

Chun Geng, Stephanie E. Muser and Paul J. Paukstelis*

Department of Chemistry and Biochemistry, Center forBiomolecular Structure and Organization, University ofMaryland, College Park, MD 20742, USA*Email: [email protected], Phone: (301) 405-9933,Fax: (301) 405-9377

DNA has proved to be a successful material for creationof nanoscale structures because of its inherent program-mability and predictable structural features. However,the assembly of periodic three-dimensional (3D) DNAcrystals is hampered by the junctions needed to connectthe inherently linear Watson–Crick duplexes. Here, weexamine how predictable noncanonical base pairingmotifs can be used in conjunction with Watson–Crickduplexes to assemble macroscopic 3D crystals withuseful nanoscale features. Parallel-stranded homopurine5′-GGA base pairs serve as a junction region in a con-tinuously base paired 13-mer DNA crystal (Paukstelis

et al., 2004). This motif is predictable and has beenused in different sequence contexts to rationally designDNA crystals with different lattice dimensions. Thesedesigned crystals have been utilized as macromolecularsieves for capturing or excluding proteins (Paukstelis,2006). Further, we have demonstrated that a proteinenzyme encapsulated in the crystal solvent channels iscapable of performing catalysis. Enzyme-infused DNAcrystals are capable of multiple cycles of catalysis fol-lowing removal of substrate and products, and may offerpotential new routes for enzyme replacement therapiesor the creation of new biodegradable solid-state catalystsand sensors. A structurally similar homoparallel region,5′-CGAA, has also been used to generate crystals thatare capable of making concerted in crystallo structuraltransitions in response to pH perturbations (Muser &Paukstelis, 2012). These studies highlight potential usesof DNA crystals as stimuli-responsive biomaterials.Despite these successes, the ability to use noncanonicalDNA motifs in crystal design is limited by both thenumber of available noncanonical DNA structures, andour understanding of how these structures self-assemble.To address this we have initiated a high-throughputcrystallization screen of short DNA oligonucleotides toidentify new noncanonical base pairing motifs and toaddress the broad question: How structurally diverse isDNA?

This research has been supported by NSF CAREERAward DMR-1149665.

ReferencesMuser, S. E., & Paukstelis, P. J. (2012). Three-dimensional DNA

crystals with pH-responsive noncanonical junctions. Journalof the American Chemical Society, 134, 12557–12564.

Paukstelis, P. J. (2006). Three-dimensional DNA crystals asmolecular sieves. Journal of the American Chemical Soci-ety, 128, 6794–6795.

Paukstelis, P. J., Nowakowski, J., Birktoft, J. J., & Seeman, N.C. (2004). Crystal structure of a continuous three-dimen-sional DNA lattice. Chemical & Biology, 11, 1119–1126.

143 Porous three-dimensional DNAcrystals as biomolecularcontainers for catalysis

Chun Geng* and Paul J. Paukstelis

Department of Chemistry & Biochemistry, Center forBiomolecular Structure and Organization, University ofMaryland, College Park, MD 20742, USA*Email: [email protected], Phone: (301) 455 3325, Fax:(301) 405 9377

The major goal of DNA nanotechnology has been thedesign and manufacture of artificial DNA structures for


technological uses. The porous three-dimensional DNAcrystals have been proposed as macromolecular scaf-folds for host–guest structure determination, as molecu-lar sieves and as molecular containers for catalysis. Byusing fluorescence dequenching technique, we havedemonstrated that a protein enzyme adsorbed in adesigned three-dimensional DNA crystal is capable ofperforming catalysis. The axially distinct aperture sizesin the crystal design allowed us to improve the enclos-ing of the enzyme with a protective protein-based“coating” cross-linked over the crystal surface. Thiscoating allows entry and exit of small moleculesthrough the crystal while restricting enzymes inside thecrystal. This enzyme-enclosed DNA crystal is capableof performing multiple cycles of catalysis and itretained its enzymatic activity over numerous days after

being protein-coated. The concepts of the enzyme-enclosed DNA crystal and the unique protein coatingtechnique provide possibilities to the development ofenzyme replacement therapies and biodegradable solid-state catalysts and biosensors.

This research has been supported by NSF CAREERaward DMR-1149665.

ReferencesPaukstelis, P. J. (2006). Three-dimensional DNA crystals as

molecular sieves. Journal of the American Chemical Society,128, 6794–6795.

Paukstelis, P. J., Nowakowski, J., Birktoft, J. J., & Seeman, N. C.(2004). Crystal structure of a continuous three-dimensionalDNA lattice. Chemistry & Biology, 11, 1119–1126.

144 Sculpting light with DNA origami

Anton Kuzyk, Robert Schreiber, Zhiyuan Fan,Günther Pardatscher, Eva-Maria Roller,Alexander Högele, Friedrich C. Simmel,Alexander O. Govorov and Tim Liedl*

Department of Physics and Center for NanoscienceLudwig-Maximilians-Universität München, München, Germany*Email: [email protected], Phone: +49 89 2180 3725,Fax: +49 89 2180 3182

We used the DNA origami method (Rothemund, 2006)for the fabrication of self-assembled nanoscopic materi-als (Seeman, 2010). In DNA origami, a virus-based8 kilobase-long DNA single-strand is folded into shapewith the help of � 200 synthetic oligonucleotides. Theresulting DNA nanostructures can be designed to adoptany three-dimensional shape and can be addressedthrough DNA hybridization or chemical modification

with nanometer precision. We have realized that complexassemblies of nanoparticles, including magnetic, fluores-cent, and plasmonic nanoparticles. Such nanoconstructsmay exhibit striking optical properties such as strongoptical activity in the visible range (Kuzyk et al., 2012).To this end, plasmonic particles were assembled in solu-tion to form helices of controlled handedness. Weachieved spatial control over particle placement betterthan 2 nm and attachment yields of 97% and above. Asa collective optical response emerging from our dis-persed nanostructures, we detected pronounced circulardichroism (CD) originating from the plasmon–plasmoninteractions in the particle helices. In recent experiments,we were able to show that the optical response of chiralbiomolecules can be transferred from the UV into thevisible region in plasmonic hotspots. Thus, sensitivedetection of chiral biomolecules may become feasible inthe near future. We also found that the orientation of thehelices in respect to the incoming light beam criticallyinfluences the resulting CD spectra. Our results can be


explained with theoretical models based on plasmonicdipole interaction and demonstrate the potential of DNAorigami for the assembly of metafluids with designedoptical properties.

This research has been supported by the VolkswagenFoundation and the Deutsche Forschungs-gemeinschaft(DFG).

ReferencesRothemund, P. W. K. (2006). Folding DNA to create nanoscale

shapes and patterns. Nature, 440, 207.Seeman, N. C. (2010). Nanomaterials based on DNA. Annual

Review of Biochemistry, 79, 12.1.Kuzyk, A., et al. (2012). DNA-based self-assembly of chiral

plasmonic nanostructures with tailored optical response.Nature, 483, 311.

145 Single-molecule observation ofenzymes and DNA structuralchanges in the DNAnanostructures

Hiroshi Sugiyama* and Masayuki Endo

Department of Chemistry, Institute for Integrated Cell-MaterialSciences (WPI-iCeMS), Graduate School of Science, KyotoUniversity, Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606 8501,Japan*Email: hs@ kuchem.kyoto-u.ac.jp, Phone: +81-75-753-4002,Fax: +81-75-753-3670

Direct observation of the movement of biomoleculesincluding enzymes and DNAs should be one of theultimate goals for investigating the detailed mechanical

behavior of the molecules during the reactions. Wedesigned various DNA nanostructures using DNA ori-gami method for the preparation of single-moleculeobservation scaffolds. Using the designed DNA scaffoldand high-speed atomic force microscopy (AFM), thesingle-molecule behaviors of the DNA-modifyingenzymes, repair enzymes, and recombinases wereobserved in the target double-stranded DNAs (dsDNAs)placed in the DNA frame structure (Endo, Katsuda,Hidaka, & Sugiyama, 2010a, 2010b). DNA structuralchanges including G-quadruplex formation (Sannohe,Endo, Katsuda, Hidaka, & Sugiyama, 2010) and B-ZDNA conformational change (Rajendran, Endo, Hidaka,& Sugiyama, 2013) were also visualized. Using thissystem, we observed the photo-induced DNA hybridiza-tion and dissociation by detecting the global structural


changes of the incorporated two dsDNAs in theDNA frame structure (Endo, Yang, Suzuki, Hidaka, &Sugiyama, 2012). A pair of azobenzene-modifiedoligonucleotides (ODNs) was employed, which formsduplex in the transform and dissociates in the cis-form.During UV-irradiation, hybridized azobenzene-modifiedODNs at the center dissociated, and the subsequentvisible-light irradiation induced the hybridization of thephotoresponsive ODNs, meaning that the reversedswitching behavior such as the hybridization and disso-ciation was directly visualized at the single-moleculelevel. These photoresponsive ODNs were also used forcontrolling assembly and disassembly of the hexagonalDNA origami structures with photoirradiation (Yang,Endo, Hidaka, & Sugiyama, 2012). The combination ofthe designed DNA scaffold modified with target DNAstrands and high-speed AFM is valuable for visualizingand analyzing the single enzymatic and chemicalreactions.

ReferencesEndo, M., Katsuda, Y., Hidaka, K., & Sugiyama, H. (2010a).

Journal of the American Chemical Society, 132, 1592–1597.

Endo, M., Katsuda, Y., Hidaka, K., & Sugiyama, H.(2010b). Angewandte Chemie International Edition, 49,9412–9416.

Endo, M., Yang, Y., Suzuki, Y., Hidaka, K., & Sugiyama, H.(2012). Angewandte Chemie International Edition, 51,10518–10522.

Rajendran, A., Endo, M., Hidaka, K., & Sugiyama, H.(2013). Journal of the American Chemical Society, 135,1117–1123.

Sannohe, Y., Endo, M., Katsuda, Y., Hidaka, K., & Sugiyama,H. (2010). Journal of the American Chemical Society, 132,16311–16313.

Yang, Y., Endo, M., Hidaka, K., & Sugiyama, H. (2012). Jour-nal of the American Chemical Society, 134, 20645–20653.

146 Amyloid fibril polymorphismprobed by advanced vibrationalspectroscopy

Igor K. Lednev* and Dmitry Kurouski

Department of Chemistry, University at Albany SUNY, Albany,NY 12222*Email: [email protected], Phone: (518) 591-8863,Fax: (518) 442-3462

Amyloid fibrils are associated with many neurodegenera-tive diseases. All known amyloids including pathogenicand nonpathogenic forms display functional and struc-tural heterogeneity (polymorphism) which determines thelevel of their toxicity. Despite a significant biological

and biomedical importance, the nature of the amyloidfibril polymorphism remains elusive. We utilized for thefirst time three most advanced vibrational techniques toprobe the core, the surface, and supramolecular chiralityof fibril polymorphs. A new type of folding, aggregationphenomenon, spontaneous refolding from one polymorphto another, was discovered (Kurouski, Lauro et al.,2010). Hydrogen–deuterium exchange deep UV reso-nance Raman spectroscopy (Oladepo, Xiong et al.,2012) combined with advanced statistical analysis(Shashilov & Lednev, 2010) allowed for structural char-acterization of the highly ordered cross-β core of amy-loid fibrils. We reported several examples showingsignificant variations in the core structure for fibril poly-morphs. Amyloid fibrils are generally composed of sev-eral protofibrils and may adopt variable morphologies,such as twisted ribbons or flat-like sheets. We discoveredthe existence of another level of amyloid polymorphism,namely, that associated with fibril supramolecular chiral-ity. Two chiral polymorphs of insulin, which can be con-trollably grown by means of small pH variations, exhibitopposite signs of vibrational circular dichroism (VCD)spectra (Kurouski, Dukor et al. 2012). VCD supramolec-ular chirality is correlated not only by the apparent fibrilhandedness but also by the sense of supramolecularchirality from a deeper level of chiral organization at theprotofilament level of fibril structure. A small pH changeinitiates spontaneous transformation of insulin fibrilsfrom one polymorph to another. As a result, fibril supra-molecular chirality overturns both accompanyingmorphological and structural changes (Kurouski, Dukoret al. 2012). No conventional methods could probe thefibril surface despite its significant role in the biologicalactivity. We utilized tip-enhanced Raman spectroscopy(TERS) to characterize the surface structure of an indi-vidual fibril due to a high depth and lateral spatial reso-lution of the method in the nanometer range (Kurouski,Deckert-Gaudig et al. 2012). It was found that the sur-face is strongly heterogeneous and consists of clusterswith various protein conformations and amino acidcomposition.

This research has been supported by grants from NIHR01 AG033719 and NSF CHE-1152752.

ReferencesKurouski, D., Deckert-Gaudig, T., Deckert, V., & Lednev, I. K.

(2012). Structure and composition of insulin fibril surfacesprobed by TERS. Journal of the American Chemical Soci-ety, 134, 13323–13329.

Kurouski, D., Dukor, R. K., Lu, X., Nafie, L. A., & Lednev, I.K. (2012a). Normal and reversed supramolecular chiralityof insulin fibrils probed by vibrational circular dichroism atthe protofilament level of fibril structure. Biophysical Jour-nal, 103, 522–531.


Kurouski, D., Dukor, R. K., Lu, X., Nafie, L. A., & Lednev,I. K. (2012b). Spontaneous inter-conversion of insulinfibril chirality. Chemical Communications, 48, 2837–2839.

Kurouski, D., Lauro, W., & Lednev, I. K. (2010). Amyloidfibrils are “alive”: Spontaneous refolding from one poly-morph to another. Chemical Communications, 46, 4249–4251.

Oladepo, S. A., Xiong, K., Hong, Z., Asher, S. A., Handen, J., &Lednev, I. K. (2012). UV resonance Raman investigations ofpeptide and protein structure and dynamics. ChemicalReviews, 112, 2604–2628.

Shashilov, V. A., & Lednev, I. K. (2010). Advanced statisticaland numerical methods for spectroscopic characterizationof protein structural evolution. Chemical Reviews, 110,5692–5713.

147 Characterizing the conformationalspace of two disordered peptidesin different solutions

Ana V. Rojasa*, David Easterhoffb, John T.M. DiMaioc,Stephen Dewhurstb, Alan Grossfieldd, Hongyu Miaoa

and Bradley L. Nilssonc

aDepartment of Biostatistics and Computational Biology;bDepartment of Microbiology and Immunology; cDepartment ofChemistry, University of Rochester, Rochester, NY 14627, USA;dDepartment of Biochemistry and Biophysics, School ofMedicine and Dentistry, University of Rochester*Email: [email protected] Phone: (585)725-0631, Fax: (585) 273-1031

Amyloid fibrils formed by peptides found in semenhave been shown to enhance HIV infectivity in vitro.The first of these peptides to be identified was the248–286 fragment of prostatic acid phosphatase(PAP248–286) (Munich et al., 2007). PAP248–286 is highlycationic, and its fibrils might facilitate infection bydecreasing the electrostatic repulsion between the nega-tively charged surfaces of the virus and the target cell.Whereas PAP248–286 can easily form fibrils in seminalfluid, it needs rapid agitation in other environments,and certain ions have been shown to be critical for itsassembly into fibrils (Olsen et al., 2012). However,mutation of the positively charged residues to alanineresults in a peptide (PAP248–286Ala) that can more eas-ily form fibrilar aggregates. We studied PAP248–286 andPAP248–286Ala fibril formation in water and water+NaCl environments. While PAP248-286Ala can effi-ciently form fibrils in both water and water +NaCl,PAP248-286 can only do so in a water +NaCl solution.The inability of PAP248–286 to form fibrils in watercould be due solely to repulsion between the positivelycharged peptides, an effect that might be diminished bythe presence of salt. However, it is also possible thatthe explanation lies in PAP248–286’s failure to populateconformations that can easily lead to ordered aggre-

gates. To answer this question, using molecular dynam-ics simulations, we characterized the ensemble ofconformations populated by the two peptides in waterand water +NaCl environments. The results indicate thatPAP248-286Ala favors contacts that stabilize a strand-turn-strand, or β-arch, motif around P31, the only pro-line residue in the sequence. Because β-arches are acommon feature in amyloid fibrils, and because it isvery unlikely that a proline residue would be in anyposition other than the β-arch, we expect the formationof this motif to be the rate-limiting step in PAP248–286Ala / PAP248–286 fibril formation. Moreover, the con-tacts stabilizing the β-arch would bring positivelycharged residues into contact in PAP248–286, which, con-sistent with the experimental results, would be facili-tated by the presence of negative ions. To summarize,we have tried to understand if the inability of PAP248–286 to efficiently form fibrils in water is only due to aslower aggregation caused by electrostatic repulsionbetween the positively charged peptides. Our data sug-gest that this effect is also due to electrostatic repulsionbetween the residues within each monomeric peptide,which prevents PAP248–286 from populating conforma-tions that would lead to ordered aggregates.

This research has been supported by grants from theNational Institute of Health (T32AI83206, R21AI094511, T32AI049815 [DE], and P30AI78498).

ReferencesMünch, J., et al. (2007). Semen-derived amyloid fibrils drasti-

cally enhance HIV infection. Cell, 131, 1059–1071.Olsen, J. S., DiMaio, J. T. M., Doran, T. M., Brown, C.,

Nilsson, B. L., & Dewhurst, S. (2012). Seminal plasmaaccelerates semen-derived enhancer of viral infection(SEVI) fibril formation by the prostatic acid phosphatase(PAP(248–286)) peptide. Journal of Biological Chemistry,287, 11842–11849.

148 Evolutionary dynamics ofconformational flexibility

Juan F. Ortiz, Patrick Masterson, Madolyn Macdonald,Vladimir N. Uversky and Jessica Siltberg-Liberles*

Department of Molecular Biology, University of Wyoming,Laramie, WY 82071, USA*Email: [email protected], Phone: 307-766-3181,Fax: 307-766-5098

A fundamental assumption in biology is that proteinstructure is more conserved than protein sequence. Thisopinion stems from observations of the fold distributionof protein structures present in the protein data bank(PDB), where homologous proteins are found to displaythe same fold. However, the set of proteins in PDB is


not representative of protein sequence space. The pre-dominant experimental method used for the structuralcharacterization of the proteins in PDB is X-ray crystal-lography. Thus, conformational flexibility is lost and astatic snapshot of a protein’s dynamic structure remains.Using structural disorder as a proxy for protein dyna-mism, it seems that at least 30% of eukaryotic proteinshave dynamic regions, that is, regions with conforma-tional flexibility. Little is known about the conservationof structurally disordered regions, except that they oftenevolve at an elevated rate compared to the structuredregions. Our preliminary results indicate that there areregions that show fast evolutionary dynamics of struc-tural disorder. A feature of structurally disordered pro-teins is their functional promiscuity; these proteinsoften interact with many different biomolecules in asignalling fashion. A central tenet of protein structureand function is that a protein’s function is given by itsstructure. Structurally disordered proteins exist as con-formational ensembles and hence, it is intuitive that afunctional ensemble would accompany the structuralensemble. The different conformations in the ensembleare rapidly interconverting over a shallow free energylandscape with multiple minima of similarly low stabil-ity. In accordance with the extended view of allostery(conformational selection), stabilizing interactions withother biomolecules can drive the population of confor-mations towards a particular functional conformation.Further, coupling the conformational ensemble freeenergy landscape to an adaptive fitness landscape forgene/protein function, we are investigating how proteinswith conformational and functional ensembles evolve.Here, a study across flaviviruses is presented. Flavivi-ruses depend on conformational flexibility at manysteps in their life cycle and the entire flavivirusproteome contains about 3400 amino acid long polypro-tein that is spliced into 11–12 proteins. Therefore, fullgenome studies are feasible. We find support formutation-driven conformational selection, as amino acidsubstitutions can increase the rate of divergence ofconformational flexibility among different homologousproteins. Some regions are more prone to shift fromorder to disorder or vice versa, and some lineages showrapid shifts from order to disorder. Lineage-specificshift between disorder and order can alter the confor-mational ensemble for the same protein in differentspecies, causing a subtle functional change. Thus, rapidevolutionary dynamics of structural disorder could be apotential driving force for biological divergence amongflaviviruses.

This work was supported by Award NumberP20RR016474 from the National Center for ResearchResources. The content is solely the responsibility of the

author and does not necessarily represent the officialviews of the National Center for Research Resources orthe National Institutes of Health.

149 Generating context-specificfunctions with intrinsicallydisordered domains

Ying Liua,b, Kathleen S. Matthewsb andSarah E. Bondosa,b*aDepartment of Biochemistry and Cell Biology, Rice University,Houston, TX 77005, ; bDepartment of Molecular and CellularMedicine, Texas A&M Health Science Center, College Station,TX 77845-1114*Email: [email protected] Phone: (979) 845-5399,Fax: (979) 847-9481,

Like many transcription factors important for animaldevelopment, members of the Hox protein familyinstigate multiple tissue-specific developmental pro-grams. In each tissue, an individual Hox protein reg-ulates a different subset of gene targets.Consequently, Hox proteins must sense positionalinformation from the cell and select different DNAbinding targets in response. Furthermore, Hox proteinsmust sense which gene it has bound and selectwhich mode of transcription regulation – activation orrepression – to implement at each site. Our studieswith the Drosophila Hox protein Ultrabithorax (Ubx)in vitro and in vivo demonstrate that interplaybetween intramolecular and intermolecular proteininteractions involving intrinsically disordered regionsplay key roles in these sense and select mechanisms.The Ubx protein comprises a structured DNA-bindinghomeodomain, which accounts for �15% of the pro-tein, short elements with predicted helical structure,and two large intrinsically disordered regions. Mostof the protein sequence influences DNA-binding affin-ity by the homeodomain. The two intrinsically disor-dered regions directly contact the homeodomain andimpact DNA-binding specificity as well. Consequently,protein interaction regions, alternatively splicedsequences, and phosphorylation sites embedded withinthese disordered regions provide an opportunity forcellular events to modulate DNA binding. In particu-lar, the C-terminal intrinsically disordered regionappears to act as a tissue identity sensor. This regionconsists of a binding motif for the Hox cofactorExd, separated from the homeodomain by alterna-tively spliced disordered segments. Intramolecularinteractions allow the motif to prevent monomer bind-ing to Ubx–Exd composite sequences. As the distancebetween the motif and the homeodomain is shortened


by splicing, DNA binding by Ubx monomers andmultimers is inhibited as well. Tissue identity deter-mines both Exd availability and the identity of theUbx splicing isoform, which together dictates whichsubset of Ubx target sequences is bound: high affin-ity monomer sites, low affinity monomer sites, and/orUbx–Exd heterodimer sites. The N-terminal disorderedregion binds transcription factors that subdivide Ubx-specified tissues, and thus may transmit positionalinformation within the tissue to the homeodomain.Conversely, DNA binding triggers a large conforma-tional change in Ubx which exposes these intrinsi-cally disordered regions to solvent. Thisconformational change in Ubx varies with the DNAsequence bound, providing a mechanism for the DNAsequence to influence protein interactions and tran-scription regulation. The reciprocal nature of theseinteractions reinforces and stabilizes “correct” Ubx-DNA complexes, generating the reliability that is anecessary part of Hox function.

ReferencesPearson, J. C., Lemmons, D., & McGinnis, W. (2005). Modu-

lating Hox gene functions during animal body patterning.Nature Reviews Genetics, 6, 893–904.

Liu, Y., Matthews, K. S., & Bondos, S. E. (2009). Internalregulatory interactions determine DNA binding specificityby a Hox transcription factor. Journal of MolecularBiology, 390, 760–774.

Liu, Y., Matthews, K. S., & Bondos, S. E. (2008). Multipleintrinsically disordered sequences alter DNA binding bythe homeodomain of the Droosphila Hox protein Ultrabi-thorax. Journal of Biological Chemistry, 283, 20874–20887.

Bondos, S. E., Tan, X. X., & Matthews, K. S. (2006). Physicaland genetic interactions link Hox function with diversetranscription factors and cell signaling proteins. Molecular& Cellular Proteomics, 5, 824–834.

150 Getting a grip on alpha-synucleinamyloid oligomers

Vinod Subramaniam*

Nanobiophysics, MIRA Institute, University of Twente,PO Box 217, 7500 AE, Enschede, The Netherlands*Email: [email protected], Phone: +31 53 4893158,Fax: +31 53 4891105

Misfolding and aggregation of proteins into nano-meter-scale fibrillar assemblies is a hallmark of manyneurodegenerative diseases. Aggregation of the humanalpha-synuclein protein is implicated in the etiology ofParkinson’s disease. A particularly relevant question is

the role of early oligomeric aggregates of alpha-synuc-lein in modulating the dynamics of protein aggrega-tion, and in the interactions with essential cellularcomponents. However, very little is known about themolecular details of these aggregate species. For largeprotein aggregates, such as alpha-synuclein oligomers,it is very difficult to determine the number of mono-mers that form an oligomer using conventional tech-niques. We have developed a method that uses sub-stoichiometric labeling; that is, only a fraction of themonomers contains a fluorescent label, in combinationwith single-molecule photobleaching to determine thenumber of monomers per oligomer (Zijlstra et al.,2012). The number of bleaching steps gives the num-ber of fluorescent labels per oligomer. Knowing theexact label density, that is, the fraction of labeledmonomers at the start of the aggregation, we can cor-relate the number of fluorescent labels per oligomer tothe total number of monomers. Using this method, wecan determine the composition, probe the distributionin the number of monomers per oligomer, and investi-gate the influence of the fluorescent label on theaggregation process. For wild-type alpha-synuclein, wefind no distribution in the number of monomers peroligomer and find a single, well-defined oligomericspecies consisting of �30 monomers per oligomer. Onthe other hand, for oligomers formed in the presenceof dopamine, we find a distinctly bimodal distributionsuggesting the existence of two populations of oligo-meric species.

This work is financially supported by the “NederlandseOrganisatie voor Wetenschappelijk Onderzoek” (NWO)through the NWO-CW TOP program number700.58.302. We further acknowledge support from the“Stichting Internationaal Parkinson Fonds.”

ReferencesZijlstra, N. C., Blum, I. M., Segers-Nolten, M. M., Claessens, A.

E., & Subramaniam, V. (2012). Molecular composition ofsub-stoichiometrically labeled alpha-synuclein oligomersdetermined by single-molecule photobleaching. AngewandteChemie (International ed. in English), 51, 8821–8824.


151 Network inference and analysis ofParkinson’s disease

Antonio Del Sol Mesa*

Luxembourg Centre for Systems Biomedicine (LCSB), Universityof Luxembourg 7, Avenue des Hauts-Fourneaux, L-4362Esch-sur-Alzette, Luxembourg*Email: [email protected], Phone: 352-466644-6982

Based on existing literature and publicly availableexpression data-sets, a map of Parkinson’s disease(PD) has been inferred in collaborative effort withother teams in LCSB and Systems Biology Institute,Tokyo, Japan. However, due to the increased com-plexity of the map, human intuition is often insuffi-cient in understanding the initiation, functionalregulation, and progression of this disease. Hence, itis necessary to mine the information content of thisnetwork to make sense of this abundance complexinformation. To this end, current work aims to ana-lyze the network topology and dynamics of the PDmap, using Boolean modeling. Grounded on perturba-tion analysis, the work also aims to obtain a systemlevel understanding of the genotype–phenotype rela-tionships to identify key components in the diseaseregulation and to generate experimentally testablehypothesis for PD susceptibility and progression.Methodology includes using existing graph theoreticalanalysis tools, as well as to develop rigorous sophisti-cated analysis tools which could be vital for under-standing the disease pathology and for successfulquantitative modeling. In general, the major focus andcontribution of this work aim a the fields of statisticalinference, graph analysis, and dynamic modeling insystems biology.

152 Order from disorder: definingstructure in disorderedproteins

Shana Elbaum-Garfinkle, Garrett Cobb, Abhinav Nathand Elizabeth Rhoades*

Department of Molecular Biophysics & Biochemistry, YaleUniversity, 266 Whitney Avenue, Bass 234, New Haven, CT06511, USA*Email: [email protected], Tel: 203-432-5342,Fax: 203-432-5175

Intrinsically disordered proteins are involved in a rangeof functional roles in the cell, as well as beingassociated with a number of diverse diseases, includingcancers, neurodegenerative disorders, and cardiac myo-pathies. We use single-molecule fluorescence approachesto characterize disordered proteins implicated in the pro-

gression of Parkinson’s and Alzheimer’s diseases. Ourgoal is to understand, how disease-associated modifica-tions to these proteins alter their conformational anddynamic properties and to relate these changes to diseasepathology.

This work is supported by NSF MCB 0919853.

153 Wonderful roles of the entropy inprotein dynamics, binding andfolding

Yue-Hui Xiea, Yan Taob and Shu-Qun Liub*aTeaching and Research Section of computer, Department ofBasic Medical, Kunming Medical College, Kunming 650031,P.R. China; bLaboratory for Conservation and Utilization ofBio-Resources & Key Laboratory for Microbial Resources ofthe Ministry of Education, Yunnan University, Kunming650091, P. R. China*Email: [email protected], Phone: (86)871-5035257

The entropy, which is central to the second law ofthermodynamics, determines that the thermal energyalways flows spontaneously from regions of highertemperature to regions of lower temperature. In theprotein–solvent thermodynamic system, the entropy isdefined as a measure of how evenly the thermalenergy would distribute over the entire system (Liu


et al., 2012). Such tendency to distribute energy asevenly as possible will reduce the state of order ofthe initial system, and hence, the entropy can beregarded as an expression of the disorder, or random-ness of the system (Yang et al., 2012). For a pro-tein–solvent system under a constant solventcondition, the origin of entropy is the thermal energystored in atoms, which makes atoms jostle aroundand bump onto one another, thus leading to vibrationsof the covalent bonds connecting two atoms (occur-ring on the fs timescale) and the rotational and trans-lational motions of amino acid side chain groups(occurring on ps timescale) and water molecules.These motions break the noncovalent bonds aroundstructural regions that are weakly constrained therebytriggering the competitive interactions among residuesor between residues and water molecules leading ulti-mately to the loop motions (occurring on ns time-scale) around the protein surface. The loop motionscan further transmit either through the water networkaround the protein surface or via specific structuralcomponents (such as the hinge-bending regions) overthe entire protein molecule leading to large concertedmotions (occurring on μs to s timescales) that aremost relevant to protein functions (Amadei, Linssen& Berendsen, 1993; Tao, Rao & Liu, 2010). Thus,the multiple hierarchies of the protein dynamics ondistinct timescales (Henzler-Wildman & Kern, 2007)are a consequence of the cascade amplification of themicroscopic motions of atoms and groups for whichthe entropy originating from atomic thermal energy ismost fundamental. In the case of protein–ligand bind-ing, the importance of the entropy is embodied in thefollowing aspects. (i) The release of the water mole-cule kinetic energy (which is a process of the solvententropy maximization) will cause Brownian motionsof individual water molecules which result in strongBrownian bombardments to solute molecules causingmolecule wanders/diffusions and subsequent accidentcontacts/collisions between proteins and ligands. (ii)Such collisions will inevitably cause water moleculedisplacement and, if the contact interfaces are prop-erly complementary, the requirement to increase thesolvent entropy would further displace the water net-work around the binding interfaces thus leading tothe formation the initial protein-ligand complex. (iii)In the initial complex, the loose association of thetwo partners provide the opportunity for protein toincrease conformational entropy, thus triggering theconformational adjustments through competitive inter-action between protein residues and ligand, leadingultimately to the formation of tightly associated com-

plex (Liu et al., 2012). In the protein folding process,the first stage, i.e. the rapid hydrophobic collapse(Agashe, Shastry & Udgaonkar, 1995; Dill, 1985), isin fact driven by the effect of the solvent entropymaximization. Specifically, the requirement to maintainas many as possible the dynamic hydrogen bondsamong the water molecules will squeeze/sequestratethe hydrophobic amino acid side chains into the inte-rior of the folding intermediates and expose the polar/charged side chains onto the intermediate surface.This will minimize the solvent accessible surface areaof the folding intermediates and as thus maximize theentropy of the solvent. The resulting molten globulestates (Ohgushi & Wada, 1983) may contain a fewsecondary structural components and native tertiarycontacts, while many native contacts, or close resi-due–residue interactions present in the native statehave not yet formed. However, the nature to increasethe protein conformational entropy can trigger a fur-ther conformational adjustment process, i.e. the con-formational entropy increase breaks the transientsecondary or tertiary contacts and triggers the compet-itive interactions among protein residues and betweenresidues and water. This process may repeat manyrounds until the negative enthalpy change resultingfrom the noncovalent formations can overcompensatefor protein conformational entropy loss. In summary,we consider that the tendency to maximize theentropy of the protein–solvent system, which origi-nates from the atomic thermal energy, is the mostfundamental driving factor for protein folding, bind-ing, and dynamics, whereas the enthalpy reduction, anopposing factor that tends to make the systembecome ordered, can compensate for the effect ofentropy loss to ultimately allow the system to reachequilibrium at the free energy minima, either globalor local.

This research is supported by NSFC (No. 31160181 and30860011) and project of innovation term of Yunnanprovince (2011CI123).

ReferencesAgashe, V. R., Shastry, M. C. R., & Udgaonkar, J. B. (1995).

Initial hydrophobic collapse in the folding of barstar.Nature, 377, 754–757.

Amadei, A., Linssen, A. B. M., & Berendsen, H. J. C. (1993).Essential dynamics of proteins. Proteins: Structure,Function, and Genetics, 17, 412–425.

Dill, K. A. (1985). Theory for the folding and stability of glob-ular proteins. Biochemistry, 24, 1501–1509.

Henzler-Wildman, K. A., & Kern, D. (2007). Dynamic person-alities of proteins. Nature, 450, 964–972.


Liu, S. Q., Xie, Y. H., Ji, X. L., Tao, Y., Tan, D. Y., Zhang, K. Q.,& Fu, Y. X. (2012). Protein folding, binding and energylandscape: A synthesis. In P. T. P. Kaumaya (Ed.), Proteinengineering (pp. 207–252). Rijeka: Intech.

Ohgushi, M., & Wada, A. (1983). 'Molten-globule state’: Acompact form of globular proteins with mobile side-chains.FEBS Letters, 164, 21–24.

Tao, Y., Rao, Z. H., & Liu, S. Q. (2010). Insight derivedfrom molecular dynamics simulation into substrate-induced changes in protein motions of proteinase K.Journal of Biomolecular Structure and Dynamics, 28,143–157.

Yang, L. Q., Sang, P., Xie, Y. H., Tao, Y., Fu, Y. X., Zhang, K. Q.,& Liu, S. Q. (in press). Protein dynamics and motions inrelation to their functions: several case studies and the under-lying mechanisms. Journal of Biomolecular Structure andDynamics.

154 Protein folding and bindingfunnels: a common driving forceand a common mechanism

Yue-Hui Xiea, Peng Sangb, Yan Taob and Shu-Qun Liub*aTeaching and Research Section of Computer, Department ofBasic Medical, Kunming Medical College, Kunming, 650031,P.R. China; bLaboratory for Conservation and Utilization ofBio-Resources & Key Laboratory for Microbial Resources ofthe Ministry of Education, Yunnan University, Kunming,650091, P.R. China*Email: [email protected], Phone: +86 871-503-5257

Under the free energy landscape theory, both the protein-folding and protein–ligand binding processes are drivenby the decrease in total Gibbs free energy of the protein-solvent or protein–ligand-solvent system, which involvesthe non-complementary changes between the entropyand enthalpy, ultimately leading to a global free energyminimization of these thermodynamic systems (Ji & Liu,2011; Liu et al., 2012; Yang, Ji & Liu, 2012). In the caseof protein folding, the lowering of the system freeenergy coupled with the gradual reduction in conforma-tional degree of freedom of the folding intermediatesdetermines that the shape of the free energy landscapefor protein folding must be funnel-like (Dill & Chan,1997), rather than non-funneled shapes (Ben-Naim,2012). In the funnel-like free energy landscape, proteinfolding can be viewed as going down the hill via multi-ple parallel routes from a vast majority of individualnon-native states on surface outside the funnel to thenative states located around the bottom of the funnel.The first stage of folding, i.e. the rapid hydrophobiccollapse process, is driven by the solvent entropymaximization. Concretely, the water molecules squeezeand sequestrate the hydrophobic amino acid side chainswithin the interior of the folding intermediates whileexposing the polar and electrostatically chargedside chains on the intermediate surface so as to minimize

the solvent-accessible surface area of the solute andthus, the minimal contacts between the folding interme-diates and the water molecules. This will maximize theentropy of the solvent, thus contributing substantially tolowering of the system free energy due to an absoluteadvantage of the solvent in both quantity and mass(Yang, Ji & Liu, 2012). The resulting molten globulestates (Ohgushi & Wada, 1983), within which a few tran-sient secondary structural components and tertiarycontacts have been formed but many native contacts orclose residue–residue interactions has yet to form, needto be further sculptured into the native states. This is arelatively slow “bottleneck” process because the competi-tive interactions between protein residues within thefolding intermediates and between residues and watermolecules may repeat many rounds to accumulate a largeenough number of stable noncovalent bonds capable ofcounteracting the conformational entropy loss of theintermediates, thus putting this bottleneck stage underthe enthalpy control (i.e. negative enthalpy change),contributing further to the lowering of the system freeenergy. Although the protein–ligand association occursaround the rugged bottom of the free energy landscape,the exclusion of water from the binding interfaces andthe formation of noncovalent bonds between the twopartners can still lower the system free energy. Inconjunction with the loss of the rotational and transla-tional degrees of freedom of the two partners as well asthe loss of the conformational entropy of the protein,these processes could merge, downwards expand, andfurther narrow the free energy wells within which theprotein–ligand binding process takes place, therebymaking them look like a funnel, which we term the bind-ing funnel. In this funnel, the free energy downhill pro-cess follows a similar paradigm to the protein-foldingprocess. For example, if the initial collisions/contactsoccur between the properly complementary interfaces ofthe protein and ligand, a large amount of watermolecules (which usually form a water network aroundthe solute surface) will be displaced to suit the need formaximizing the solvent entropy. This process is similarto that of the hydrophobic collapse during proteinfolding, resulting in a loosely associated protein–ligandcomplex that needs also to be further adapted into a tightcomplex, i.e. the second step which is mainly driven bythe negative enthalpy change through intermolecularcompetitive interactions to gradually accumulate thenoncovalent bonds and ultimately, to stabilize thecomplex at a tightly bound state. Taken together,we conclude that whether in the protein-folding or inthe protein–ligand binding process, both the entropy-driven first step and the enthalpy-driven second stepcontribute to the lowering of the system free energy,resulting in the funnel-like folding or binding freeenergy landscape.


This research is supported by NSFC (No. 31160181 and30860011) and project of innovation term of Yunnanprovince (2011CI123).

ReferencesBen-Naim, A. (2012). Levinthal’s question revisited, and

answered. Journal of Biomolecular Structure and Dynam-ics, 30, 113–124.

Dill, K. A., & Chan, H. S. (1997). From levinthal to pathwaysto funnels. Nature Structural Biology, 4, 10–19.

Ji, X. L., & Liu, S. Q. (2011). Thinking into mechanism ofprotein folding and molecular binding. Journal of Biomo-lecular Structure and Dynamics, 28, 995–996.

Liu, S. Q., Xie, Y. H., Ji, X. L., Tao, Y., Tan, D. Y., Zhang, K.Q., & Fu, Y. X. (2012). Protein folding, binding and energylandscape: A synthesis. In P. T. P. Kaumaya (Ed.), Proteinengineering (pp. 207–252). Rijeka: Intech.

Ohgushi, M., & Wada, A. (1983). ‘Molten-globule state’: Acompact form of globular proteins with mobile side-chains.FEBS Letters, 164, 21–24.

Yang, L. Q., Ji, X. L., & Liu, S. Q. (2012). The free energylandscape of protein folding and dynamics – a global view.Journal of Biomolecular Structure and Dynamics, in press.

155 3D QSAR and protein–proteininteraction studies onneuraminidase againstClostridium perfringens: Anapproach toward targetidentification using structure-based drug designing

Pallavi Somvanshia*, Suruchi Raib andBhartendu Nath Mishrab

aDepartment of Biotechnology, TERI University, 10 InstitutionalArea, Vasant Kunj, New Delhi, 110070, India; bDepartment ofBiotechnology, Institute of Engineering and Technology, G.B.Technical University, Sitapur Road, Lucknow 226021, India*Email: [email protected], Phone: +91 11-261-22222,Fax: +91 11-261-22874

The rapid onset of resistance to new drugs andemergence of antibiotic resistant bacteria has led toresurgence in life-threatening bacterial infections.These problems have revitalized interest in antibioticsand lead to new research. To gain further insightbetween structural and biological activity of Clostrid-ium perfringens, a gram-positive anaerobe responsiblefor food poisoning, mynecrosis in wound infections,and enterotoxemia in humans. We have consideredvarious in silico approaches for developing new drugleads, based on small ligand structure. The importanceof neuraminidases in the virulence of C. perfringensmakes it a potent target for the studies of drugdesigning against this microbe. Natural products ortheir direct derivatives play crucial roles in many dis-eases. In the present study, 3D QSAR analysis usingkNN-MFA method was performed on a series ofpterocarpan derivatives as Clostridial neuraminidaseinhibitors. Twenty-five compounds using random selec-tion and sphere exclusion method for the division ofdataset into training and test set were chosen. kNN-MFA methodology with stepwise, simulated annealingand genetic algorithm was used for model buildingand four predictive models have been generated. Themost significant model has a high internal predictivityof 64.80% (q2 = 0.6480) and an external predictivityof 95.46% (r2 = 0.9546). Model showed that electro-static and steric interactions play important role indetermining neuraminidase inhibitory activity. ThekNN-MFA plots provide further understanding of therelationship between structural features of substitutedpterocarpan derivatives and their activities which wereapplied for designing new compounds as inhibitors.The drug likeliness of these compounds was checkedthrough ADME property prediction and their interac-tion with the neuraminidase was checked by molecu-lar docking studies. Moreover, analysis of protein–protein interaction network of NanI in C. perfringenswas done.


156 A cutting-edge to drug discoveryin Cancer; Cyclins as novel,targets - an in silico technique

P. Sarita Rajender, K. Bhargavi, D. Ramasree andV. Uma*

Molecular Modelling Research Laboratory, Department ofChemistry, Nizam College, Osmania University, Basheerbagh,Hyderabad 500 001, India.*Email: [email protected].

Cyclins are requisite factors operating in eukaryotic cellcycle. Disorientation of their function leads to tumourgen-esis (M. Stamatakos et al., 2010). Various cyclins bind tocyclin-dependent kinases (CDK/ cyclin complexes) as cru-cial regulators during transcription and mRNA processingthat operates between G0, G1, S, G2 and M phases of cellcycle (Loyera et al., 2005). The overview on the role andfunction of the cyclins in the cell cycle is presented in thecurrent article. The cyclins – Cyclin C and Cyclin D2 –were taken up to arrest the initial stages of cell cycleprogression by designing new chemical entities for can-cer therapy. The structure of the cyclin proteins havebeen modelled by homology modelling and validated.Active site for the cyclin structures were identified andsubjected to virtual screening using various databanks. Aset of new lead molecules were identified (Sarita Rajend-er et al., 2011, Bhargavi et al., and 2010). The leadswere prioritised, synthesised and tested for activity. Thenew leads showed promising cancer activity.

ReferencesBhargavi, K., Chaitanya, K. P., Ramasree, D., Vasavi, M., Mur-

thy, D. K., & Uma, V. (2010). Homology modeling anddocking studies of human Bcl-2L10 protein. Journal ofBiomolecular Structure and Dynamics, 28, 379–391.

Loyera, P. B., Trembleya J. H., Katonac R., Kidda V. J., & LahtiaJ. M. (2005). Role of CDK/cyclin complexes in transcriptionand RNA splicing. Cellular Signalling, 17, 1033–1051.

Sarita Rajender, P., Vasavi, M., & Vuruputuri, U. (2011).Identification of novel selective antagonists for cyclin Cby Homology modeling and virtual screening. Interna-tional Journal of Biological Macromolecules, 48, 292–300.

Stamatakos, M., Palla, V., Karaiskos, I., Xiromeritis, K., Alex-iou, I., Pateras, I., & Kontzoglou, K. (2010). Cell cyclins:Triggering elements of cancer or not? World Journal ofSurgical Oncology, 8, 111–119.

157 Identification of intein inhibitorsas novel anti-microbials withrelevance to tuberculosis

Seth Pearsona, Brian Callahanb, Georges Belforta andMarlene Belfortb

aHoward P. Isermann Department of Chemical andBiological Engineering, The Center for Biotechnology andInterdisciplinary Studies, Rensselaer Polytechnic Institute,Troy, NY 12180, USA; bDepartment of Biological Sciences,The RNA Institute, University at Albany, Albany, NY 12222,USA*Email: [email protected], Phone: +1 518-591-8833,Fax: +1 518-437-4445

Inteins are naturally occurring protein elements thatautocatalytically excise themselves from a nonfunc-tional precursor and ligate the flanking proteinsegments with a peptide bond, resulting in a func-tional protein. Inteins interrupt three proteins essentialfor the viability of Mycobacterium tuberculosis. Pre-venting intein splicing, and thus, the formation offunctional post-processed proteins suggests that inteininhibition may be used as a novel antimycobacterialstrategy (M. Belfort, US Patent, 5795,731). Due tothe growing problem of multiple drug-resistant tuber-culosis infections, such alternatives to traditional anti-biotic regimens are especially appealing. It has beenshown that cisplatin, an FDA approved anticancerdrug, is a potent inhibitor of intein splicing, bothin vitro and in vivo (Zhang et al., (2011) JBC, 286,1277). Due to its high toxicity, however, cisplatinhas limited clinical value as an antimycobacterial.Several cisplatin analogs were selected for furtherstudy using an in vitro fluorescent reporter splicingassay in an effort to identify compounds that retainedpotent inhibitory activity while minimizing the toxic-ity associated with cisplatin. An in vitro inhibitor,more potent than cisplatin, was identified. Structuraland biochemical experiments are ongoing to gaininsight into the mechanism of the action of theseplatinum compounds which will lay the groundworkfor a potential de novo design of novel antimicrobi-als.


158 A molecular dynamics simulation-based prediction of deleteriousangiogenin mutations causingamyotrophic lateral sclerosis

Aditya K. Padhia, B. Jayarama,b,c and James Gomesa

aIndian Institute of Technology Delhi, Kusuma School ofBiological Sciences, Hauz Khas, New Delhi, 110016, India;bDepartment of Chemistry, Indian Institute of TechnologyDelhi, Hauz Khas, New Delhi, 110016, India; cSupercomputingFacility for Bioinformatics and Computational Biology, IndianInstitute of Technology Delhi, Hauz Khas, New Delhi, 110016,India*Email: [email protected], [email protected],[email protected]

Amyotrophic lateral sclerosis (ALS) is a fatalneurodegenerative disorder characterized by the selec-tive death of motor neurons leading to paralysis anddeath between 3–5 years of diagnosis. Through wholegenome association studies, several single nucleotidepolymorphisms (SNPs) encoding missense mutations inangiogenin (ANG) protein have been identified as oneof the primary factors causing ALS. Structural studiesof ANG show that catalytic triad comprising His13,Lys40, and His114 residues imparts ribonucleolyticactivity while nuclear localization signal residues31RRR33 are responsible for nuclear translocation activ-ity. Loss of either ribonucleolytic activity or nucleartranslocation activity or both of these functions due tomutations cause ALS. However, the mechanisms ofloss-of-functions of ANG mutants are not completelyunderstood. Here, we present a cohesive and compre-hensive picture of functional loss mechanisms of allknown ALS-associated ANG mutants by extensivemolecular dynamics (MD) simulations (Padhi, Kumar,Vasaikar, Jayaram, & Gomes, 2012; AK, 2013). Ourstudies show that conformational switching of catalyticresidue His114 is responsible for the loss of ribonucle-olytic activity while reduction in solvent-accessible sur-face area (SASA) of 31RRR33 as a result of localfolding is responsible for the loss of nuclear transloca-tion activity (Padhi et al., 2012; AK, 2013). Our pre-diction of loss-of-functions of 17 ANG mutantscorrelated positively with the reported experimentalresults. We have subsequently developed a fast molec-ular dynamics method based on certain global attri-butes / dynamic markers that can be used to determinewhether a mutation is deleterious or benign. To makeour method accessible to researchers and clinicians, wecreated a web server-based tool, ANGDelMut, freelyavailable at http://bioschool.iitd.ernet.in/research.htm,where a user can submit new mutations to ascertainwhether they cause ALS. We hope that our methodwill benefit the community at large and will pave the

way for the development of a successful therapy forpatients suffering from ALS.

ReferencesPadhi, A. K., Kumar, H., Vasaikar, S. V., Jayaram, B., &

Gomes, J. (2012). Mechanisms of loss of functions ofhuman angiogenin variants implicated in amyotrophiclateral sclerosis. PLoS One, 7(2), e32479.

Padhi, A.K., Jayaram, B., & Gomes, J. (accepted for publica-tion). Prediction of functional loss of human angiogeninmutants associated with ALS by molecular dynamicssimulations. Scientific Reports (NPG).

159 Computational approach foroptimizing inhibitors of 1HWK(HMG-CoA reductase)

Ramakrishna Annabhimoju and Uma Vuruputuri*

Molecular Modelling Research Laboratory, Department ofChemistry, Nizam College, Osmania University, Basheerbagh,Hyderabad, 500 001, India*Email: [email protected]

Coronary heart disease is a leading cause of death, andrepresents an increasing burden on healthcare resourcesworldwide. It is one of the most investigated diseasesin Medicinal Chemistry. HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-CoA reductase or HMGCR)is the rate-controlling enzyme of the mevalonate path-way, the metabolic pathway that produces cholesteroland other isoprenoids (Istvan et al., 2001). Normallyin mammalian cells this enzyme is suppressed by cho-lesterol derived from the internalization and degrada-tion of low density lipoprotein (LDL) via LDLreceptor as well as oxidized species of cholesterol.This enzyme is thus the target of the widely availablecholesterol-lowering drugs collectively known as statins(Da Silva et al., 2008). In the present study, crystalstructure of HMG-coA 1HWK was prepared. Activesite was identified. A virtual screening was performed(Schrödinger, LLC, New York, NY) against liganddata-set prepared from different literature search andZINC (Irwin et al., 2012) database to identify the leadmolecules. These lead molecules were optimized usingmolecular dynamics and show good docking scorethan statins.

ReferencesDa Silva, V. B., Taft, C. A., & Silva, C. H. (2008). Use of vir-

tual screening, flexible docking, and molecular interactionfields to design novel HMG-CoA reductase inhibitors forthe treatment of hypercholesterolemia. Journal of PhysicalChemistry, 112, 2007–2011.


Irwin, Sterling, Mysinger, Bolsatd, & Coleman. (2012). ZINC:A free tool to discover chemistry for biology. Journal ofChemical Information.

Istvan, E. S., & Deisenhofer, J. (2001). Structural mechanismfor statin inhibition of HMG-CoA reductase. Science, 292,1160–1164.

Schrödinger, L. L. C. (2011). Glide, version 5.7. New York, NY.

160 A dynamic model for the linkerhistone

Stefjord Todolli* and Wilma K. Olson

BioMaPS Institute for Quantitative Biology, Rutgers University,Piscataway, NJ 08854, USA*Email: [email protected], Phone: +1 732-445-4619

Linker histones play an important role in the packingof chromatin. This family of proteins generally consistsof a short, unstructured N-terminal domain, a centralglobular domain, and a C-terminal domain (CTD). TheCTD, which makes up roughly half of the protein, isintrinsically disordered in solution but adopts a specificfold upon interaction with DNA (Fang et al., 2012).While the globular domain structure is well character-ized, the structure of the CTD remains unknown.Sequence alignment alone does not reveal any signifi-cant homologs for this region of the protein. Construc-tion of a model thus requires additional information.For example, the atomic model for the rat histone H1dCTD, proposed over a decade ago, used novel bioin-formatics tools and biochemical data (Bharath et al.,2002). New fluorescence resonance energy transfer(FRET) studies of the folding of the CTD in the pres-ence of linear DNA, single nucleosomes, and oligonu-cleosomal arrays (Caterino et al., 2011; Fang et al.,

2012) have stimulated our interest in constructing adynamic model of the protein. We have obtained preli-minary information about the structure and dynamicsof the linker histone CTD through ab initio foldingsimulations using the Rosetta modeling package (Rohlet al., 2004). By analyzing a large number of confor-mations sampled through a Monte Carlo procedure, weget a clearer picture of the preferred states of the pro-tein and its dynamics. Our results show that the CTDmay frequently adopt a structure with 3–5 helices andhelix-turn-helix motifs in specific regions. Some of thebest scoring structures show high similarity with theHMG-box-containing proteins previously used as tem-plates by Bharath et al. Further clustering analysis ofour results hints of a preferred set of conformationsfor the CTD of the linker histone. Comparison ofthese models with distances measured by FRET mayhelp account for the distinct structures of the CTDobserved upon binding to different macromolecularpartners.

ReferencesBharath, M. M., Chandra, N. R., & Rao, M. R. (2002). Predic-

tion of an HMG-box fold in the C-terminal domain of his-tone H1: Insights into its role in DNA condensation.Proteins, 49, 71–81.

Caterino, T. L., Fang, H., & Hayes, J. J. (2011). Nucleosomelinker DNA contacts and induces specific folding of theintrinsically disordered H1 carboxyl-terminal domain.Molecular Cell Biology, 31, 2341–2348.

Fang, H., Clark, D. J., & Hayes, J. J. (2012). DNA andnucleosomes direct distinct folding of a linker histone H1 C-terminal domain. Nucleic Acids Research, 40, 1475–1484.

Rohl, C. A., Strauss, C. E. M., Misura, K. M. S., & Baker,D. (2004). Protein structure prediction using Rosetta.Methods in Enzymology: Numerical Computer Methods,383, 66–93.


161 Discovery of potent KdsAinhibitors of Leptospirainterrogans through homologymodeling, docking, and moleculardynamics simulations

Dibyabhaba Pradhan*, Vani Priyadarshini,Manne Munikumar, Sandeep Swargam andAmineni Umamaheswari

Department of Bioinformatics, Bioinformatics Centre,Sri Venkateswara Institute of Medical Science University,Tirupati, Andhra Pradesh 517507, India*Email: [email protected], Phone: +91 877-2287727

Leptospira interrogans is the foremost cause of humanleptospirosis. Discovery of novel lead molecules forcommon drug targets of more than 250 Leptospiraserovars is of significant research interest. Lipopolysac-charide (LPS) layer prevent entry of hydrophobicagents into the cell and protect structural integrity ofthe bacterium. KDO-8-phosphate synthase (KdsA) cata-lyzes the first step of KDO biosynthesis that leads toformation of inner core of LPS. KdsA was identifiedas a potential drug target against Leptospira interro-gans through subtractive genomic approach, metabolicpathway analysis, and comparative analysis (Amineniet al., 2010). The present study rationalizes a system-atic implementation of homology modeling, docking,and molecular dynamics simulations to discover potentKdsA inhibitors (Pradhan et al., 2013; Umamaheswariet al., 2010). A reliable tertiary structure of KdsA incomplex with substrate PEP was constructed based onco-crystal structure of Aquifex aeolicus KdsA synthasewith PEP using Modeller9v10. Geometry-based analogsearch for PEP was performed from LigandInfo data-base to generate an in house library of 352 ligands.

The ligand data-set was docked into KdsA active sitethrough three-stage docking technique (HTVS, SP, andXP) using Glidev5.7. Thirteen lead molecules werefound to have better binding affinity compared to PEP(XP Gscore =�7.38 kcal/mol; Figure 1). The best leadmolecule (KdsA- lead1 docking complex) showed XPGscore of �10.26 kcal/mol and the binding interactions(Figure 2) were correlated favorably with PEP–KdsAinteractions (Figure 1). Molecular dynamics simulationsof KdsA– lead1 docking complex for 10 ns hadrevealed that the complex (Figure 3) remained stablein closer to physiological environmental condition. Thepredicted pharmacological properties of lead1 werewell within the range of a drug molecule with goodADME profile, hence, would be intriguing towardsdevelopment of potent inhibitor molecule against KdsAof Leptospira.

This research is supported by DBT, Ministry of Science& Technology, BIF program, Govt. of India (No. BT/BI/25/001/2006).

ReferencesAmineni, U., Pradhan, D., & Marisetty, H. (2010). In silico

identification of common putative drug targets in Lepto-spira interrogans. Journal of Biological Chemistry, 3, 165–173.

Pradhan, D., Priyadarshini, V., Munikumar, M., Swargam, S.,Umamaheswari, A., & Aparna, B. (in press). Para-(ben-zoyl)-phenylalanine as a potential inhibitor against LpxC ofLeptospira spp.: Homology modeling, docking andmolecular dynamics study. Journal of Biomolecular Struc-ture Dynamics. http://dx.doi.org/10.1080/07391102.2012.758056.

Umamaheswari, A., Pradhan, D., & Hemanthkumar, M.(2010). Identification of potential Leptospira phosphohep-tose isomerase inhibitors through virtual high-throughputscreening. Genomics Proteomics Bioinformatics, 8, 246–255.


162 Dominant forces in proteinfolding

Arieh Ben-Naim*

Department of Physical Chemistry, The Hebrew University ofJerusalem, Jerusalem, 91904, Israel*Email: [email protected], Phone: (00972) 2-6454554

In the first part of this talk, I will discuss the need fora paradigm shift from hydrophobic (H/O) to a hydro-philic ((H/I) based theory of protein folding. Next, Iwill discuss the various types of solvent-induced forcesthat are exerted on various groups on the protein. It isargued, both theoretically and by simulations, that theH/I–H/I solvent-induced forces are likely to be thestrongest. Therefore, it is suggested that these forcesare also the forces that force the protein to fold, in ashort time, along a narrow range of pathways. Thisparadigm shift also answers Levinthal’s question aboutthe factors that “speed” and “guide” the folding ofproteins.

ReferencesBen-Naim, A. (2011). Molecular theory of water and aqueous

solutions; part II: The role of water in protein folding selfassembly and molecular recognition. Singapore: World Sci-entific.

Ben-Naim, A. (2012). Levinthal’s question revisited, andanswered. Journal of Biomolecular Structure and Dynam-ics, 30, 113–124. doi:10.1080/07391102.2012.674286.

Ben-Naim, A. (2013). The protein folding problem and its solu-tions. Singapore: World Scientific.

Levinthal, C. (1969). How to fold graciously. Mössbauer Spec-troscopy in Biological Systems Proceedings, 67, 22–26.

163 Enhanced sampling of peptidesand proteins with a new biasingreplica exchange method

Katja Ostermeir* and Martin Zacharias

Physics Department T38, Technische Universität München,James-Franck-Str.1, Garching 85748, Germany*Email: [email protected], Phone: +49 89-289–12731,Fax:+49 89-289-12444

Classical MD simulations (cMD) are limited by thesampling of relevant states of the peptides. Replicaexchange (REMD) methods aim to search the confor-mational space of proteins more efficiently (reviewed inOstermeir & Zacharias, 2013). We have developed aHamiltonian REMD method that takes advantage of anintrinsic property of proteins, the specific Φ ϕ dihedralangle combinations along the polymer backbone. Byemploying a coupled two-dimensional biasing potential

the energy barriers along the polymer backbone arereduced more effectively than by a previous approachbased on a one-D biasing potential (Kannan & Zacha-rias, 2007). Thus, adjacent amino acids along the poly-mers backbone can easily switch between favourableregions in the Ramachandran plot. Additionally, energybarriers of rotameric states of amino acid side chains ofproteins are also biased in the replica runs. The methodimproves the sampling of conformational substates ofproteins at a modest number of replicas (nine replicasin the standard set-up with one replica running withoutbiasing potential) compared to much larger numbersnecessary in the case of standard temperature (T)-REMD simulations. A further improvement is achievedby a dynamical adjustment of the penalty potential lev-els in the replicas such that high exchange rates andimproved mixing of conformations between differentreplicas are guaranteed. The biasing potential (BP)-REMD method turns out to be suitable to speed upboth the folding of spaghetti-like test peptides and therefinement of loop decoy structures. Starting fromextended structures, an α-helical oligo-alanine and β-hairpin chignolin and the Trp-cage protein fold morerapidly in near-native structures than in cMD simula-tions. The BP-REMD simulations not only acceleratethe folding process of test proteins but also enlarge thevariety of sampled configurations in conformationalspace. Since flexible parts of the protein can be penal-ized selectively, this method provides a precise tool toinvestigate regions of interest of the protein.

ReferencesGabrielian, A., Bocharova, T. N., Smirnova, E. A., Volodin, A.

A., & Harutjunyan, G. (2011). Strand exchange reactionbetween short oligonucleotides promoted by a derivative of1,3-diazaadamantane. Journal Biomolecular Structure &Dynamics, 27(6), 1124–1125.

Kim, W. J., Cato, Y., Akaike, T., & Maruyama, A. (2003). Cat-ionic comb-type copolymers for DNA analysis. NatureMaterials, 2, 815–820.

Bocharova, T. N., Smirnova, E. A., Volodin, A. A. (2012). Lin-ker histone H1 stimulates DNA strand exchange betweenshort oligonucleotides retaining high sensitivity to heterol-ogy. Biopolymers, 97, 229–239.

164 Extracting dynamics informationfrom multiple structures

Robert L. Jernigan*, Jie Liu, Kannan Sankar, Kejue Jiaand Michael T. Zimmermann

Department of Biochemistry, Biophysics and Molecular Biology,Baker Center for Bioinformatics and Biological Statistics, IowaState University, Room 115, Office and Lab Building, Ames, IA,50011-3020, USA*Email: [email protected], Phone: +1 515-294-3833,Fax: +1 412-294-3841


Meaningful dynamics information can be extracted frommultiple experimental structures of the same, or closelyrelated, proteins or RNAs. The covariance matrix ofatom positions is decomposable into its principal compo-nents, and in this way, it is possible to rank-order thechanges in the set of structures, and to determine whatthe most significant changes are. Usually, only a fewprincipal components dominate the motions of thestructures, and these usually relate to the functionaldynamics. This dynamics information provides strongevidence for the plasticity of protein and RNA struc-tures, and also suggests that these structures almost

always have a highly limited repertoire of motions. Insome cases, such as HIV protease, the dominant motionsare opening and closing over the active site. For myoglo-bin, the changes are much smaller, reflecting in part thesmall changes in sequence, but nonetheless they showcharacteristic details that depend on the species. Sets ofstructures can also be used to derive the effective micro-scopic forces that are forcing a given conformationaltransition.

This research is supported by NIH R01GM072014.

165 Protein domains: athermodynamic definition

Lauren L. Portera,b and George D. Rosea*aDepartment of Biophysics, Johns Hopkins University,3400 N. Charles Street, Baltimore, MD 21218, USA; bPotomacAffinity Proteins, Institute for Bioscience and BiotechnologyResearch, 9600 Gudelsky Dr, Rockville, MD 20850,USA*Email: [email protected], Phone: +1 410-516-7244,Fax: +1 410-516-4118

Protein domains are conspicuous structural units inglobular proteins, and their identification has been atopic of intense biochemical interest dating back to theearlier crystal structures. Numerous disparate domainidentification algorithms have been proposed, all

involving some combination of visual intuition and/orstructure-based decomposition. Instead, we present arigorous thermodynamically based approach that rede-fines domains as cooperative chain segments. In greaterdetail, most small proteins fold with high cooperativity,meaning that the equilibrium population is dominatedby completely folded and unfolded molecules, with anegligible subpopulation of partially folded intermedi-ates. Here, domains are equated to chain segments thatretain full cooperativity when excised from their parentstructures. Implementing this approach computationally,the domains in a large representative set of proteinswere identified; all exhibit consistency with experimen-tal findings. Our reframed interpretation of a proteindomain transforms an indeterminate structural phenome-non into a quantifiable molecular property, grounded insolution thermodynamics.


166 Millisecond-long moleculardynamics simulations ofproteins on a special-purposemachine

David E. Shaw

Research and Center for Computational Biology and Bioinfor-matics, Columbia University, 120 W. 45th Street, 39th Floor,New York, NY 10036, USA*Email: [email protected],Phone: +1 212-478-0260, Fax: +1 212-845-1286

Molecular dynamics (MD) simulation has long beenrecognized as a potentially powerful tool for understand-ing the structural, dynamic, and functional characteristicsof proteins at an atomic level of detail. Many biologi-cally important phenomena, however, occur over time-scales that have previously fallen far outside the reach ofMD technology. We have constructed a specialized,massively parallel machine, called Anton, that is capableof performing all-atom simulations of proteins in anexplicitly represented solvent environment at a speedroughly two orders of magnitude beyond that of theprevious state of the art. Using novel algorithms devel-oped within our lab, the machine has now simulated thebehavior of a number of proteins for periods as long astwo milliseconds – approximately 200 times the lengthof the longest such simulation previously published.Such simulations have allowed us to observe and analyzekey characteristics of the dynamics of proteins (includingcentral elements of the process of protein folding) thatwere previously inaccessible to both computational andexperimental study.

167 Network properties of decoy andCASP predicted models: acomparison with native proteinstructures

S. Chatterjee, S. Ghosh and S. Vishveshwara*

Molecular Biophysics Unit, Indian Institute of Science, Bangalore,560012, India*Email: [email protected], Phone: +91 80-22932611,Fax: +91 80-23600535

Principles that govern protein folding still remainelusive. Given the huge sequence space, it is reasonableto assume that sequences follow a particular pattern toattain one of the folds already defined in the relativelysmall structural space. In this study, we have used

protein structure networks at different interactionstrengths of non-covalent interactions (Imin) (Brinda &Vishveshwara, 2005; Kannan & Vishveshwara, 1999), toidentify patterns that can distinguish a native proteinfrom decoy/modelled structures. This is a rigorous exten-sion of an earlier study performed at IminP 0% (Chatter-jee, Bhattacharyya et al., 2012). Network properties suchas the size of the largest cluster (SLClu), largest k-2communities (ComSk2) and clustering coefficients(CCoe) are analysed for 5422 native structures and29543 decoy/modelled structures. Steeper transition pro-file of the native structures as a function of Imin is con-sistently observed (see Figure)

The network properties generated at different Imin andmain-chain hydrogen bonds (MHB) are integrated intosupport vector machine to build a classifier, giving anaccuracy of 94.11%. The uniqueness of the proteinstructures through side-chain interactions are capturedby the network parameters, while MHB represents thebackbone packing. Quality predictions for the recentlyconcluded CASP 10 predicted models are also per-formed using the model with the selected ones show-ing RMSD values < 2.5Å with respect to the nativestructures. Amongst the network properties, ComSk2 ismaximally able to capture the transition properties ofthe structures. Importance of ComSk2 has earlier beenimplicated to capture the percolating behaviour of aprotein structure (Deb & Vishveshwara, 2009). Overall,a robust classifier is obtained, and patterns specific tonative structures have been analysed and discussed.The study highlights the importance of side-chaininteractions at different Imins, along with backbonelevel interactions.

S.V acknowledges the Council of Scientific and Indus-trial Research (CSIR, India) for Emeritus professorship.


ReferencesBrinda, K., & Vishveshwara, S. (2005). A network representa-

tion of protein structures: Implications for protein stability.Biophysical Journal, 89, 4159.

Chatterjee, S., Bhattacharyya, M., et al. (2012). Network prop-erties of protein-decoy structures. Journal of BiomolecularStructure and Dynamics, 29, 1110–1126.

Deb, D., & Vishveshwara, S. (2009). Understanding proteinstructure from a percolation perspective. Biophysical Jour-nal, 97, 1787–1794.

Kannan, N., & Vishveshwara, S. (1999). Identification ofside-chain clusters in protein structures by a graphspectral method. Journal of Molecular Biology, 292,441–464.

168 NMR study of RQC domain ofBLM protein

Vee Vee Cheong, Brahim Heddi and Anh Tuân Phan

School of Physical and Mathematical Sciences, NanyangTechnological University, Singapore 637371*Email: [email protected], **[email protected],Phone: +65 6514-1915, Fax: +65 6795-7981

BLM, a member of the RecQ helicase associated withthe Bloom’s syndrome human genetic disorder, hasbeen found to bind to noncanonical DNA with highaffinity via its RecQ C-terminal domain (RQC). Usingmulti-dimensional NMR spectroscopy, we have deter-mined the solution structure of BLM RQC, and foundthat BLM RQC retains the overall winged-helix motifpreviously observed for other RQC proteins. Compari-son between BLM RQC and the RQC domain of itshomologue, Werner syndrome protein (WRN RQC),revealed two major structural differences. Firstly, BLMRQC contains an extended 14-residue insertion forminga flexible loop between two first α-helices, only foundin BLM RQC and not other RQC proteins. Secondly,in contrast to the third α-helix of WRN RQC, anunstructured loop was observed for this region ofBLM RQC.

169 Polymorphic assembly ofcomputationally designedhydrophobic-rich collagenpeptides

Kenneth N. McGuinness* and Vikas Nanda

Department of Biochemistry, UMDNJ/Rutgers University,Center for Advanced Biotechnology and Medicine, 679 HoesLane, Piscataway, NJ 08854, USA*Email: [email protected],Phone: +1 928-925-7693,Fax: +1 732-235-5318

We seek to understand how the position and length ofhydrophobic content within a collagen peptidesequence dictates morphology of self-assembly. Wemodeled collagen assembly using diffusion limitedaggregation1 (DLA) (Parkinson et al. 1995). of discret-ized, rigid rods composed of hydrophilic and hydro-phobic spheres. Simulations predicted that the inclusionof short hydrophobic domains should direct the assem-bly of lamellar structures. We designed a set of colla-gen peptide sequences with six, five and fourcontiguous nonpolar residues. Electron microscopy ofaggregates revealed the peptide with six nonpolar resi-dues self-assembled into uniform fibrils and the pep-tide with five residues assembled into both fibrils andplates, while including four hydrophobic residues thatformed only plates. This polymorphic behavior can beexplained by packing models of rod vs. screw-like-par-ticles

This research has been supported by NSF DMR-0907273and NIH DP2-OD-006478-1.

ReferenceParkinson, J., Kadler, K. E., & Brass, A. (1995). Simple physi-

cal model of collagen fibrillogenesis based on diffusionlimited aggregation. Journal of Molecular Biology, 247,823–831.

four aliphatic amino acidsfive nonpolar residuessix hydrophobic side chains


170 GMPC model and the helix–coiltransition in biopolymers

V.F. Morozova*, Sh.A. Tonoyana, L.V. Melkonyana

N.L. Poghosyana and Sh.A. Sargsyanb

aDepartment of Molecular Physics, Yerevan State University,Yerevan, Armenia; bDepartment of Medical Physics, YerevanState Medical University, Yerevan, Armenia*Email: [email protected]

The Hamiltonian of the generalized model of polypeptidechain (GMPC) is introduced to describe the system inwhich the conformations are correlated over some dimen-sional range Δ. The Hamiltonian does not contain anyparameter designed especially for helix–coil transition anduses pure molecular microscopic parameters (the energyof hydrogen bond formation, reduced partition function ofrepeated unit, the number of repeated units fixed by onehydrogen bond, the flexibility of chain, the energies ofinteraction between the repeated units and the solvent mol-ecules) (Badasyan et al., 2005, 2004). We evaluate the par-tition function using transfer-matrix approach. Wedescribe the influence of solvent interaction with biopoly-mer, both with competing and noncompeting for hydrogenbond formation ways. On handling the problem of solventinfluence on helix–coil transition, we obtained dependenceon energy of solvent–macromolecule interaction, how sol-vents change correlation length, transition temperature,and interval. We obtained that two type interaction of sol-vent results in low temperature coil–helix transition, whichwe connect with cold denaturation. A consistent inclusionof osmotic pressure effects in a description of helix–coiltransition for poly(L-glutamic acid) in solution with poly-ethylene glycol can offer an explanation of the experimen-tally observed linear dependence of transition temperatureon osmotic pressure as well as the concurrent changes inthe cooperativity of the transition (Badasyan et al., 2012).We also took into account two biopolymers’ side-by-sideinteractions. In the case of effective repulsion; the shape ofthe melting curve is two-phase with high and wide correla-tion length in a plateau on denaturation curve (Badasyanet al., 2009). We also took into account structural hetero-geneity of biopolymers using constrained annealingapproximation (Serva & Paladin, 1993).

ReferencesBadasyan, A. V., Grigoryan, A. V., Mamasakhlisov, E. Sh.,

Benight, A. S., & Morozov, V. F. (2005). The helix-coiltransition in heterogeneous double stranded DNA: Microca-nonical method. Journal of Chemical Physics, 123, doi:10.1063/1.2727456

Badasyan, A. V., Hayrapetyan, G. N., Tonoyan, Sh. A., Mam-asakhlisov, Y. Sh., Benight, A. S., & Morozov, V. F.(2009). Intersegment interactions and helix-coil transitionwithin the generalized model of polypeptide chainsapproach. Journal of Chemical Physics, 131, doi: 10.1063/1.3216564

Badasyan, A. V., Tonoyan, Sh. A., Giacometti, A., Podgor-nik, R., Parsegian, V. A., Mamasakhlisov, Y. Sh., &Morozov, V. F. (2012). Osmotic Pressure Induced Cou-pling between Cooperativity and Stability of a Helix-CoilTransition. Physical Review Letters, 109, doi: 10.1103/PhysRevLett.109. 068101

Morozov, V. F., Badasyan, A. V., Grigoryan, A. V., Sahakyan,M. A., & Mamasakhlisov, Y. Sh. (2004). Stacking andhydrogen bonding: DNA cooperativity at melting. Biopoly-mers, 75, 434–439.

Serva, M., & Paladin, G. (1993). Gibbs thermodynamic poten-tials for disordered systems. Physical Review Letters, 70,105–108.

171 Protein structure prediction withlimited data

Alberto Perez, Justin MacCallum and Ken Dill

Laufer Center for Physical and Quantitative Biology, StonyBrook University, Stony Brook, NY 11794, USA*Email: [email protected]

Proteins need to interact with other molecules in orderto carry out their biological role. Knowing the proteinstructure is crucial to study these interactions and it canfor example lead to drug design. However, the cost ofdetermining the structure of a protein with currentexperimental techniques is very high – both in timeand money. In the absence of experimental structures,current computational tools are not always able tocorrectly predict the native fold. We are using a physicsbased computational framework to determine the struc-ture of proteins. The pipeline is designed to handlesparse data coming from evolution, bioinformatics orexperiments (solid state NMR, cross linking, …). Thedata is transformed into a set of restraints used in ourphysical simulations. However, we require that only asubset of the input information is satisfied. This is doneto account for uncertainties in the input data. Theframework uses a Hamiltonian-temperature replicaexchange formalism that allows the system to choosewhat data is compatible with the physics of the system.I will show some results on how this methodology canhelp us in both protein structure refinement and proteinstructure prediction.


172 Role of conserved water moleculartriad in the recognition of IMP,NAD+ with Asp 274, Asn 303, Arg322, and Asp 364 in both theisoform of hIMPDH

Deepak K. Mishra, Hridoy R. Bairagya andBishnu P. Mukhopadhyay*

Department of Chemistry, National Institute of Technology,Durgapur 713209, India*Email: [email protected], Phone: +91 343-254-6397,Fax: +91 343-254-7375

Inosine monophosphate dehydrogenase (IMPDH) playsan important role in the Guanosine monophosphate(GMP) biosynthesis pathway. As hIMPDH-II is involvedin CML-Cancer, it is thought to be an active target forleukemic drug design. The importance of conservedwater molecules in the salt-bridge-mediated interdomainrecognition and loop-flap recognition of hIMPDH hasalready been indicated in some simulation studies (Bai-ragya et al., 2009, 2011a, 2011b, 2012; Mishra et al.,2012). In this work, the role of conserved water mole-cules in the recognition of Inosine monophosphate (IMP)and NAD+ (co-factor) to active site residues of both theisoforms has been investigated by all atoms MD-Simula-tion studies. During 25-ns dynamics of the solvated hIM-PDH-II and I (1B3O and 1JCN PDB structures), theinvolvement of conserved water molecular triad (WM, WL

and WC) in the recognition of active site residues (Asp274, Asn 303, Arg 322, and Asp 364), IMP and NAD+

has been observed (Figure 1). The H-bonding co-ordina-

tion of all three conserved water molecular centers iswithin 4–7 and their occupation frequency is 1.0. The H-bonding geometry and the electronic consequences ofthe water molecular interaction at the different residues(and also IMP and NAD+) may put forward somerational clues on antileukemic agent design

This research has been supported by Department ofBiotechnology, Govt. of India.

ReferencesMishra, D. K., Mukhopadhyay, B. P., & Bairagya, H. R.

(2012). Molecular modeling of Inosine 5’- monophosphate-dehydrogenase-II (human) structure using MD-simulation-method. International Journal of Pharmaceutical BioScience, 3, (B) 102–120.

Bairagya, H. R., & Mukhopadhyay, B. P. (2012). An insight tothe dynamics of conserved water-mediated salt bridge inter-action and interdomain recognition in hIMPDH isoforms.Journal of Bimolecular Structure and Dynamics. doi:10.1080/07391102.2012.712458

Bairagya, H. R., Mukhopadhyay, B. P., & Bera, A. K. (2011a).Role of salt bridge dynamics in inter domain recognition ofhuman IMPDH isoforms: an insight to inhibitor topologyfor isoform-II. Journal of Bimolecular Structure andDynamics, 29, 441–462.

Bairagya, H. R., Mukhopadhyay, B. P., & Bera, A. K. (2011b).Conserved water mediated recognition and the dynamics ofactive site Cys 331 and Tyr 411 in hydrated structure of humanIMPDH-II. Journal of Molecular Recognition, 24, 35–44.

Bairagya, H. R., Mukhopadhyay, B. P., & Sekar, K. (2009). Aninsight to the dynamics of conserved water molecular triadin IMPDH II (human): recognition of cofactor and substrateto catalytic Arg 322. Journal of Bimolecular Structure andDynamics, 27, 149–158.


173 Role of free cysteines in leptinreceptor

R. Karthicka, Gokul Raghunathb, Sharmila Anishettyb

and R. Malathia*aDepartment of Genetics, University of Madras, Chennai,600113, India; bDepartment of Biotechnology, Anna University,Chennai, 600025, India*Email: [email protected], Phone: +91 44-2454-7062,Fax: +91 44-2454-0709

Leptin, a 16-kDa adipocytic peptide hormone (product ofob gene), is known to play a key role in the control ofbody weight and exerts its influence by binding to itslong-form receptor (Ob-Rb). Ob-Rb belongs to class Icytokine receptor superfamily and consists of an extracel-lular, transmembrane, and an intracellular domain. Cyste-ines including free and disulphide-bonded are known toplay a significant role in recognition of leptin by itsreceptor and are known to be highly conserved in differ-ent organisms including human, macaca, mouse, dog,sheep, zebrafish, and medaca. Recently, the crystal struc-ture of leptin-binding domain of human leptin receptorhas been determined (1). Using the structural data, weanalyzed the role of free cysteines in leptin-bindingdomain of leptin receptor through docking studies usingRosettadock. The conserved free cysteines namely Cys-604 and Cys-613 were mutated to alanines and thisresulted in drastic change in the binding orientation ofleptin and its receptor. Based on computational analysis,we propose that cysteines either free or involved in disul-phide bridges might play a crucial role during signalingand might be the primary determinant of leptin-receptorinteractions, the details of which will be discussed. Cur-rently, understanding the structural basis of leptin and itsbinding to leptin receptor gains much significance since itmight pave the way for designing inhibitors that might beused in controlling obesity.

ReferenceCarpenter, B., Hemsworth, G. R., Wu, Z., Maamra, M.,

Strasburger, C. J., Ross, R. J., & Artymiuk, P. J. (2012).Structure of the human obesity receptor leptin-bindingdomain reveals the mechanism of leptin antagonism by amonoclonal antibody. Structure, 20, 487–497.

174 Self-association prompts proteinsfor new function: The role ofaltered dynamic properties

Shoshana J. Wodaka*, Michael Gartonb,Anatoly Malevanetsa and Stephen McKinnonc

aHospital for Sick Chikdren, Toronto, Canada; bUniversity ofNottingham, UK; cUniversity of Toronto, Canada*Email: [email protected]

Local backbone flexibility along the chain was analyzed inpairs of monomer and homodimeric protein structures withidentical sequence. We identified pairs, where highly flexi-ble regions (corresponding to prominent peaks in rmsd’s orcrystallographic B-factor values) clearly ‘migrate’ to a newlocation along the polypeptide in the homodimer. We pres-ent several systems, where the new flexible region can belinked to the recruitment of a 3rd binding partner, andwhere this recruitment is not reported for the correspondingmonomer. Conformational sampling of these systems overmicrosecond timescales further confirms the flexibilitymigration phenomenon. Compelling evidence is providedthat altered backbone flexibility in the homodimer stateenables specific recruitment of the heterologous bindingpartner through the process of conformational selection.Our findings lend support to conformational selection as ageneral mechanism for protein–ligand binding and highlightthe regulatory allosteric role played by the homomeric asso-ciation event itself. There are many examples of allostericbehavior in homo-oligomers, but this is the first time thathomomer association is shown to be the binding event thatalters binding properties elsewhere in the protein.

175 Structure-based engineering togenerate high-affinityimmunotherapy for the drugof abuse

Shraddha Thakkar, Nisha Nanaware-Kharade,Guillermo Gonzalez III, Reha Celikel, Eric Petersonand Kottayil I. Varughese*

University of Arkansas for Medical Sciences, Little Rock,AR 72205, USA*Email: [email protected], Phone: +1 501-686-7160

Methamphetamine (METH) abuse is a major threat inthe USA and worldwide without any FDA approvedmedications. Anti-METH antibody antagonists block orslow the rate of METH entry into the brain and haveshown efficacy in preclinical studies (Peterson, Laurenz-ana, Atchley, Hendrickson, & Owens, 2008). A keydeterminant of the antibody’s efficacy is its affinity forMETH and we attempted to enhance the efficacy bydesigning mutations to alter the shape or the electrostaticcharacter of the binding pocket. Towards this goal, wedeveloped a single chain anti-METH antibody fragment(scFv6H4) from a parent IgG (1). The crystal structureof scFv-6H4 in complex with METH was determined(Celikel, Peterson, Owens, & Varughese, 2009). Basedon its elucidated binding interactions, we designed pointmutations in the binding pocket to improve its affinityfor METH and amphetamine (AMP), the active metabo-lite of METH. The mutants, scFv-S93T,-I37M and -Y34M were cloned, expressed in yeast and tested for


affinity against METH and AMP. Two mutants showedenhanced binding affinity for METH: scFv-I37M by1.3-fold and scFv-S93T by 2.6-fold. Additionally, all themutants showed increase in affinity for AMP: scFv-I37M by 56-fold, scFv-S93T by 17-fold and scFvY34Mby 5-fold. Crystal structure for one of the high-affinitymutant, scFv-S93T, in complex with METH was deter-mined (Figure 1). Binding pocket of the mutant is morehydrophobic in comparison with the wild type. ScFv-6H4 binds METH in a deep pocket containing twowater molecules. The substitution of a serine residue bya threonine leads to the expulsion of a water molecule(Figure 2), relieving some unfavorable contacts betweenthe hydrocarbon atoms of METH and the water mole-cule and increasing the affinity to sub-nanomolar range.

Therefore, the present study shows that efficacy couldbe enhanced by altering the hydrophobicity or the shapeof the binding pocket.

ReferencesCelikel, R., Peterson, E. C., Owens, S. M., & Varughese, K. I.

(2009). Crystal structures of a therapeutic single chain anti-body in complex with two drugs of abuse-Methamphet-amine and 3,4-methylenedioxymethamphetamine. ProteinScience, 09, 2336–2345.

Peterson, E. C., Laurenzana, E. M., Atchley, W. T., Hendrickson,H. P., & Owens, S. M. (2008). Development and preclinicaltesting of a high-affinity single-chain antibody against (+)-methamphetamine. Journal of Pharmacology and Experi-mental Therapeutics, 08, 124–133.

176 Structure-based virtual screeningtowards identification of potentialFabH inhibitors

Vani Priyadarshini, Dibyabhaba Pradhan,Manne Munikumar, Sandeep Swargam andAmineni Umamaheswari*

Bioinformatics Centre, Department of Bioinformatics,Sri Venkateswara Institute of Medical Sciences University,Tirupati, Andhra Pradesh 517507, India*Email: [email protected], Phone: +91-877-2287727

Infective endocarditis (IE) is a serious form of micro-bial infection of the endocardial surface, lining of theheart chambers and heart valves with a high mortalityrate. Through comparative genomics, subtractivegenomics, and metabolic pathway analysis, 18 commondrug targets were identified (Priyadarshini et al., 2013).In the present study, β-Ketoacyl-acyl carrier proteinsynthase III (FabH), a common protein among eightselected pathogens of IE, was selected for the study.FabH catalyzes the initiation of fatty acid elongation bycondensing malonyl-ACP with acetyl-CoA. FabH is an

essential enzyme for bacterial viability, because of itspivotal roles in both initiation and regulation of thefatty acid biosynthesis. Experimentally determined ter-tiary structure of FabH of Streptococcus mitis (referenceorganism) was not reported yet. Therefore, molecularmodeling of FabH in complex with 2-({[4-bromo-3-(diethylsulfamoyl) phenyl] carbonyl} amino) benzoicacid (B82) was constructed using Modeller9v10 (Fig-ure 1). An in-house library consisting of 23969 struc-tural analogs from 60 available FabH inhibitors wascompiled from Ligand.Info database. Structure-basedvirtual screening was performed through three-stagedocking technique (HTVS, SP, and XP) using Glidev5.7 led to identification of seven lead molecules withbetter binding affinity compared to published inhibitor(XP Gscore �8.268 kcal/mol). Lead1 showed the lowestXP Gscore of �9.953 kcal/mol with strong bindinginteractions with FabH. Molecular dynamic (MD) simu-lations (Priyadarshini et al., 2011) for FabH–lead1docking complex were performed using Desmond v3.0for 10 ns. It revealed that the complex (Figure 1)remained structurally and energetically stable in all2084 trajectories. The docking interactions were also


reproduced during MD simulations. Therefore, lead1would be a potent inhibitor of FabH and ideal fordesigning drug for IE.

VP is highly thankful to ICMR, Ministry of Health &Family Welfare, Govt. of India, for sanctioning seniorresearch fellowship (No. 45/18/2011-BIF/BMS). Authorsare thankful to DBT, Ministry of Science & Technology,Govt. of India, for providing infrastructure through BIFprogram (No. BT/BI/25/001/2006).

ReferencesPriyadarshini, V., Pradhan, D., Munikumar, M., Swargam, S.,

Umamaheswari, A., & Rajasekhar, D. (2011). Docking andmolecular dynamic simulations of Legionella pneumophilaMurB reductase for potential inhibitor design. Biochemistryand Analytical Biochemistry, 1, 101.

Priyadarshini, V., Pradhan, D., Munikumar, M., Swargam, S.,Umamaheswari, A., & Rajasekhar, D. (2013). In silico drugtargets for infective endocarditis. Online Journal of Bioin-formatics, 14, 32–50.

177 T-cell vaccine design forStreptococcus pneumoniae: an insilico approach

Manne Munikumar, Vani Priyadarshini,Dibyabhaba Pradhan, Sandeep Swargam andAmineni Umamaheswari*

Bioinformatics Centre, Department of Bioinformatics, SriVenkateswara Institute of Medical Sciences University, Tirupati517507, Andhra Pradesh, India*Email: [email protected], Phone: +91-877-2287727

Streptococcus pneumoniae (pneumococcus) remains animportant cause of meningitis, bacteremia, acute otitismedia, community acquired pneumonia associatedwith significant morbidity, and mortality world wide.Conjugated polysaccharide, glycoconjugated, and capsu-lar polysaccharide based vaccines were existent forpneumococcal disease but are still specific and restricted

to serotypes of S. pneumoniae. Proteome of eight sero-types of S. pneumoniae was retrieved and identified incommon proteins (Munikumar et al., 2012). 18 mem-brane proteins were distinguished from 1657 commonproteins of eight serotypes of S. pneumoniae. Implement-ing comparative genomic approach and subtractivegenomic approach, three membrane proteins werepredicted as essential for bacterial survival and non-homologous to human (Munikumar et al., 2012; Umama-heswari et al., 2011). ProPred server was used to proposefour promiscuous T-cell epitopes from three membraneproteins and validated through published positive control,SYFPEITHI and immune epitope database (Munikumaret al., in press). The four epitopes docked into peptidebinding region of predominant HLA-DRB alleles withgood binding affinity in Maestro v9.2. The T-cell epitope89-VVYLLPILI-97 and HLA-DRB5⁄0101 docking com-plex was with best XPG score (�13.143 kcal/mol). Fur-ther, the stability of the complex was checked through


molecular dynamics simulations in Desmond v3.3. Thesimulation results had revealed that the complex was sta-ble throughout 5000 ps (Munikumar et al., in press).Thus, the epitope would be the ideal candidate for T-celldriven subunit vaccine design against selected serotypesof S. pneumoniae.

This research was supported by DBT, Ministry ofScience & Technology, BIF program, Govt. of India(No. BT/BI/25/001/2006).

ReferencesMunikumar, M., Priyadarshini, V., Pradhan, D., Swargam, S.,

Umamaheswari, A., & Vengamma, B. (2012). In silicoidentification of common putative drug targets among thepathogens of bacterial meningitis. Biochemistry and Analyt-ical Bioichemistry, 1, 123.

Munikumar, M., Priyadarshini, V., Pradhan, D., Umamahes-wari, A., & Vengamma, B. (in press). Computationalapproaches to identify common subunit vaccine candidatesagainst bacterial meningitis. Interdisciplinary Sciences:Computational Life Sciences.

Umamaheswari, A., Pradhan, D., & Hemanthkumar, M. (2011).Computer aided subunit vaccine design against pathogenicLeptospira serovars. Interdisciplinary Sciences: Computa-tional Life Sciences, 4, 38–45.

178 Targeting “Beta lactamase-C” indevelopment of novel anti-tuberculosis therapeutics

Saugata Hazra* and John S. Blanchard

Department of Biochemistry, Albert Einstein College ofMedicine, Bronx, NY 10461, USA*Email: [email protected], Phone: +1-312-401-5319

Tuberculosis is a common, and in many cases lethal,infectious disease caused by various strains of Mycobac-terium, usually Mycobacterium tuberculosis. (Kumaret al., 2007) In addition, co-infection with Mycobacte-rium tuberculosis and HIV (TB/HIV), especially inAfrica, and multidrug resistant and extensively drug-resistant tuberculosis in all regions, (WHO, 2010) makesit important to develop novel therapeutics against thisbacterium. Penicillin like β-Lactam antibiotics are amongthe most clinically prescribed drugs for anti-bacterialtherapeutics. The general mechanism of action involvedthe inhibition of enzyme d,d-transpeptidases, which takespart in the biosynthesis of the bacterial cell wall (Heese-mann, 1993). A major strategy of bacterial resistance toβ-lactams is the production of β-lactamases that catalyzethe hydrolysis of β-lactams, leading to the inactivation ofthe antibiotics. β-Lactams have not been used in clinicalpractice to treat TB infections, because an active penicil-linase was reported in M. tuberculosis (Lessel, 1996).

BlaC is a class A β-lactamase that contains a nucleo-philic serine residue (Ser70) and shares sequence homol-ogy with the penicillin-binding protein domain of theancestral d,d-transpeptidases. Recent studies includingour lab show that β-lactam drugs like Clavulanate, Carb-apenem, and Meropenem are used primarily against thistype of resistant bacteria (Hugonnet et al., 2009). β-lacta-mase induces the same acetylating reaction with all ofthese drugs but cannot induce deacetylation. As a result,those drugs remain attached with β-lactamase even afterthe distortion of their β-lactam ring. At this time, second-ary treatment has been done by applying previously usedpotent penicillin like β-lactam drugs with this primarilytreated β-lactamase. In current study, we conductedkinetic and mass spectrometric analysis of different BlaCinhibitors, like NXL104 (Xu et al., 2012) and showedthat how they quantitatively inactivates BlaC by forminga carbamyl linkage with the enzyme. In addition, wedetermined the three-dimensional structures of the differ-ent reactive forms of these drugs for better understandingthe undergoing mechanisms involved in this inhibitionprocess. Based on our understanding, we are trying todevelop novel small molecules with better inhibitory pro-cess.

ReferencesHeesemann J. (1993). Mechanisms of resistance to beta-lactam

antibiotics. Infection 21, S4–S9. Review. German.Hugonnet, J. E., Tremblay, L. W., Boshoff, H. I., Barry, C. E. 3rd.,

& Blanchard, J. S. (2009). Meropenem-clavulanate is effectiveagainst extensively drug-resistant Mycobacterium tuberculo-sis. Science, 323, 1215–1218.

Kumar, V., Abbas, A. K., Fausto, N., Mitchell, R. N. (2007).Robbins Basic Pathology (8th ed.). Saunders Elsevier. 516–522.

Lessel, J. (1996). Penicillin–binding protein: The target forbeta-lactam antibiotics, beta-lactamases and their inhibitors.Pharmazie in Unserer Zeit, 25, 17–27.

WHO. (2010). Multidrug and extensively drug-resistant TB (M/XDR-TB): 2010 global report on surveillance andresponse.

Xu, H., Hazra, S., & Blanchard, J. S. (2012). NXL104 irreversiblyinhibits the β-lactamase from Mycobacterium tuberculosis.Biochemistry, 51, 4551–4557.

179 Impact of cancer mutations onprotein binding and activity

Anna Panchenko*

National Center for Biotechnology Information, NationalLibrary of Medicine, National Institutes of Health, Bethesda,MD, USA*Email: [email protected]

Missense mutations play an important role in carci-nogenesis, however, their effect on biomolecular


interactions remains unclear. We describe a newframework that uses experimental evidence on struc-tural complexes, the atomic details of binding inter-faces, and evolutionary conservation to map the setof human protein–biomolecular interactions (Shoe-maker et al., 2012; Tyagi et al., 2012). To analyzethe impact of missense cancer mutations on proteininteractions, we model the affected protein com-plexes and estimate the change in binding energyupon mutations. We find that although some mis-sense mutations overstabilize protein complexes,overall, they are destabilizing mostly affecting theelectrostatic component of binding energy. Our anal-ysis allows to stratify cancer-related interactions,identify potential driver genes, and propose twodozen additional cancer biomarkers. Furthermore, weobserve that interactions of proteins with mutationsmapped on interfaces have higher bottleneck proper-ties compared to interactions with mutationselsewhere on the protein. This suggests that geneswith mutations directly affecting protein-bindingproperties are preferably located in central networkpositions and may influence critical nodes and edgesin signal transduction networks. Next, we study themechanisms of aberrant activation of receptor tyro-sine kinases in cancer and estimate the effects ofsingle and double cancer mutations on the stabilityof active and inactive states (Hashimoto et al.,2012). We show that singleton cancer mutationsdestabilize active and inactive states, however, inac-tive states are destabilized more than the activeones potentially leading to kinase activation. Inaddition, more frequent mutations have a higheractivating effect. The activation mechanisms of dou-ble mutations are found to be quite different fromthose of single mutations.

ReferencesHashimoto, K., Rogozin, I., & Panchenko, A. R. (2012). Effect

of cancer single and double somatic mutations on stabilityand activity of receptor tyrosine kinases. Human Mutation.doi: 10.1002/humu.22145

Shoemaker, B. A., Zhang, D., Tyagi, M., Thangudu, R.R., Fong, J. H., Marchler-Bauer, A., Bryant, S. H.,Madej, T., & Panchenko, A. R. (2012). IBIS reports,predicts and integrates multiple types of conservedinteractions for proteins. Nucleic Acids Research, 40,D834–D840.

Tyagi M., Hashimoto K., Shoemaker B., Wuchty S., & Panc-henko A. R. (2012). Large-scale mapping of human proteininteractions using structural complexes. EMBO Reports, Jan20. doi: 10.1038

180 Integrating structural and systemsbiology: structure-basedprediction of protein–proteininteractions on a genome-widescale

Qiangfeng Cliff Zhang, Donald Petrey and Barry Honig*

Department of Biochemistry and Molecular Biophysics, Centerfor Computational Biology and Bioinformatics, ColumbiaUniversity, 1130 St. Nicholas Ave, New York, NY 10032, USA*Email: [email protected], Phone: +1-212-851-4651,Fax: +1-212-851-4650

The genome-wide identification of pairs of interactingproteins is an important step in the elucidation of cellregulatory mechanisms. To date, structural informationhas had only limited impact on genome-scale efforts topredict protein–protein interactions (PPIs). A newalgorithm, PrePPI, will be introduced that combinesstructural information with nonstructural clues and that iscomparable in accuracy to high-throughput experiments.The surprising effectiveness of three-dimensional struc-tural information can be attributed to the use of homol-ogy models and the exploitation of both close andremote geometric relationships between proteins. Moregenerally, the “structural BLAST” approach encapsulatedin PrePPI significantly expands the range of applicationof protein structure in the annotation of protein function.

This research has been supported by NIH GM030518,GM094597, and CA121852.

ReferencesZhang, Q. C., Petrey, D., Deng, L., Qiang, L., Shi, Y., Thu, C. A.,

… Honig, B. (2012). Structure-based prediction of protein–protein interactions on a genome-wide scale. Nature, 490,556–560.

Zhang, Q. C., Petrey, D., Garzon, J. I., Deng, L., & Honig, B.(2013). PrePPI: A structure-informed database of protein–protein interactions. Nucleic Acids Research, 41, D828–833.

181 Integrative analysis of geneexpression and protein–proteininteraction networks inGlioblastoma

Seema Mishra*

Department of Biochemistry, School of Life Sciences, Universityof Hyderabad, Hyderabad, A.P. 500046, India*Email: [email protected], [email protected],Phone: 23134705


Glioblastoma multiforme (GBM) is the most malignant ofall the brain tumors with very low median survival time ofone year, as per Central Brain Tumor Registry of the USA,2001. Efforts are ongoing to understand this disease patho-genesis in complete details. Global gene expressionchanges in GBM pathogenesis have been studied by sev-eral groups using microarray technology (e.g. Carro et al.,2010). One of the many approaches to ‘understand thecontrol mechanisms underlying the observed changes inthe activity of a biological process’ (Cline et al., 2007) isintegration of gene expression and protein–protein interac-tions (PPI) datasets. Among several examples, aberrantactivation of Wnt/β-catenin signaling pathway as well assonic hedgehog (SHH) signaling pathway is reported inGBMs (Klaus & Birchmeier, 2008). Further, these twopathways are also involved in proliferation and clonoge-nicity of glioma cancer stem cells (Li et al., 2009), whichare thought to play a role in glioma initiation, proliferation,and invasion, and are one of the important points of inter-vention. Hedgehog–Gli1 signaling is also found to regulatethe expression of stemness genes. In this paper, analyses ofthe relationship between the significant differential expres-sion of these and other genes and the connectivity as wellas topological features of a PPI network would be dis-cussed. This way, genes potentially overlooked when rely-ing solely on expression profiles may be identified whichcan be biologically relevant as possible drug target/s ordisease biomarker/s.

ReferencesCarro, M. S., Lim, W. K., Alvarez, M. J., Bollo, R. J., Zhao,

X., Snyder, E. Y., … Iavarone, A. (2010). The transcrip-tional network for mesenchymal transformation of braintumours. Nature, 463, 318–325.

Cline, M. S., Smoot, M., Cerami, E., Kuchinsky, A., Landys, N.,et al. (2007). Integration of biological networks and geneexpression data using Cytoscape. Nature Protocols, 2, 2366–2382.

Klaus, A., & Birchmeier, W. (2008). Wnt signalling and itsimpact on development and cancer. Nature Reviews Cancer,8, 387–398.

Li, Z., Wang, H., Eyler, C. E., Hjelmeland, A. B., & Rich, J.N. (2009). Turning cancer stem cells inside out: an explora-tion of glioma stem cell signaling pathways. Journal ofBiological Chemistry, 284(25), 16705–16709.

182 Investigating the mode of actionof an essential OBG GTPase,CgtA in bacteria

Ananya Chatterjee and Partha P. Datta*

Department of Biological Science, Indian Institute of ScienceEducation and Research-Kolkata, Mohanpur 741252, WB,India*Email: [email protected], Phone: +91-3473-279137, +91-9051-636690, Fax: +91-33-25873020

CgtA is an essential OBG GTPase (Trach & Hoch,1989) highly conserved from bacteria to eukaryotes. Itis a multifunctional protein, involved in DNA replica-tion, chromosome partitioning (Slominska et al., 2002),nutritional stress response, initiation of sporulation,ribosome maturation, etc. Despite being a multifunc-tional essential protein, its mode of action is not well-characterized and key question remains: how does thisprotein work in wide varieties of cellular function? Theexpression of cgtA-mRNA increases on the onset ofnutritional stress. Purified CgtA protein shows increasedGTPase activity in the presence of ribosome. Ourexperiment with thiostrepton reveals that, although ribo-some is able to trigger the GTPase activity of CgtA, itsprobable site of GTPase inducing activity is differentfrom other regular translation factors like EF-G, thatuses GTP. For structure function study we have gener-ated an energy minimized homology model of the Vib-rio cholerae CgtA protein, which reveals two largedomains, an OBG-fold and a GTP– hydrolysis domain,with an extended C-terminal part. We compared theamino acid sequence of CgtA across various species inthe database, and found that its Glycine98 and theTyrosine195 residues are 100% conserved in prokary-otes. These amino acids are highly conserved ineukaryotes as well. Gly98 and Tyr195 are located onthe hinge region of CgtA comprising of portions of theOBG and the GTP–hydrolysis domains, respectively. Todecipher the mode of actions of CgtA and the role ofthe conserved Gly98 residue, we have replaced the Glywith a relatively larger amino acid, i.e. Asp. Our studyreveals that the mutant CgtA(G98D) shows a reducedGTPase activity in presence of ribosome compared tothe wild type. This indicates a restricted inter-domainmovement of CgtA due to the above point mutation.To understand this phenomenon we are using MD sim-ulations. We will discuss results from MD simulationsand other mutation studies as well. Our results indicatethat ribosome acts as a modulator for increasing theGTPase activity of CgtA. The perfect conservation ofG98 residue is important for the proper functionality ofCgtA.

Our lab’s research activity is currently supported by IIS-ER-K and DBT.

ReferenceTrach, K., & Hoch, J. A. (1989). The Bacillus subtilis spoOB

stage 0 sporulation operon encodes an essential GTP-bindingprotein. Journal of Bacteriology, 171, 1362–1371.

Slominska, M., Konopa, G., Webgrzyn, G., & Czyz, A. (2002).Impaired chromosome partitioning and synchronization ofDNA replication initiation in an insertional mutant in theVibrio harveyi cgtA gene coding for a common GTP-bindingprotein. Biochemical Journal, 362, 579–584.


183 Isolation and partialcharacterization of a generalhormone transporting bloodprotein complex

Margarita I. Garipova* and Rita R. Usmanova

Bashkirian State University, Ufa, Z. Validy, 32 450076*Email: [email protected], Phone: 347-2299671,Fax: 347-273-67-78

Using immobilized insulin, human blood serum insulin-binding proteins were isolated at physiological pH valuesand zinc concentrations. By means of enzyme-linkedimmunoassay, albumin (26.7 ± 1.75%), α-fetoprotein (2± 0.61%), transferin (10 ± 0.73%), retinol-binding protein(4 ± 0.75%), and immunoglobulin G (20.3 ± 0.95%) wereidentified in isolated protein complex from human bloodserum. Gel filtration of isolated proteins on SephadexG-75 has demonstrated formation of a supramolecularcomplex under pH 7.2 and zinc concentration of 1mg/mL (Garipova et al., 2010). The possible existence ofsuch complex is supported by the presence of lipocalinglycoproteins such as retinol-binding protein andtransferrin in obtained proteins; lipocalins form theprotein family, which is capable to form intermolecularcomplexes to ensure the delivery of hormones to targetcells through their own cell receptors (Flower, 1996). Ithas been shown that the isolated protein complex, alongwith insulin, transports other hydrophilic hormones. Bymeans of ELISA, protein hormones such as luteinizing(1mU/mg), thyreotropic (0.03mM/mg) hormones, andprolactin (0.5mU/mg) were identified. In addition, thecomplex has included hydrophobic hormones: thyroxin(11.0 ± 0.5 nmol/mg), triiodothyronine (1.3 ± 0.06 nmol/mg), and testosterone (7.0 ± 0.03 nmol/mg). Based onthese data, we have proposed an existence in humanblood of a general transporting complex that is commonfor both hydrophobic and hydrophilic hormones. Thecore of this complex is formed by transporting proteins,including those relating to lipokalins. This complextransfers thyroxin, triiodothyronine, testosterone, thyroid-stimulating hormone, prolactin, and luteinizing hormone,as well as triglycerides and cholesterol to the tissues.The composition of insulin-binding protein complex wassignificantly changed in the insulin-dependent diabetesmellitus patient’s blood. Albumin and α-fetoprotein,which are present in a normal complex, are revealedonly in trace amounts in samples isolated from thediabetic blood and are replaced by α1-acid glycoprotein,and possibly other unidentified proteins. Disturbance ofhormone-transporting complex protein composition inthe diabetic blood may be a reason of insulin deliverydisruption. Perhaps, for the same reason, delivery totarget cells of not only insulin, but also other members

of the hormone-transporting complex, is disrupted in thediabetic blood.

ReferencesFlower, D. R. (1996). The lipocalin protein family: Structure

and function. Biochemical Journal, 318, 1–14.Garipova, M. I., Morugova, T. V., Kireeva, N. A., Ibragimov,

R. I., Pershina, A. S., Eliseeva, O. S., & Baranova, M. V.(2010). Affinity extraction and study of insulin-bindingprotein of human serum. Problems of Biological, Medicinaland Pharmaceutical Chemistry, 8, 40–44.

184 Small molecule identificationagainst novel MDRA protein ofMycobacterium tuberculosis

M. Kiran Kumara, M. Vasavia,C. Venkataramana Reddyb and V. Umab*aDepartment of Chemistry, Molecular Modelling ResearchLaboratory, Nizam College, Osmania University, Basheerbagh,500 001Hyderabad; bDepartment of Chemistry, JNTUH-CEH,Jawaharlal Nehru Technology University, Kukatpally,500 085 Hyderabad*Email: [email protected]; [email protected]

Mycobacterium tuberculosis (Mtb) is an obstinate patho-gen causing tuberculosis (TB) in Homo sapiens. Onethird of the World population is affected by Mtb (Jameset al., 2008). The multidrug-resistant protein-A (MDRA)belongs to ABC transporter family. The protein MDRAand the membrane integral protein MDRB together formthe efflux pump (MDRA2B2 complex) that confers resis-tance by transport of the drugs out of the cell. TheMDRB protein expression depends on the expression ofMDRA (Baisakhee et al., 2002). In the present study,MDRA 3-D model (Figure) was generated with the helpof comparative homology modeling techniques usingpair-wise sequence alignment. The predicted 3-D modelwas subjected to refinement and validated. The activesite of the protein was predicted. The virtual screening(VS) studies were performed at MDRB binding site withan in-house library of small molecules to identify a leadmolecule that can inhibits the MDRA protein. The


results of VS project competitive inhibitors of MDRB,for its binding with MDRA, and its drug-resistant activ-ity. Hence, the MDRA protein may be treated as a noveltarget for the development of new chemical entities fortuberculosis therapy (Bhargavi et al., 2010; Malkhedet al., 2011).

ReferencesJames, C. S., Eric, J. R., & Joel, S. F. (2008). Drugs versus

bugs: in pursuit of the persistent predator Mycobacteriumtuberculosis. Nature Reviews Microbiology, 6, 41–52.

Baisakhee, S. C., Sanjib, B., Rajib, B., Joyoti, B., Manikuntala,K., & Parul, C. (2002). Overexpression and functionalcharacterization of an ABC (ATP-binding cassette) trans-porter encoded by the genes drrA and drrB of Mycobacte-rium tuberculosis. Biochemical Journal, 367, 279–285.

Malkhed, V., Gudlur, B., Kondagari, B., Dulapalli, R., &Vuruputuri, U. (2011). Study of interactions between Myco-bacterium tuberculosis proteins: SigK and anti-SigK. Jour-nal of Molecular Modeling, 17, 1109–1119.

Bhargavi, K., Chaitanya, K. P., Ramasree, D., Vasavi, M., Mur-thy, D. K., & Uma, V. (2010). Homology modeling anddocking studies of human Bcl-2L10 protein. Journal ofBiomolecular Structure and Dynamics, 28, 379–391.

185 Vascular endothelial growthfactor receptor-2 (VEGFR2):structure and functionalrelationship

Jhansi Rani Nathan, Shilpi Shika and Malathi Ragunathan*

Department of Genetics, Dr. ALM PG IBMS, University ofMadras, Taramani, Chennai, 600 113, India*Email: [email protected], Phone: +91-44-24547062,Fax: +91-44-24540709

Vascular endothelial growth factor (VEGF), is expressed inthe vicinity of sprouting vessels and its receptor (VEGF-R2/Flk-1/kdr) on the angioblasts and new vessels, and both arerequired for vasculogenesis and angiogenesis. VEGFR2,also called as KDR or Flk-1, is identified as an early markerfor endothelial cell progenitors, whose expression isrestricted to endothelial cells in vivo. VEGFR2 consists ofextracellular (7-Ig-like sub-domains), transmembrane andcytoplasmic domains. In order to understand the structure–functional relationship and signal transduction process ofVEGFR2, we have examined their amino acid sequences

from a wide range of species including mammals, birds,Zebrafish and also computed the phylogenetic tree, second-ary and domain structures. Phylogeny constructed usingMaximum Parsimony tree software MEGA-5 versionsuggested an interesting sequence similarity between Zebra-fish and Gallus, closeness between human, rat, horse andpig. Strong homology in amino acids sequences wasobserved between the species, such as human, Macaca mul-atta, gorilla, etc, and small variations in Zebrafish andzebrafinch. The Arg and Asp residues which are involved informing salt bridges are evolutionarily conserved from Zeb-rafish to human in D7 domain of VEGFR2, indicating theirfunctional importance in VEGFR activity. Amino acids,tyrosine in the extracellular loops and cysteines involved indisulphide bridges of VEGFR2, are highly conserved sug-gesting their importance during ligand binding, the detailsof which will be discussed.

ReferencesCarmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S., Kieckens,

L., Gertsenstein, M., Fahrig, M., Vandenhoeck, A., Harpal,K., Eberhardt, C., et al. (1996). Abnormal blood vesseldevelopment and lethality in embryos lacking a singleVEGF allele. Nature, 380, 435–439.

Leung, D., Cachianes, G., Kuang, W. J., Goeddel, D. V., &Ferrara, N. (1989). Vascular endothelial growth factor is asecreted angiogenic mitogen. Science, 246, 1306–1309.

Shalaby, F., Rossant, J., Yamaguchi, T. P., Gertsenstein, M.,Wu, X. F., Breitman, M. L., & Schuh, A. C. (1995). Fail-ure of blood-island formation and vasculogenesis in flk-1deficient mice. Nature, 376, 62–66.

186 Molecular dynamics and ligandbased studies for the validation ofpotential inhibitors for SrtAagainst Bacillus anthracis

Sanjeev Kumar Singh* and Chandrabose Selvaraj

Computer Aided Drug Design and Molecular Modeling Lab,Department of Bioinformatics, Alagappa University, Karaikudi630003, India*Email: [email protected], Phone: +91-4565-230725,Fax: +91-4565-255202

SrtA enzymes cleave the sorting signals of secretedproteins to form isopeptide (amide) bonds between thesecreted proteins and peptidoglycan or polypeptides tofunction as the principal architects of the Bacillus anthra-cis. Inhibition of SrtA can completely eradicate thegrowth of B. anthracis due to the lack of signals and sothe SrtA is universally accepted as the drug target for allgram positive pathogens. Here, with the reported com-pounds, the pharmacophore-based virtual screening proto-col has been used to obtain similar pharmacological


The structure of MDRA

property that derived new compounds to inhibit the SrtAstructure. New compounds on AAAHR Pharmacophorehypothesis screening were treated with four phase ofdocking protocols with combined Glide-QPLD dockingapproach and charge accuracy variations were dominatedby QM/MM approach. Finally, the compounds of 19941,14704, 121962, 20137, and 19533 from binding db,Chembridge db, and Toslab were succeeded from thescreening; these compounds are having better scoring,energy parameters, ADME physio/chemical properties,tendency to transfer the electrons between the protein–ligand interactions, and these compounds also predicthaving cell adhesion inhibitory activity. These screenedcompounds have better stability at active site loop regionswith strong bonding interactions and these compounds on

clinical trials will definitely emerge as better SrtA inhibi-tor against the B. anthracis.

ReferencesFazil, M. H., et al. (2012). Journal of Biomolecular Structure

and Dynamics, 30, 574–584.Pagano, B., et al. (2011). International Journal of Biological

Macromolecules, 49, 1072–1077.Sakkiah, S., et al. (2012). Acta Pharmacologica Sinica, 33,

964–978.Suree, N., et al. (2009). Journal of Biological Chemistry, 284,

24465–24477.Weiner, E. M., et al. (2010). Journal of Biological Chemistry,

285, 23433–23443.

187 Plucking the high hanging fruit: asystematic approach for targetingprotein interfaces

Paramjit S. Arora*

Department of Chemistry, New York University, New York, NY10003, USA*Email: [email protected], Phone: +1-212-998-8470

Development of specific ligands for protein targets thathelp decode the complexities of protein–protein interac-tion networks is a key goal for the field of chemicalbiology. Despite the emergence of powerful in silico andexperimental high-throughput screening strategies, thediscovery of synthetic ligands that selectively modulateprotein–protein interactions remains a challenge for thechemical biologists. Proteins often utilize small foldeddomains for recognition of other biomolecules. The basichypothesis guiding our research is that by mimickingthese domains, we can modulate the function of a partic-ular protein with metabolically-stable synthetic molecules

(Raj et al., 2013). This presentation will discuss compu-tational approaches (Bullock et al., 2011; Jochim & Aro-ra, 2010) to identify targetable interfaces along withsynthetic methods (Patgiri et al., 2008; Tosovska & Aro-ra, 2010) to develop protein domain mimics (PDMs) asmodulators of intracellular protein–protein interactions.(Henchey et al., 2010; Patgiri et al., 2011).


This research has been supported by NSF CHE-1151554and NIH (GM073943).

ReferencesBullock, B. N., Jochim, A. L., & Arora, P. S. (2011). Assessing

Helical Protein Interfaces for Inhibitor Design. Journal ofAmerican Chemical Society, 133, 14220–14223.

Henchey, L. K., Kushal, S., Dubey, R., Chapman, R. N.,Olenyuk, B. Z., & Arora, P. S. (2010). Inhibition ofHypoxia inducible factor 1–transcription coactivator inter-action by a hydrogen bond surrogate alpha-helix. Journalof American Chemical Society, 132, 941–943.

Jochim, A. L., & Arora, P. S. (2010). Systematic Analysisof Helical Protein Interfaces Reveals Targets for SyntheticInhibitors. ACS Chemical Biology, 5, 919–923.

Patgiri, A., Jochim, A. L., & Arora, P. S. (2008). A hydrogenbond surrogate approach for stabilization of short peptidesequences in alpha-helical conformation. Accounts ofChemical Research, 41, 1289–1300.

Patgiri, A., Yadav, K. K., Arora, P. S., & Bar-Sagi, D. (2011).An orthosteric inhibitor of the Ras-Sos interaction. NatureChemical Biology, 7, 585–587.

Raj, M., Bullock, B. N., & Arora, P. S. (2013). Plucking thehigh hanging fruit: A systematic approach for targetingprotein-protein interactions. Bioorganic and MedicinalChemistry, 21, Online article.

Tosovska, P., & Arora, P. S. (2010). Oligooxopiperazines asNonpeptidic Alpha-Helix Mimetics. Organic Letters, 12,1588–1591.

188 Using CG modeling in exploringthe role of dynamical effects incatalysis and in simulatingbiological molecular machines

Arieh Warshel*

Department of Chemistry, University of Southern California,Los Angeles, CA 90089*Email: [email protected], Phone: +1-213-740 4114,Fax: +1-213-740 2701

Studies of structure–function correlation of biologicalmolecules involve in some cases the need to explorelong-time processes and to sample complex multidimen-sional landscapes. Here, we review advances in suchstudies, starting by considering the proposal that enzymecatalysis involves dynamical effects (see Kamerlin &Warshel, 2010 for discussion and analysis). In order toexplore the validity of this dynamical proposal, we usedour renormalization approach that allows us to simulatethe very long-time coupling between catalysis and con-formational changes. The corresponding simulations haveproved that dynamical effects cannot change the rate ofthe chemical steps in enzymes, as long as the chemistryis the rate-limiting step. The same analysis is then beapplied to allosteric transitions and to the control of rep-lication fidelity, reaching the same conclusions. Next, we

describe the use of the renormalization method and elec-trostatic-based coarse-grained (CG) models to simulatethe action of various challenging complex systems. It isshown that our CG model produces, for the first time,realistic landscapes for vectrorial process such as theactions of F1 ATPase (Mukherjee and Warshel, 2011)and F0 ATPase (Mukherjee and Warshel, 2012) . It isalso shown that such machines are working by exploitingfree energy gradients and cannot just use Brownianmotions as the vectroial driving force. Finally, we outlinea recent simulation of the tag of war between staledelongated peptide in the ribosome and the translocon.

ReferencesKamerlin S. C. L., & Warshel A. (2010). At the dawn of the

21st century: Is dynamics the missing link for understand-ing enzyme catalysis? PROTEINS: Structure, Function, andBioinformatics (INVITED REVIEW), 78, 1339–1375.

Mukherjee S., & Warshel A. (2011). Electrostatic origin of themechanochemical rotary mechanism and the catalytic dwellof F1-ATPase. Proceedings of National Academy of ScienceUSA, 108, 20550–20555.

Mukherjee S., & Warshel A. (2012). Realistic simulations ofthe coupling between the protomotive force and themechanical rotation of the F0-ATPase. Proceeding ofNational Academy of Science USA, 109, 14876–14881.

189 Structural articulation ofbiochemical reactions usingaltered bonds and iterativetopology switching

Swati R. Manjari and Nilesh K. Banavali*

Division of Genetics, Wadsworth Center, New York StateDepartment of Health, CMS 2008, 150 New Scotland Ave,Albany, NY 12208, USA*Email: [email protected], Phone: +1-518-474-0569,Fax: +1-518-402-4623

Structural and dynamic trajectories of biochemical reac-tions catalyzed by large macromolecular complexes aredifficult and expensive to characterize experimentally.Predicting them thoroughly using quantum mechanics/molecular mechanics (QM/MM) approaches is possible,but requires significant computational resources. Atomicdetail structural snapshots of individual states obtainedthrough crystallography may also not always be in aconformation conducive to the reaction, presenting a hur-dle for choosing the appropriate starting structures forthe QM/MM calculations. The geometric displacementsfor nuclei that change their bonding during chemicalreactions are usually not large, when starting from appro-priate reactive conformations. If we hypothesize thatthere is no need for these rearrangements to be repre-sented in electronic detail to capture the response of thesurrounding macromolecular environment, MM energy


functions should provide a relatively accurate descriptionof this structural reorganization. In the present study, astrategy called altered bonds and iterative topologyswapping with MM models is used to obtain reasonableatomic detail trajectories for complex biochemical reac-tions. The implementation, advantages, and disadvan-tages of this strategy are illustrated using a symmetricmodel proton transfer reaction. This application isexpanded to describe a similar proton-transfer reaction ina complex macromolecular environment. The strategy isthen utilized to visualize a complex biochemical reactionrepresenting a critical step in the central dogma of biol-ogy, namely addition of a deoxyribonucleotide to a DNAstrand by a DNA polymerase. This strategy reduces thecomputational cost of obtaining reasonable biochemicalreaction trajectories within complex macromolecularenvironments by orders of magnitude as compared to

QM/MM simulations, thus rapidly identifying goodstructural starting points for such simulations. Its easyimplementation allows the direct application of mostcomputational methods designed for MM modelstowards understanding reactions involving covalentchanges. It also enables a reasonable structural predictionfor all reactant, transition, intermediate, and productstates for any biochemical reaction for which only oneof these states is known. Its high-throughput applicationoffers the promise of delineating, dynamically and inatomic detail, any biochemical reaction catalyzed by amacromolecular catalyst whose structure is either knownor can be accurately modeled.

This research has been supported by new investigatorfunds from the Wadsworth Center.

190 The peculiarities of regulation ofYarrowia lipolytica yeast and citricacid overproduction

Igor G. Morgunov* and Svetlana V. Kamzolova

G.K. Skryabin Institute of Biochemistry and Physiology ofMicroorganisms RAS, Pushchino, Moscow 142290, Russia*Email: [email protected], Phone: +7-4967-318660,Fax: +7-495-9563370

The growth of Yarrowia lipolytica yeast as well thebiosynthesis of citric acid on rapeseed oil were stud-ied. It was indicated that the initial step of assimilationof rapeseed oil in the yeast Y. lipolytica is their hydro-lysis by extracellular lipases with the formation ofglycerol and fatty acids, which appear in the mediumin the phase of active growth. The concentrations ofthese metabolites change insignificantly upon furthercultivation. Lipase and the key enzymes of glycerolmetabolism (glycerol kinase) and the glyoxylate cycleresponsible for the metabolism of fatty acids (isocitratelyase and malate synthase) are induced just at the

beginning of the growth phase and remain active inthe course of further cultivation. These results, takentogether, suggest that glycerol and fatty acids accord-ing in the medium do not suppress the metabolism ofeach other. The fact that glycerol and fatty acids canbe consumed simultaneously is of special importancefor the development of the efficient regime of oil feed-ing, Y. lipolytica produced citric acid (175 g/L) with ayield of 150%. It should be noted that the simulta-neous utilization of two different substrates is nottypical of micro-organisms, which first assimilate oneof the two available substrates (commonly, a carbohy-drate), whereas the assimilation of the other substratestarts only after the first substrate is fully consumedfrom the medium. Indeed, upon the cultivation of Y.lipolytica on the mixture of glucose and oleic acid,the latter substrate began to be utilized only when theconcentration of glucose decreased. The glycolyticenzyme pyruvate dehydrogenase was induced from thefirst hours of cultivation and remained at high levelsuntil the exhaustion of glucose in the medium. At thesame time, the activities of isocitrate lyase and malate


synthase were very low during the metabolism of glu-cose, but were rapidly induced (approximately in 10times) after the exhaustion of glucose in the medium.When Y. lipolytica was grown on the mixture of glu-cose and hexadecane, the dynamics of growth andsubstrate consumption was typical of the diauxie phe-nomenon: the utilization of hexadecane began only inseveral hours after the time when glucose was com-pletely exhausted in the cultivation medium. In thiscase, the exhaustion of glucose arrested growth andthe culture resumed growth only after a lag period.The assay of enzymes showed that the glycolyticenzyme pyruvate dehydrogenase was active during thephase of growth on glucose, whereas the enzymes ofthe glyoxylate cycle, isocitrate lyase and malate syn-thase were active during the phase of growth on hexa-decane. In recent years in the literature, there are datathat the different sugars produce signals which modifythe conformation of certain proteins that, in turn,directly or through a regulatory cascade affect theexpression of the genes subject to catabolite repression.These genes are not all controlled by a single set ofregulatory proteins (Cho et al. 2009), but there are dif-ferent circuits of repression for different groups ofgenes (Gancedo 1990). We will discuss the possiblemetabolic regulation in the case of Y. lipolytica.

ReferencesCho, I. H., Lu, Z. R., Yu, J. R., Park, Y. D., Yang, J. M., Hahn,

M. J., & Zou, F. (2009). Towards profiling the gene expres-sion of tyrosinase-induced melanogenesis in HEK293 cells:a functional DNA chip microarray and interactomics stud-ies. Journal of Biomolecular Structure & Dynamics, 27,331–345.

Gancedo, J. M. (1998). Yeast carbon catabolite repression.Microbiology and Molecular Biology Reviews, 62, 334–361.

191 Point mutations in the yeast Pma1H+-ATPase affect polyphosphate(PolyP) distribution

Alexandr A. Tomashevski and Valery V. Petrov*

Institute of Biochemistry and Physiology of Microorganisms,RAS, 142290 Pushchino, Russia*Email: [email protected]; [email protected], Phone: +7 496 731 8698, Fax: +7 495 956 3370

Yeast plasma membrane Н+-ATPase (Pma1) is a keyenzyme of the yeast cell metabolism. It generateselectrochemical Н+ gradient providing energy for oper-ating the secondary solute transport systems andmaintaining intracellular pH and ion homeostasis.

Most of the enzyme molecule anchored in the plasmamembrane by M1–M10 segments is located in cyto-sole and membrane; less than 5% of the Pma1 faceextracellular space. Membrane domain contains aminoacid residues, which form H+ transport pathway; cyto-sole parts house the enzyme active center and cyto-solic C-terminal tail has regulatory function. Theenzyme function and regulation are tightly connectedto glucose metabolism: its fermentation triggers activa-tion of Pma1 function, structurally accompanied bythe enzyme multiple phosphorylation during intracellu-lar traffic on route to plasma membrane. There areca. 10 phosphorylation sites; only 3 of them are iden-tified: one single and two tandemly located sites arein the C-terminal tail. Both ATP and PolyP can beused to phosphorylate amino acid residues; however,there are little data on the interactive metabolism ofATP and PolyP. Most of phosphorylable Ser, Thr,Asp, Glu, and Tyr residues are located in the innerparts of the enzyme; however, there are several suchresidues in the Pma1 outer parts: D714, S716, D718,and D720 in M5–M6 loop and S846, E847, T850,and D851 in M9–M10 loop, close to the enzyme reg-ulatory C-tail. It seems reasonable that multiple phos-phorylation of Pma1 goes subsequently, and first ofsuch sites could be located in the enzyme extracyto-solic part. The M5–M6 loop phosphorylable residues,except D714, were found to be unimportant for theenzyme structure-function relationship; D714A mutantwas poorly expressed and inactive (Petrov, 2011).However, D714N did not disturb the enzyme func-tioning, thus excluding the role of D714 in theenzyme phosphorylation. Therefore, we choose tostudy further residues in the M9–M10 loop by replac-ing them with Ala. The ATPase activity of thesemutants ranged from the wild-type level (S846A) to2- (E847A) to 3-fold (T850A) drop. Changes ofactivity were accompanied by changes in PolyP frac-tions (Figure 1), which were most significant forS846A and T850A. S846A had 1.5–1.7-fold increasein PolyP1 (found mostly in cytosole and vacuoles)and PolyP3 (localized near the cell surface) and dra-matic 3-fold decrease in PolyP4-5 fractions (associatedwith cell wall), while T850A had stable 1.5-foldincrease in all PolyP but PolyP4-5 fractions. Bothmutants also had 20% (S846A) to 37% (T850A)increase in total PolyP. These data may point to lackof one or more phosphorylation sites and/or participa-tion of PolyP in the Pma1 ATPase phosphorylation.Possibly, the sites at S846 and T850 act jointly, simi-larly to tandemly located and acting S911 and T912in the regulatory C-tail (Lecchi et al., 2007). Furtherstudy of these mutants, although methodologically


challenging, seems certain to yield more usefulinsights into functioning and regulation of the Pma1ATPase as well as into the interactive mechanisms ofATP and PolyP metabolism.

The study was supported in part by Russian Foundationfor Basic Research (RFBR) grant 13-04-02031a.

ReferencesLecchi, S., Nelson, C. J., Allen, K. E., Swaney, D. L., Thompson,

K. L., Coon, J. J., … Slayman, C. W. (2007). Tandemphosphorylation of Ser-911 and Thr-912 at the C terminus of yeastplasma membrane H+-ATPase leads to glucose-dependent activa-tion. Journal of Biological Chemistry, 282, 35471–35481.

Petrov, V. V. (2011). Role of M5–M6 loop in the biogenesis andfunction of the yeast Pma1 H+-ATPase. Journal ofBiomolecular Structure and Dynamics, 28, 1024–1025.

Figure 1. Effect of the Pma1 point mutations on the ATPase activity and PolyP distribution.

192 Computer-aided search for novelanti-HIV-1 agents presentingpeptidomimetics of broadlyneutralizing antibodies againstthe virus envelope GP120 V3loop

Alexander M. Andrianova*, Ivan A. Kashynb andAlexander V. Tuzikovb

aInstitute of Bioorganic Chemistry, National Academy ofSciences of Belarus, Kuprevich Street 5/2, 220141, Minsk,Republic of Belarus; bUnited Institute of Informatics Problems,National Academy of Sciences of Belarus, Surganov Street 6,220012, Minsk, Republic of Belarus*Email: [email protected]

Computer-aided search for novel anti-HIV-1 agentsthat are able to imitate the pharmacophore propertiesof the antigen-binding site of a broadly neutralizingmAb 3074 against the envelope gp120 V3 loop wascarried out followed by evaluation of their potentialinhibitory activity by molecular modeling. In doingso, the following problems were solved: (1) the mAb

3074 amino acid residues responsible for specificbinding to the HIV-1 V3 loop were identified fromthe X-ray structures of this antibody Fab in com-plexes with the MN, UR29, and VI191 V3 peptides(Jiang et al., 2010); (2) using these data, 2039 possi-ble mAb-3074 peptidomimetics were found by pep-MMsMIMIC presenting a public, web-oriented virtualscreening platform (Floris et al., 2011); (3) the com-plexes of these compounds with the above V3 pep-tides were built by molecular docking and, based ontheir analysis, the four molecules exhibiting a highaffinity to V3 in the in silico studies were selectedas the most probable peptidomimetics of mAb 3074(Figure 1); and (4) stability of the complexes of thesemolecules with the MN, UR29, and VI191 V3 pep-tides was estimated by molecular dynamics and freeenergy simulations. As a result, a key role in specificbinding of the selected compounds to the V3 loopwas shown to belong to π-π interactions between theiraromatic rings and the conserved Phe20 and/or Tyr21of the V3 immunogenic crown. Similarly to mAb3074, these compounds were found to block the tipof the V3 loop forming its invariant structural motif,


0

100

S846A E847A T850AATPase PP1 PP2 PP3 PP4-5

which contains residues critical for cell tropism(Andrianov et al., 2011; Andrianov et al., 2012). Inaddition, the complexes of interest do not undergosignificant changes within the molecular dynamics cal-culations, exhibiting the low values of free energy oftheir formation. In this context, the compounds givenin Figure 1 are considered as the promising basicstructures for the design of novel, potent, and broadanti-HIV-1 drugs.

ReferencesAndrianov, A. M., Anishchenko, I. V., & Tuzikov, A. V.

(2011). Discovery of novel promising targets for anti-AIDSdrug developments by computer modeling: application tothe HIV-1 gp120 V3 loop. Journal of Chemical Informa-tion and Modeling, 51, 2760–2767.

Andrianov, A. M., Kornoushenko, Yu. V., Anishchenko, I. V.,Eremin, V. F., & Tuzikov, A. V. (2012). Structural analysisof the envelope gp120 V3 loop for some HIV-1 variantscirculating in the countries of Eastern Europe. Journal ofBiomolecular Structure Dynamics10.1080/07391102.2012.706455:1-19.

Floris, M., Masciocchi, J., Fanton, M., & Moro, S. (2011). Swim-ming into peptidomimetic chemical space using pepMMs-MIMIC. Nucleic Acids Research, 39, 261–269.

Masciocchi, J., Frau, G., Fanton, M., Sturlese, M., Floris,M., Pireddu, L., Palla, P., Cedrati, F., Rodriguez-Tome,P., & Moro, S. (2009). MMsINC: A large-scale chemoin-formatics database. Nucleic Acids Research, 37, D284–D290.

Jiang, X., Burke, V., Totrov, M., Williams, C., Cardozo, T.,Gorny, M. K., Zolla-Pazner, S., & Kong, X. P. (2010).Conserved structural elements in the V3 crown of HIV-1gp120. Nature Structural & Molecular Biology, 17, 955–961.

Figure 1. 3D structures of the compounds presenting the most probable peptidomimetics of mAb 3074. The molecules are denotedaccording to their codes in the MMsINC database (Masciocchi et al., 2009).


193 Computer-aided design of novelHIV-1 entry inhibitors based onglycosphingolipids

Alexander M. Andrianova*, Yuri V. Kornoushenkoa,Ivan A. Kashynb and Alexander V. Tuzikovb

aInstitute of Bioorganic Chemistry, National Academy ofSciences of Belarus, Kuprevich Street 5/2, 220141, Minsk,Republic of Belarus; bUnited Institute of Informatics Problems,National Academy of Sciences of Belarus, Surganov Street 6,220012 Minsk, Republic of Belarus*Email: [email protected]

Novel HIV-1 entry inhibitors targeting the envelopegp120 V3 loop were designed by computer modelingbased on glycosphingolipid β-galactosylceramide (β-Gal-Cer) forming on the surface of some susceptible hostcells the primary receptor for HIV-1 alternative to CD4,which is used by the virus to enter macrophages and T-lymphocytes (e.g. Fantini et al., 1993). To achieve thisgoal, 3D structures of the twelve water-soluble analogsof β-GalCer containing different substitutes of its fattyacid residue were determined by quantum chemical cal-culations and evaluation of their potential anti-HIV-1activity was carried out by molecular docking, moleculardynamics, and free energy simulations. Analysis of thestructural complexes of these β-GalCer derivatives withthe HIV-1 V3 loops from the five diverse viral strainsmakes it clear that, in all of the cases of interest, thethird variable domain of gp120 forms two potentialbinding sites for glycolipids concerning the immuno-genic tip and the base of V3. At the same time, noncon-ventional XH … π hydrogen bonds between XH sugargroups (X designates C or O) and overlapping π-orbitalsof the conserved Phe-20, Tyr-21, and His-34 residues ofthe V3 loop were shown to play a key role in specificbinding of the designed glycosphingolipids to the aboveconserved structural motifs of V3 that include residuescritical for cell tropism (Andrianov et al., 2011; Andria-nov, Kornoushenko, et al., 2012). These findings testify-ing to the ability of the simulated chemicals tospecifically and effectively interact with the functionallyimportant sites of V3 were confirmed by those onmolecular dynamics and calculating the free energy offormation of the complexes for these β-GalCer analogswith the HIV-1 V3 loops from different viral modifica-tions. Finally, the majority of the designed moleculeswere found to form much more stable complexes withV3, compared to that of the β-GalCer-based anti-HIV-1agent developed previously (Andrianov, Anishchenko,et al., 2012) and used in the calculations as a control. Inthe light of the data obtained, these potential HIV-1entry inhibitors present the promising basic compoundsfor the development of novel, potent, and broad antiviraldrugs.

ReferencesAndrianov, A. M., Anishchenko, I. V., Kisel, M. A.,

Kornoushenko, Yu. V., Nikolayevich, V. A., Eremin, V. F.,… Tuzikov, A. V. (2012). Computer-aided design of novelHIV-1 entry inhibitors blocking the virus envelope gp120V3 loop. Biopolymers and Cell, 28, 468–476.

Andrianov, A. M., Anishchenko, I. V., & Tuzikov, A. V.(2011). Discovery of novel promising targets for anti-AIDSdrug developments by computer modeling: Application tothe HIV-1 gp120 V3 loop. Journal of Chemical Informa-tion and Modeling, 51, 2760–2767.

Andrianov, A. M., Kornoushenko, Yu. V., Anishchenko, I. V.,Eremin, V. F., & Tuzikov, A. V. (2012). Structural analysisof the envelope gp120 V3 loop for some HIV-1 variantscirculating in the countries of Eastern Europe. Journal ofBiomolecular Structure and Dynamics, 1–19. doi: 10.1080/07391102.2012.706455.

Fantini, J., Cook, D. G., Nathanson, N., Spitalnik, S. L., & Gonz-alez-Scarano, F. (1993). Infection of colonic epithelial celllines by type 1 human immunodeficiency virus is associatedwith cell surface expression of galactosylceramide, a poten-tial alternative gp120 receptor. Proceedings of the NationalAcademy of Sciences, 90, 2700–2704.

194 Energetics and dynamics ofadenine protonation in HIV-1’sdimerization initiation site

Timothy J. Robbins, Nicole A. Whitakera andYongmei Wang*

Department of Chemistry, University of Memphis, Memphis, TN38152*Email: [email protected], Phone: +1-901-678-2621,Fax: +1-901-678-3447

HIV-1, like most retroviruses, packages two homologouscopies of its RNA genome. The two RNA strands arenon-covalently linked near their 5’ends. The proposeddimerisation initiation site is a 35-nucleotide (nt) stemloop capable of forming loop–loop interactions (a kissingdimer) via its highly conserved 6-nt loop palindrome. Ina structural transformation affected by temperature, saltconcentration, and by the HIV-1 nucleocapsid protein,the initial, kinetically-stable kissing dimer (KD) convertsto a thermodynamically-stable extended dimer. It hasbeen suggested that this in vitro observed rearrangementis associated with the in vivo viral genome maturation.Mihailescu et al. demonstrated enhanced rearrangementdynamics triggered by the protonation of a specificadenine residue at the genome sequence location 272(A272) (Mihailescu, 2004). They suggested that the localenvironment of A272 caused its N1 atom’s pKa to shiftupwards (more basic) by approximately 2.5 pH unitswhen compared with 5’-adenosine monophosphate (5’-AMP). In this work, we investigated the dynamics andenergetics of protonating A272’s N1 atom with explicitsolvent molecular dynamics (MD) simulations, Thermo-


dynamic Integration (TI), and the Poisson–Boltzmannequation (PB). Two initial structures were used, an NMRsolution structure (PDB ID: 2D19) and an X-ray crystalstructure (PDB ID: 1XP7), where A272 was found inside(NMR) and outside (X-ray) the helical axis respectively.MD simulations showed when A272 started,it remainedinside the KD’s axis, and when outside, it attempted toinsert itself within the axis. Calculated pKa shiftsobtained from solving the PB equation were approxi-mately + 2.2 and + 1.0 pH units for the NMR and X-raystructures respectively; while TI calculations performedwith the NMR structure yielded a shift of approximately+ 2.5 pH units. Our simulations confirmed the stronginfluence of A272's local environment on its calculatedpKa when inserted in the helical axis. A272's N1 atomwas approximately 200 times more likely protonatedwhen compared with 5'-AMP. Also, protonated A272swere more energetically favoured in the kissing dimerversus an isolated monomer due to a diminished positiveelectrostatic potential near A272, while in the dimer.Overall, our computational investigations affirm theexperimental suggestion that A272 in the kissing dimeris more likely to be protonated at physiological condi-tions and this protonation may trigger the structural rear-rangement of the initial kissing dimer to form theextended dimer.

This research has been supported by ORAU/ORNL HighPerformance Computing Award and NSF TennesseeEPSCOR funding (grant EPS-1,004,083).

ReferenceMihailescu, M. R., & Marino, J. P. (2004). A proton-coupled

dynamic conformation switch in the HIV-1 dimerizationinitiation site kissing complex. PNAS, 101, 1189–1194.

195 In silico study on HIV-PRIssubstructures to terminateproteolytic activity in HTLV

Poonam Singha*, Sanjeev Kumar Singhb,Chandrabose Selvarajb and Rama Kant Singha

aToxicology Division, Central Drug Research Institute,Lucknow 226 001, Uttar Pradesh, India; bComputer AidedDrug Design and Molecular Modeling Lab, Department ofBioinformatics, Alagappa University, Karaikudi 630003,Tamilnadu, India*Email: [email protected],Phone: +91-522-2612411, Fax: +91-522-2623405

Human T-lymphotropic virus (HTLV) is RNA retrovi-rus, which causes CD3 + and CD4+T-cell type leuke-mia and demyelinating diseases, like tropical spastic

myelopathy. The replicative stage of the virus is one ofthe critical stages for the development of the disease.At present, there are no approved therapeutic agents tar-geting HTLV. The HTLV mechanism of malignant cellgrowth in adult T-cell leukemia (ATL)/lymphoma, andthe HTLV-PR has been an attractive target for antican-cer drug design. In comparison with other retroviruses,HTLV also encodes protease (PR) enzyme which isessential for maturation. Both the HIV and HTLV prote-ases show high structural similarity but known inhibi-tors of HIV-PR are not able to inhibit the HTLV-PR,while comparing the binding pocket of both proteases,MET37 of HTLV shows repulsive role with knownHIV inhibitors. Functional analysis of M37A mutationclearly shows that MET37 is highly important for theprotease function. Available inhibitors were testedagainst the HTLV-PR binding pocket and failed tointeract with MET37. Screening of similar libraries ofknown compounds provides better interactions withMET37 and further validation with in vivo and in vitrostudies on these screened compounds will provide morestrength in discovering potent inhibitor for HTLV-PR.

ReferencesDas, D., et al. (2009). Prediction of potency of protease inhibi-

tors using free energy simulations with polarizable quantummechanics-based ligand charges and a hybrid water model.Journal of Chemical Information and Modeling, 49, 2851–2862.

Kadas, J., et al. (2004). Total chemical synthesis of human T-cellleukemia virus type 1 (HTLV-1) protease via native chemicalligation. Chemical Biology, 279, 27148–27157.

Shuker, S. B., et al. (2003). Understanding HTLV-I protease.Chemical Biology, 10, 373–380.

Zhang, M., et al. (2008). Locking the two ends of tetrapeptidicHTLV-I protease inhibitors inside the enzyme. Bioorganicand Medicinal Chemistry, 16, 6880–6890.

196 Mechanisms of viral dsRNAdetection and antiviral signalactivation by MDA5 filament

Bin Wu, Alys Peisley and Sun Hur*

Department of Biological Chemistry and MolecularPharmacology, Harvard Medical School, Boston, MA 02115, USA*Email: [email protected], Phone: +1-617-713-8250,Fax: +1-617-713-8260

MDA5, a viral double-stranded RNA (dsRNA) receptor,shares sequence similarity and interferon signalingpathways with RIG-I, yet plays essential functions inantiviral immunity by recognizing largely distinct groupsof viruses and viral RNAs. We have previously shownthat MDA5 forms novel filamentous oligomers by stack-ing monomers along the length of dsRNA head-to-tail,


and that such filament formation is distinct from mono-meric binding observed with RIG-I. We have recentlydetermined the first crystal structure of MDA5 in com-plex with dsRNA, which revealed the molecular basis forfunctional divergence between RIG-I and MDA5, and inparticular, the divergent mechanism for dsRNA recogni-tion and filament formation. We have further demon-strated that MDA5 filament formation is (1) required forhigh affinity interaction with dsRNA, (2) provides amolecular framework to measure dsRNA length that isorders of magnitude larger than the individual proteinitself, and finally, (3) brings the signaling domain, tandemCARD (2CARD), into proximity to promote oligomeriza-tion of 2CARD, which in turn activates interferon signal-ing pathways by inducing MAVS filament formation.Interestingly, during each of these processes, ATP playscritical roles by allowing conformational change and olig-omerization of 2CARD within the filament, while trigger-ing dissociation of MDA5 from filament ends which thenregulates the global stability of the filament (and 2CARDoligomers) in a dsRNA length-dependent manner. Ourdiscoveries of the novel roles of ATP hydrolysis, filamentdynamics, and the structure of the MDA5:dsRNA com-plex together provide comprehensive understanding ofthe complex molecular mechanisms of MDA5 functionduring self and non-self RNA discrimination, and offernovel insights into the divergent evolution of MDA5 andRIG-I.

ReferencesPeisley, A., Jo, M. H., Lin, C., Wu, B., Orme-Johnson, M.,

Walz, T., … Hur, S. (2012). Kinetic mechanism for viraldsRNA length discrimination by MDA5 filament. Proceed-ings of the National Academy of Sciences U.S.A, 109,E3340–9. PMID: 23129641.

Peisley, A., Lin, C., Wu, B., Orme-Johnson, M., Liu, M., Walz,T., & Hur, S. (2011). Cooperative assembly and dynamicdisassembly of MDA5 filaments for viral dsRNA Recogni-tion. Proceedings of the National Academy of Sciences U.S.A, 108, 21010-5. PMID: 22160685.

Peisley, A., & Hur, S. (2012). Multi-level regulation of cellularrecognition of viral dsRNA. Cellular and Molecular LifeSciences, PMID: 22960755.

Wu, B., Peisley, A., Richards, C., Yao, H., Zeng, X., Lin, C.,… Hur, S. (2012). Structural basis for dsRNA recognition,filament formation and antiviral signaling by MDA5. Cell.Epub, PMID: 23273991.

197 Modeling the maturation pathwayof the HIV-1 5’-UTR RNA dimer

Nikolai B. Ulyanov*, Christophe Guilbert, RichardTjhen and Thomas L. James

Department of Pharmaceutical Chemistry, University ofCalifornia, San Francisco, CA 94158-2517, USA*Email: [email protected], Phone: +1-650-872-9107

HIV-1 genomic RNA is packaged as a dimer into thevirions. The initial metastable RNA dimer is believedto be formed by virtue of “kissing interactions”between two copies of the palindromic apical loops ofstem-loop SL1 of the 5’-untranslated region (5’-UTR)of the genomic RNA. Viral nucleocapsid protein NCp7promotes maturation of the RNA dimer into more sta-ble form, which involves extended or linear form ofSL1 dimer (reviewed in Paillart et al., 2004; Moore &Hu, 2009; Lu et al., 2011). In vitro experiments haveshown that this conversion occurs at stoichiometricamounts of NCp7 without breaking interactions betweenthe two copies of the SL1 apical loops (Mujeeb et al.,2007). We have proposed a hypothetical pathway andcalculated models of the intermediate structures for theSL1 stem-loop dimer maturation that does not requiresimultaneous dissociation of all base pairs in SL1stems; this pathway involves formation of an RNA ana-log of the Holliday junction intermediate between thetwo stems of the SL1 dimer and a following branchmigration towards the palindromic duplex (Ulyanovet al., 2011). Here, we extend these models to thedimer of the 1–344 fragment of HIV-1 RNA, whichincludes all of the 5’-UTR and the gag start AUGcodon region, and show that the branch-migrationmechanism of the dimer maturation is also feasible forthe full 5’-UTR RNA. All RNA models have been cal-culated with the miniCarlo program (Zhurkin et al.,1991).

This research was supported in part by the CaliforniaHIV/AIDS Research Program award ID09-SF-030.


ReferencesLu, K., Heng, X., & Summers, M. F. (2011). Journal of Molec-

ular Biology, 410, 609–633.Moore, M. D., & Hu, W. -S. (2009). AIDS Review, 11, 91–

102.Mujeeb, A., Ulyanov, N. B., Georgantis, S., Smirnov, I.,

Chung, J., Parslow, T. G., & James, T. L. (2007). NucleicAcids Research, 35, 2026–2034.

Paillart, J. -C., Shehu-Xhilaga, M., Marquet, R., & Mak, J.(2004). Nature Reviews Microbiology, 2, 461–472.

Ulyanov, N. B., Tjhen, R., Guilbert, C., & James, T. L. (2011).Journal of Biomolecular Structure And Dynamics, 28,1082–1083.

Zhurkin, V. B., Ulyanov, N. B., Gorin, A. A., & Jernigan, R. L.(1991). Proceedings Of The National Academy Of SciencesUSA, 88, 7046–7050.

198 MS-based approaches for thestructural determination ofretroviral ribonucleoproteins

Dan Fabris*, Matteo Scalabrin, Papa Nii Asare-Okai,Maria Basanta-Sanchez and Jennifer Lippens

The RNA Institute, University at Albany, Albany, NY 12222,USA*Email: [email protected], Phone: +1-518-437-4464,Fax: +1-518-442-3462

The interactions between the nucleocapsid (NC) domainof the Gag polyprotein and the 5′-untranslated region (5′-UTR) of viral RNA play multifaceted roles in the lifecy-cle of HIV-1. Owing to the well-known chaperone activ-ity of NC, such interactions may induce remodeling ofRNA structure which results in either exposing or con-cealing RNA signals responsible for different viral func-tions. For this reason, mapping the sequences bound by

NC must be followed by the characterization of theunderlying RNA structure to fully understand the biolog-ical significance of the specific interactions. We havedeveloped complementary approaches based on massspectrometry (MS) which enable the identification ofprotein-binding sites and the investigation of theirstructural context. One of them involves the applicationof bifunctional alkylating reagents to form irreversiblecross-links between protein and RNA moieties that areplaced within mutual striking distance by the ribonucleo-protein fold. The specific protein–RNA and RNA–RNAcontacts are characterized by protease/nuclease digestionto isolate the conjugated products followed by massmapping and sequencing. The analysis of NC·5′-UTRcomplexes in samples containing up to 10:1 protein toRNA ratios revealed the presence of six high-affinitysites, while a few others became detectable only after theionic strength was decreased to strengthen protein–RNAbinding. The major sites mapped to the trans-activationresponse element, the primer binding site, the primeractivation signal, and the packaging signal (Ψ-RNA). Insome instances, the RNA–RNA cross-links detected inthe same experiments revealed discrepancies with theleading secondary structures proposed for 5′-UTR consis-tent with possible structure remodeling mediated byNC. The significance of these findings is currently beingexplored by utilizing a series of relatively short (i.e. 8–12 nt) antisense oligonucleotides that are complementaryto the affected regions. Their specific binding is beingmonitored directly by MS and ion mobility spectrometrywhich enable one to observe the effects of ligands on theoverall topology and conformation of biomolecularcomplexes. The results are expected to provide theboundaries of the specific NC sites and to addresspossible long-range effects of binding on the entire foldof 5′-UTR. Further, this information will guide the


design of mutants lacking determinant features necessaryto foster NC-binding. Supported by MS detection, thecombination of cross-linking and antisense probingpromises to offer new insights necessary to understandthe structural rearrangements involved with 5′-UTR pro-cesses, which could provide the keys for developing newantiviral strategies.

Research supported by The RNA Institute of UAlbanyand the National Institutes of Health (GM064328-12).

199 Network analysis of inhibitionand resistance mechanisms inviral polymerases

Ashutosh Srivastavaa* and Somdatta Sinhab

aCSIR-Centre for Cellular and Molecular Biology, Hyderabad,AP, India; bIndian Institute of Science Education and Research(IISER), Mohali, Punjab, India*Email: [email protected]

Replication of viral genomes is one of the mostimportant steps in the infection process, as virusesneed to replicate inside the host cell in order to makemany copies. Therefore, traditionally, this has been animportant stage for drug targeting in antiviral therapies.For their replication, viral genes can encode polymer-ase enzymes like reverse transcriptase (RT) in retrovi-ruses, and RNA dependent RNA polymerase in RNAviruses. These polymerases have been important drugtargets against several viruses (Walker & Hong (2002),Sarafianos et al. (2009)). However, a major challengehas been the emergence of resistance owing to muta-tions in the genes coding for these enzymes, therebyrendering these drugs useless. Unravelling the mecha-nisms of inhibition and resistance is an important areaof research that contributes to both basic understandingof the processes and clinical applications. In this work,we have attempted to study the structural basis of themechanisms of inhibition and resistance in reversetranscriptase in human immunodeficiency virus (HIV-1), and compared that to the RNA dependent RNA

polymerase in Hepatitis C Virus (HCV), using a net-work-based approach. Though structure-based proteincontact networks (PCN) have been used to understandthe structure-function relationship in different proteins(Bagler & Sinha (2007), del Sol & O’Meara (2005)),using this approach to study such interactions has notbeen done before.

The PCNs of HIV-1 RT and HCV NS5B in inhibi-tor-bound and unbound states were developed in bothcoarse grained scale (Figure (a)), and by consideringthe side chains of the constituent amino acids. Thecontact patterns for all cases were analysed along withtheir network parameters. Though inhibitor binding andresistance mutations in RT cause significant functionalchanges, they are known to cause only subtle changesin the protein structures. Our results show that theycan be easily identified by changes in the contact pat-terns of the active site and inhibitor binding site resi-dues. Analysis of different network parameters (e.g.shortest paths, cliques and communities) provide aninsight into the communication pathways between theinhibitor binding region and the active site. Study ofthe allosteric communication pathways, considering thedynamics of the proteins (Figure (b)), shows that smallconformational fluctuations cause important changes inthe communication pathways (Figure (c)), thus contrib-uting to the overall effect of inhibition and resistance.These analyses can help in understanding the crucialresidues involved in resistance mutation directly orindirectly in the mechanism of inhibition and evolutionof resistance to drugs.

ReferencesBagler, G., & Sinha, S. (2007). Assortative mixing in protein

contact networks and protein folding kinetics. Bioinformat-ics (Oxford, England), 23, 1760–1767.

del Sol, A., & O’Meara, P. (2005). Small-world network approachto identify key residues in protein–protein interaction.Proteins, 58, 672–682.

Sarafianos, S. G., Marchand, B., et al. (2009). Structure andfunction of HIV-1 reverse transcriptase: Molecular mecha-nisms of polymerization and inhibition. Journal of MolecularBiology, 385, 693–713.

Walker, M. P., & Zhi, H. (2002). HCV RNA-dependent RNApolymerase as a target for antiviral development. CurrentOpinion in Pharmacology, 2, 534–540.


200 Potent inhibition of HIVreplication by RNA-bindingpeptide mimetics withunprecedented specificity

Gabriele Varani*

Department of Biochemistry and Department of Chemistry,University of Washington, Seattle, WA 98195-1700, USA*Email: [email protected], Phone: (206) 543 7113

RNA provides an inviting target for pharmaceuticalintervention in both infectious and chronic diseases,but it has so far been impossible to identify drug-likemolecules with sufficient potency to lead to the suc-cessful clinical applications. We have developed anew class of structurally constrained cyclic peptidesto target the interaction between the human immuno-deficiency virus (HIV-1) transactivator protein Tat andits response element TAR (1,2), which plays anessential role in viral replication. Many previousattempts to inhibit this interaction have failed to yieldmolecules with sufficient potency and specificity towarrant pharmaceutical development. The peptidicmimics of Tat that are pM inhibitors of the Tat-TARinteraction and discriminate > 1000 fold between clo-sely related RNAs. They are potent inhibitors of viral

replication (tens of nM) with no cytotoxicity and effi-cient cell penetration which specifically inhibit TAR-dependent reverse transcription as well as activationof transcription, and repress replication of a widevariety of viral strains representing all the major HIVclades in primary human lymphocytes (3). Thepotency and selectivity observed for this family ofpeptides is unprecedented among Tat inhibitors andsuggest that peptides of this class may be morewidely useful for the pharmacological inhibition ofother protein–RNA interactions.

ReferencesDavidson, A., Leeper, T. C., Athanassiou, Z., Patora-Komisarska,

K., Karn, J., Robinson, J. A., & Varani, G. (2009). Simulta-neous recognition of HIV-1 TAR RNA bulge and loopsequences by cyclic peptide mimics of Tat protein. Proceed-ings of the National Academy of Sciences USA, 106, 11931–11936.

Davidson, A., Patora-Komisarska, K., Robinson, J. A., & Varani,G. (2011). Essential structural requirements for specificrecognition of HIV TAR RNA by peptide mimetics of Tatprotein. Nucleic Acids Research, 39, 248–256.

Lalonde, M. S., Lobritz, M. A., Ratcliff, A., Chamanian, M., Ath-anassiou, Z., Tyagi, M. J. W, … Varani, G. (2011). Inhibitionof both HIV-1 reverse transcription and gene expression by acyclic peptide tht binds the Tat-Transactivation response ele-ment (TAR) RNA. PLoS pathogens, 7, e1002038.

201 The ESCRT pathway in HIVbudding and cell division

Wesley I. Sundquist*

Department of Biochemistry, University of Utah, Salt Lake City,UT 84112-5650, USA*Email: [email protected], Phone: (801) 585-5402,Fax: (801) 581-7959

The endosomal sorting complexes required for trans-port (ESCRT) pathway mediates membrane fissionreactions during intraluminal endosomal vesicle forma-tion, budding of HIV-1 and other enveloped viruses,and the final abscission step of cytokinesis in mam-

mals and archaea. Current models hold that ubiquitin-binding ESCRT factors act early in the pathway toregulate factor recruitment and assembly, whereas thelate acting ESCRT-III proteins form filaments that drawthe membranes together and mediate fission, possibly,in collaboration with VPS4-ATPases. I will discuss ourcurrent understanding of the structures and functionsof the different ESCRT factors in HIV budding andabscission with a particular focus on our studies aimedat understanding: (1) how ubiquitin regulates ESCRTrecruitment during HIV-1 budding and (2) the struc-tures and membrane-binding properties of ESCRT-IIIsubunits and filaments.


202 Structural aspects of the infectioncycles of giant viruses

Abraham Minsky* and Yael Mutsafi

Department of Structural Biology, The Weizmann Institute ofScience, Rehovot, 76100, Israel*Email: [email protected], Phone: 972-8-9342003,Fax: 972-8-9344142

With a particle size of �800 nm and a DNA genome of1.2M base-pairs, the recently discovered amoeba-infecting virus Mimivirus is the largest virus identified,blurring the established division between viruses and sin-gle-cell organisms. Such unusual parameters raise funda-mental questions related to the physical aspects of theMimivirus infection cycle. These include the mechanismsthat promote entry of the huge Mimivirus genome intohost cells and its trafficking within the highly crowdedhost cytoplasm, virion assembly, and genome packaging.Our studies indicate that, in contrast to all other DNAviruses, the Mimivirus releases its genome into the hostcytoplasm in a single step that is promoted by a large-scaleconformational change of the viral capsid (Figure). Thisprocess is followed by the assembly of a large and highlyelaborate viral factory in the host cytoplasm within whichmultiple viral progeny are rapidly generated in a pathwaythat is independent of the host nucleus (Zauberman et al.,2008). The transactions that occur in the viral factory,including replication, transcription, translation, membranebiogenesis, and capsid assembly are well-coordinated intime and space (Mutsafi et al., 2010; Mutsafi et al., submit-ted), thus, providing an unusual and exciting case study inself-assembly. Moreover, the transactions that lead to thegeneration of viral factories raise the intriguing notion thatsuch factories might have acted as precursors to eukaryoticnuclei. The implications of these findings on the evolutionof viruses and the role viruses might have played in theemergence of eukaryotic cells will be discussed.

References

Zauberman, N., et al. (2008). Distinct DNA exit and packagingportals in A. polyphaga Mimivirus. PLoS Biology, 6, 1104–1114.

Mutsafi, Y., et al. (2010). Vaccinia-like cytoplasmic replicationof the giant Mimivirus. Proceedings of the National Acad-emy of Science USA, 107, 5978–5982.

Mutsafi, Y. et al., (submitted). Membrane assembly during theinfection cycle of the giant Mimivirus.

203 Polymers through pores: single-molecule experiments with nucleicacids, polypeptides andpolysaccharides

Hagan Bayley*

Department of Chemistry, University of Oxford, Oxford,OX1 3TA, UK*Email: [email protected], Phone: +44 1865285101

The translocation of polymers through pores has beenexamined for almost two decades with an emphasis onnucleic acids. There are also interesting circumstances inbiology where polypeptides and polysaccharides passthrough transmembrane pores, and our laboratory hasbeen investigating examples of them. Single-moleculenucleic acid sequencing by nanopore technology is anemerging approach for ultrarapid genomics. Strandsequencing with engineered protein nanopores is a viabletechnology which has required advances in four areas:nucleic acid threading, nucleobase identification,controlled strand translocation, and nanopore arrays(Bayley, 2012). The latter remain a pressing need andour attempts to improve arrays will be described. In sev-eral physiological situations, folded proteins pass throughtransmembrane pores. We have developed a model sys-tem comprising mutant thioredoxins as the translocatedproteins, and staphylococcal alpha-hemolysin, as thepore. Our findings support a mechanism in which thereis local unfolding near the terminus of the polypeptidethat enters the pore. The remainder of the protein thenunfolds spontaneously and diffuses through the pore intothe recipient compartment (Rodriguez-Larrea & Bayley,2013). We have also examined the pore formed by theE. coli outer membrane protein Wza, which transportscapsular polysaccharide from its site of synthesis to theoutside of the cell. We made mutant open forms of thepore and screened blockers for them by electrical record-ing in planar bilayers. The most effective blocker bindsin the alpha-helix barrel of Wza, a site accessible fromthe external medium, and therefore, a prospective targetfor antibiotics (Kong et al., 2013).


ReferencesBayley, H. (2012). Are we there yet? Physics of Life Reviews,

9, 161–163.Kong, L., Harrington, L., Li, Q., Cheley, S., Davis, B. G., &

Bayley, H. (in press). Single-molecule interrogation of abacterial sugar transporter allows the discovery of an extra-cellular inhibitor. Nature Chemistry.

Rodriguez-Larrea, D., & Bayley, H. (in press). Multistepprotein unfolding during nanopore translocation. NatureNanotechnology.

204 Polymer translocation

Murugappan Muthukumar*

University of Massachusetts, Amherst, MA 01003, USA*Email: [email protected], Phone: (413)577-1212,Fax: (413)545-0082

The translocation of a single macromolecule through aprotein pore or a solid-state nanopore involves threemajor stages: (1) approach of the macromolecule towardsthe pore, (2) capture/recognition of the macromolecule atthe pore entrance, and (3) threading through the pore(see the Figure) (Muthukumar, 2011). All of these stagesare controlled by conformational entropy of the macro-molecule, charge decoration, and the geometry of thepore, hydrodynamics, and electrostatic interactions. Chiefamong the contributing factors are the entropic barrierpresented by the pore to the penetration of the macro-molecule, pore–polymer interactions, electro-osmoticflow, and the drift-diffusion of the macromolecule in

electrolyte solutions. A unifying theory of these contrib-uting factors will be described in the context of a fewillustrative experimental data on DNA translocation andprotein translocation through protein pores and solid-state nanopores. Future challenges to specific biologicalsystems will be briefly discussed.

ReferenceMuthukumar, M. (2011). Polymer translocation. CRC Press.

205 Ultrathin nanopores for structuralanalysis of small nucleic acids

Meni Wanunu*

Department of Physics and Chemistry/Chemical Biology,Northeastern University, Boston, MA 02115, USA*Email: [email protected], Phone: (617) 373-7412,Fax: (617)373-2940

Pinpointing the mechanisms behind function in biologi-cal macromolecules is essential for understanding theemerging and evolving nature of life. Biological mac-romolecules have evolved over billions of years tofunction efficiently in the highly heterogeneous,crowded environment of a cell. In particular, non-cod-ing ribonucleic acids (RNAs) and deoxyribonucleicacids (DNAs) are omnipresent in cells, preformingboth regulatory and catalytic functions by virtue oftheir structure. One class of such nucleic acids, ironi-cally termed “junk DNA,”1 is significantly involved inorchestrating gene regulation. The RNA subunits ofthe ribosome2 are an integral part of the translationalmachinery for protein synthesis. Many ribozymes andother viral RNAs such as the hammerhead ribozymeand the canonical internal ribosome entry site3 exhibitenzymatic activity. Although the exact mechanismsremain unclear, the uniting theme in these non-codingnucleic acids is that their tertiary (spatial) structureyields specific chemical activities and functions. Whileexisting techniques (e.g. nuclear magnetic resonance,X-ray crystallography) provide detailed structural infor-mation, inherent drawbacks such as ensemble averag-


ing errors, crystallization artifacts, low time resolutions,and the need for ample amounts of material limit theavailability and relevancy of the obtained structuralinformation. Bioinformatics-based tools aim to bridgethe gap in knowledge by proposing a homology-basedapproach to structural prediction, although viableexperimental techniques are required in order tounequivocally support and improve such predictions. Inthis talk, I will present our group's efforts to fabricatenanoscale pore devices for extracting useful structuralinformation about nucleic acids that display in vivofunction. Electron-beam irradiation of various thin free-standing membranes affords nanopores with controlleddimensions and interfacial properties, to a quality levelthat allows highly sensitive analysis of individualnucleic acids in solution at high-throughput. I willdescribe the properties of our nanopores, as well assome of our recent explorations that have permittedthe analysis of DNA and RNA structures.

This research is supported by NHGRI grant # R01HG006321, NHGRI grant # R21 HG006873.

ReferencesBiemont, C., & Vieira, C. (2006). Genetics - Junk DNA as an

evolutionary force. Nature, 443(7111), 521–524.Schluenzen, F., et al. (2000). Structure of functionally activated

small ribosomal subunit at 3.3 angstrom resolution. Cell,102(5), 615–623.

Spahn, C. M. T., et al. (2001). Hepatitis C virus IRES RNA-induced changes in the conformation of the 40S ribosomalsubunit. Science, 291(5510), 1959–1962.

206 An artificial active DNAtransporter from biologicalbuilding blocks

Lorenzo Franceschini, Misha Soskineand Giovanni Maglia*

Department of Chemistry, University of Leuven, Leuven, 3001,Belgium*Email: [email protected], Phone: +32(016) 327 696

The selective transport of molecules across membranepores is an essential biological process that occurs in allliving organisms. Examples include the chaperone ofproteins and mRNA in and out the cell nucleus mediatedby the nuclear pore complex and the transport of specificmolecules across a biological membrane mediated byactive and passive transporters. Here, I describe an artifi-cial DNA transporter that is formed by incorporating aDNA carrier element into a biological nanopore imbed-ded in a lipid bilayer. This device is able to transportspecific DNA strands across a biological membrane med-

iated by a simple reaction mechanism based on DNAstrand displacement. Similar to biological secondaryactive transporters, this system uses an electrochemicalpotential difference to pump a specific substrate across abiological membrane at a constant transmembrane poten-tial. Our DNA actuator might be used to separate orconcentrate nucleic acids, or to vehicle genetic informa-tion across the biological membranes.

This research has been supported by the EuropeanResearch Council (European Commission’s SeventhFramework Programme, project no. 260884).

207 Nanopore immobilization of DNApolymerase enhancessingle-molecule sequencing

Joseph Larkina*, Matieu Foquetb, Jonas Korlachb andMeni Wanunua

aDepartment of Physics, Northeastern University, 360Huntington Ave, Boston, MA 02115, USA; bPacific Biosciences,Menlo Park, CA, USA*Email: [email protected], Phone: (617) 373-7818,Fax: (617) 373-2943

A zero-mode waveguide (ZMW) is a nanoscale opti-cal waveguide driven at a frequency below its cut-off.In this mode, the electric field, instead of travelingdown the axis of the conducting cavity, decays expo-nentially. By fabricating waveguides with sub-wave-length diameters and illuminating them with laserlight, the electric field in the waveguide is confinedenough to enable single-molecule optical detection atmicromolar concentration [1]. Immobilizing singleDNA polymerases in ZMWs and using special phos-phate-fluorescently labeled dNTPs form the basis for


single-molecule real-time DNA sequencing, one of themost promising next-generation sequencing platforms[2]. In this method, the polymerase replicates thesample DNA, and as it incorporates new bases intothe product strand, the labeled dNTPs emit a burst oflight before the phosphate is cleaved off. Thesequence of colors corresponds to the DNA sequence(see Figure 1 below from Eid et al., 2009). Becausethe ZMW aperture’s diameter is sub-diffraction-limit,it is impossible to optically distinguish one polymer-ase in a ZMW from two. Having only one polymer-ase in each waveguide is critical to sequencingaccuracy. In its present state, experimenters use diffu-sion to fill ZMWs with polymerases, resulting in aPoisson distribution for filling ZMWs, and conse-quently a theoretical limit of 36.8% of ZMWs havingonly one polymerase [2]. We achieve full polymeraseoccupancy of ZMWs by fabricating the structures onan ultrathin silicon nitride membrane and drilling ananopore at the base of each waveguide with an ionbeam. A short DNA fragment with biotin on eitherend is conjugated to a streptavidin and then drawninto the nanopore with a voltage bias. There is thena free biotin at the base of the ZMW. A polymerase–streptavidin complex can diffuse into the ZMW andbind to the exposed biotin. Because the nanopore istoo small to fit more than one molecule, only oneZMW will bind to a biotin in the nanopore. Uponflushing the ZMW chamber, the biotin-bound poly-merase will remain trapped in the pore, and only asingle polymerase will remain at the base of eachwaveguide.

ReferencesLevene, M. J., et al. (2003). Zero-mode waveguides for single-

molecule analysis at high concentrations. Science, 299, 682–686.

Eid, J., et al. (2009). Real-time DNA sequencing from singlepolymerase molecules. Science, 323, 133–138.

208 Electrophoresis and capture ofDNA into a nanopore

Alexander Y. Grosberg*

Department of Physics and Center for Soft Matter research,New York University, New York, NY 10003, USA*Email: [email protected], Phone: (212) 992-9574,Fax: (212) 995-4016

DNA capture into a nanopore is driven by the electricfield which extends out of the pore and is not subject toDebye screening, because it is maintained by the exter-nally applied voltage (M.Wanunu et al., 2010). Thisoutside field is closely related to the pore access resis-tance (Kowalczyk et al., 2011). DNA motion in thishighly nonuniform field is coupled to both electric andhydrodynamic currents. To understand this complexdynamics, we develop a set of scaling arguments. Firstwe re-examine the classical problem of DNA electropho-resis in a uniform field and rederive the known resultsfor DNA electrophoretic mobility which does not dependon DNA length. Second, we generalize these scalingarguments for the nonuniform field. We show that thedistance between DNA end and the pore entrance can beviewed as a reaction coordinate describing the captureprocess, and this description remains marginally applica-


ble up until DNA entrance into the pore. This descriptionnaturally couples to the nonequilibrium description ofDNA translocation based on the concept of iso-fluxtrumpet (Rowghanian & Grosberg, 2011).

This research was sponsored in part by the US-IsraelBinational Science Foundation.

ReferencesKowalczyk, S. W., Grosberg, A. Y., Rabin, Y., & Dekker, C.

(2011). Modeling the conductance and DNA blockade ofsolid-state nanopores. Nanotechnology, 22, 315101.

Rowghanian, P., & Grosberg, A. Y. (2011). Force driven poly-mer translocation through a nanopore: an old problemrevisited. Journal of Physical Chemistry B, 115, 14127–14135.

Wanunu, M., Morrison, W., Rabin, Y., Grosberg, A. Y., & Meller,A. (2010). Electrostatic focusing of unlabeled DNA intonanoscale pores using a salt gradient. Nature Nanotechnol-ogy, 5, 160–165.

209 Designing stiff protein nanoporesfor challenging tasks in biosensing

L. Movileanu*

Department of Physics, Syracuse University, 201 Physics Bldg,Syracuse, NY 13244-1130, USA*Email: [email protected], Phone: (315) 443-8078,Fax: (315) 443-9103

Protein nanopore-based sensing elements represent apressing need in molecular biomedical diagnosis (Bayley& Cremer, 2001). However, the integration of proteinnanopores with other nanofluidic devices is a challengingtask. This is especially true, if we consider that isolatedsingle proteins are in general fragile and unstable underharsh conditions of detection. Here, I will present a strat-egy for improving the stability of the open-state currentof a redesigned nanopore using ferric hydroxamateuptake component A (FhuA), a beta-barrel membraneprotein channel of E. coli (Mohammad et al., 2012). Theprimary function of FhuA is to facilitate the energy-dri-ven, high-affinity Fe3+ uptake complexed by the sidero-phore ferrichrome (Paweleket al., 2006). The keyingredient of this strategy was the coupling of directgenetic engineering of FhuA with a fast-dilution refold-ing approach to obtain an unusually stable protein nano-pore under a broad range of experimentation. Theseadvantageous characteristics were recently demonstratedby examining proteolytic activity of an enzyme at ahighly acidic pH, a condition at which majority of beta-barrel protein nanopores are normally gated or unfolded.Future membrane protein design work will not onlyreveal a better understanding of the processes employedin membrane protein folding and stability, but will alsoserve as a platform for the integration of robust protein

components into devices (Astier, Bayley, & Howorka,2005).

This research has been supported in part by the grantsNSF DMR-1006332 and NIH GM088403.

ReferencesAstier, Y., Bayley, H., & Howorka, S. (2005). Protein

components for nanodevices. Current Opinion in ChemicalBiology, 9, 576–584.

Bayley, H., & Cremer, P. S. (2001). Stochastic sensors inspiredby biology. Nature, 413, 226–230.

Mohammad, M. M., Iyer, R., Howard, K. R., McPike, M. P.,Borer, P. N., & Movileanu, L. (2012). Engineering a rigidprotein tunnel for biomolecular detection. Journal of theAmerican Chemical Society, 134, 9521–9531.

Pawelek, P. D., Croteau, N., Ng-Thow-Hing, C., Khursigara,C. M., Moiseeva, N., Allaire, M., & Coulton, J. W. (2006).Structure of TonB in complex with FhuA, E. coli outermembrane receptor. Science, 312, 1399–1402.

210 Computational study of substratesand mediators features of lacasses

Azar Delavari and Juan J. Perez*

Department of Chemical Engineering, Univeristat Politecnicade Catalunya, 08028 Barcelona, Spain*Email: [email protected], Phone: 0034-93-4016679,Fax: 0034-93-4017150

Laccases are enzymes of the family multicopperoxidases, being widely used for biotechnological appli-cations (Canas & Camarero, 2010). The enzymes’ cata-lytic cycle consists of the oxidation of the substratewith the concomitant reduction of molecular oxygen towater. In this process, the substrate is converted to afree radical, that can oxidize larger substrates acting asa mediator or it can undergo polymerization. Substratebinding is not specific, and there is a large diversity ofsubstrates for laccases. Moreover, the binding siteshows important differences among diverse species. Thegoal of the present work is to characterize the laccasebinding pocket of different species, in order to establishtheir common pharmacophoric characteristics. For thispurpose, we have carried out docking studies with asubset of substrates, covering the diversity of substratesusing the Glide program (Friesner et al., 2004). Wehave also analyzed the characteristics of the bindingsite using diverse probes. We further have rationalizedthe differential values of km found among diverse spe-cies for a specific substrate. Finally, special attentionhas been devoted to the binding of the mediator 2,2′-azido-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS),


commonly used in industrial processes. Figure 1 shows,ABTS docked onto the fungal laccase, whereas Figure 2shows ABTS docked onto the bacterial laccase. Theanalysis of the protein–ligand complex together with thecorresponding optimized geometries of the possible sub-strate species carried out using DFT suggest that thebound species is the protonated form of ABTS as previ-ously suggested (Enguita et al., 2004). Furthermore, theresults of this study also suggest that its mechanism ofoxidation occurs in a similar way to the rest of sub-strates/mediators, in contrast to previous suggestions(Fabbrini et al., 2002).

This research has been supported by Bioprocel, Spain.

ReferencesCanas, A. I., & Camarero, S. (2010). Laccases and their nat-

ural mediators: Biotechnological tools for sustainableeco-friendly processes. Biotechnology Advances, 28, 694–705.

Enguita, F. J., Marçal, D., Martins, L. O., Grenha, R., Henri-ques, A. O., Lindley, P. F., & Carrondo, M. A. (2004).Substrate and dioxygen binding to the endospore coatlaccase from Bacillus subtilis. Journal of BiologicalChemistry, 279, 23472–23476.

Fabbrini, M., Galli, C., & Gentili, P. (2002). Comparing thecatalytic efficiency of some mediators of laccase. Journalof Molecular Catalysis B, 16, 231–240.

Friesner, R. A., Banks, J. L., Murphy, R. B., Halgren, T. A.,Klicic, J. J., Mainz, D. T., … Shenkin, P. (2004). Glide: Anew approach for rapid, accurate docking and scoring. 1.Method and assessment of docking accuracy. Journal ofMedicinal Chemistry, 47, 1739–1749.

211 Synthesis of informationalpolymers by guidedpolymerization reactions in liquidcrystalline matrices of lipids

Chaitanya Mungi and Sudha Rajamani*

Department of Biology, Indian Institute of Science Educationand Research (IISER), Pune, Maharashtra 411021, India*Email: [email protected], Phone: 91-20-25908061

The fundamental question of how life originated is one ofthe greatest scientific mysteries. In particular, the pro-cesses by which polymers are capable of catalysis andreplication that emerged on early Earth are still elusive.We investigated the guided polymerization reactionswhich can occur in organized liquid crystalline matricesof amphiphilic lipids. These result from spontaneous self-assembly of amphiphiles that form orderly structures likemultilamellar phases, which can concentrate reactants,yet also permit diffusional mobility. These lamellaeenhance reaction rates by promoting self-aggregation ofreactants in configurations that enhance polymerization,and prevent side reactions (Wächtershäuser, 1988).Monomers form highly ordered 2D films between thelamellae, thereby overcoming entropic barriers. The con-ditions also produce a chemical potential that reduceswater activity and shifts reaction equilibrium towardscondensation. Using relatively simple amphiphilic lipids,we are discerning the process underlying oligomerizationof biologically relevant monomers like nucleotides,amino acids, etc, which might have resulted in the forma-tion of complex mixtures of oligomers under prebioticconditions. I will present some preliminary data towardsthe end. Delineating the underlying mechanisms, lipid-


catalyzed reactions has implications for emergence ofinformational molecules on prebiotic earth. It is also rele-vant for gaining an understanding of how amphiphilicboundary structures contributed to the emergence ofcellular life as it has been postulated that, on early Earth,encapsulated compartments containing evolving replicat-ing molecules might have existed in the form of stablemicelles (Orgel, 2004). Additionally, it is also believedthat lipids might have played a potentially importantmetabolic role in the emergence of life on Earth (Segréet al., 2001).

ReferencesOrgel, L. E. (2004). Prebiotic chemistry and the origin of the

RNA World. Critical Reviews in Biochemistry and Molecu-lar Biology, 39, 99–123.

Segré, D., Ben-Eli, D., Deamer, D. W., & Lancet, D. (2001).The Lipid World. Origins of Life and Evolution of theBiosphere, 31, 119–145.

Wächtershäuser, G. (1988). Before enzymes and templates:Theory of surface metabolism. Microbiology Reviews, 452–484.

212 Shifting interfaces: changes inprotein–protein and protein–DNAinterfaces probed via moleculardynamics

Lacra Negureanu and Freddie Salsbury Jr.*

Department of Physics, Wake Forest University, Winston-Salem,NC 27109, USA*Email: [email protected], Phone: 336-758-4975

Over the past decade, there has been a growing interestin studying the binding of DNA to the MutSalpha pro-tein complex. This heterodimeric protein complex, theMsh2/Msh6 complex in humans, is the initial complex

that binds mismatched DNA and other DNA defects thatoccur during replication. This complex has also beenshown to bind at least some types of damaged DNA,such as the cross-linked adducts due to the chemothera-peutics cisplatin and carboplatin, or the incorporation ofthe chemotherapeutic, FdU. As a result of this interest,multiple studies have contrasted the interactions ofMutSalpha with its normal mismatched substrate andwith the interactions of MutsSalpha with the DNAdamaged by chemotherapeutic cisplatin. To complementthese studies, we examine the interaction betweenMutSalpha and the DNA damaged by carboplatinvia all-atom molecular dynamics simulations. Thesesimulations provide evidence for subtle changes in theprotein–DNA and protein–protein interfaces. The inter-faces shifts found are broadly similar to those found inbinding with adduct from cis-platin, but have distinctdifferences. These subtle differences may play a role inthe way of the different damages and mismatched DNAare signaled by MutSalpha, and suggest a signalingmechanism for DNA damage that chiefly involves shiftsin protein–protein interactions as opposed to changes inprotein conformation.

ReferencesNegureanu, L., & Salsbury, F. R. Jr. (2012a). Insights into

protein–DNA interactions, stability and allosteric communi-cations: A computational study of Mutsalpha-DNA recogni-tion complexes. Journal of Biomolecular Structure &Dynamics, 29, 757–779.

Negureanu, L., & Salsbury, F. R. Jr. (2012b). The molecularorigin of the MMR-dependent apoptosis pathway fromdynamics analysis of the MutSalpha-DNA complexes.Journal of Biomolecular Structure & Dynamics, 30, 347–361.

Salsbury, F. R. Jr., Clodfelter, J. E., Gentry, M. B., Hollis, T.,& Scarpinato, K. D. (2006). The molecular mechanism ofDNA damage recognition by MutS homologs and its con-sequences for cell death response. Nucleic Acids Research,34, 2173–2185.


213 Structures and interactions ofproteins involved in ER-associatedprotein degradation

Udo Heinemanna,b*, Anup Arumughana,b,Jennifer Hannaa,b, Yvette Roskea,b, Anja Schütza,b andErich E. Wankera,b

aMax-Delbrück-Centrum für Molekulare Medizin, Berlin-Buch,Germany; bInstitut für Chemie und Biochemie, Freie Universität,Berlin, Germany*Email: [email protected], Phone: 0049-30-9406-3420, Fax: 0049-30-9406-2548

Proteins are translocated into the endoplasmic reticulum(ER) of cells in an unfolded state, and acquire theirnative conformation in the ER lumen after signal peptidecleavage. ER-associated degradation (ERAD) of folding-incompetent protein chains is mediated by the proteincomplexes residing in the ER membrane. We study thearchitecture and function of one of these, the HRDcomplex assembled around the E3 ubiquitin ligase Hrd1.The recognition of ERAD substrates is linked to thematuration of their carbohydrate structures. The HRDcomplex-associated lectin Yos9 is involved in ERADsubstrate recognition by binding carbohydrates throughits mannose-6-phosphate receptor homology (MRH)domain. We have determined the crystal structure of acentral domain of Yos9, adjacent to the MRH domain,which was previously annotated as interaction regionwith the HRD subunit Hrd3 (Hanna et al., 2012). Wefind that this domain does not support Hrd3 associationwhich we map to the N-terminal half of Yos9 instead. Incontrast, the domain has a function in Yos9 dimerizationas seen in the crystal structure, in various solution exper-iments and as supported by mutagenesis of dimer inter-face residues. The dimerization of the ER-luminal Yos9,in conjunction with studies of the cytosolic domain ofthe HRD component Usa1 (Horn et al., 2009) and other

biochemical data thus supports a model of a HRDcomplex that exists and functions as a dimer or a highermultimer. The delivery of ubiquitinated ERAD substratesto the proteasome is mediated by the cytosolic AAAATPase Cdc48 (p97 in mammalian cells). The p97(VCP) serves a wide variety of cellular functions in addi-tion to its role in ERAD, including organelle membranefusion, mitosis, DNA repair, and apoptosis. These differ-ent functions are linked to the binding of adaptorproteins to p97, many of which contain ubiquitin regula-tory X (UBX) domains. One of these adaptors, ASPL(alveolar soft part sarcoma locus), uses a substantiallyextended UBX domain for binding to the N domain ofp97 where a lariat-like, mostly α-helical extension wrapsaround one subunit of p97. By this binding ASPL trig-gers the dissociation of functional p97 hexamers leadingto partial inactivation of the AAA ATPase. To the bestof our knowledge, this is the first time that the structuralbasis for adaptor protein-induced inactivation by hexa-mer dissociation of p97 and, indeed, any AAA ATPasehas been demonstrated. This observation has far reachingimplications for AAA ATPase-regulated processes.

This research has been supported by the DeutscheForschungsgemeinschaft through CRG 740.

ReferencesHanna, J., Schütz, A., Zimmermann, F., Behlke, J., Sommer,

T., & Heinemann, U. (2012). Structural and biochemicalbasis of Yos9 protein dimerization and possible contribu-tion to self-association of 3-hydroxy-3-methylglutaryl-coen-zyme A reductase degradation ubiquitin-ligase complex.Journal of Biological Chemistry, 287, 8633–8640.

Horn, S. C., Hanna, J., Hirsch, C., Volkwein, C., Schütz, A.,Heinemann, U., … Jarosch, E. (2009). Usa1 functions as ascaffold of the HRD-ubiquitin ligase. Molecular Cell, 36,782–793.


AUTHOR INDEX

Abe, N. 43Achalere, A. 41Adesoba, V.T. 84Agris, P.F. 17, 71Agudelo, D. 85Aguilera, A. 81Akhoury, J. 31Aksenova, A. 84Alber, F. 64Aldersley, M.F. 5,6Alexandrov, A.A. 35Alexandrov, B.S. 49Aloyan, L.R. 56Amirbekyan, K.Yu. 59An, N. 30Ananyan, G.V. 55Andrianov, A.M. 124, 126Andriasyan, V.K. 55Anishetty, S. 112Annabhimoju, R. 103Antaramian, A. 79Antonyan, A.P. 58, 59Appella, E. 41Arakelyan, H.V. 55Arakelyan, V.B. 55, 58Arora, P.S. 120Arumughan, A. 139Arya, G. 33Asare-Okai, P.N. 25, 129Attwater, J. 7Aytenfisu, A.H. 18

Bairagya, H.R. 111Bajaj, C. 19, 20Bamburg, V. 35Banas, P. 25, 70Banavali, N.K. 121Barkhudaryan, V.G. 56Basanta-Sanchez, M. 129Bashan, A. 1Bashkin, J.K. 44Bathaie, S.Z. 27, 62Bayley, H. 132Bazantova, P. 40Beglov, D. 57Belfort, G. 102Belfort, M. 15, 102Bellaousov, S. 18Belotserkovskii, B.P. 83Ben-Naim, A. 106

Beno, I. 37Bernard, S.E. 76Best, R.B. 25Bhargavi, K. 102Bilbille, Y. 17Bilotti, K. 66Birktoft, J.J. 84Birney, E. 49Bishop, A.R. 49Bishop, T.C. 35Blanchard, J.S. 115Bobay, B.G. 17Bocharova, T.N. 82Bogdanove, A. 64Bohn, M.-F. 74Bohnuud, T. 57Bondos, S.E. 96Botta, G. 6Brazdova, M. 40Brenke, R. 57Brewster, A. 78Brown, W. 74Bulyk, M.L. 45Burrows, C.J. 30Buss, S.N. 19, 20Bussemaker, H.J. 65Byrnes, J. 80

Caetano-Anolles, G. 14Cafferty, B.J. 12Callahan, B. 102Cantara, W.A. 71Carneiro, K.M.M. 87Carpenter, M.A. 74Carr, S.B. 50Carter, Jr., C.W. 8Cassidy, L. 26Castellano-Pozo, M. 81Castor, K.J. 31, 88Celikel, R. 112Chaikin, P.M. 84Chalamcharla, V.R. 15Chandrasekaran, A.R. 84, 85Chandrasekaran, A. 84Chatterjee, A. 117Chatterjee, S. 108Cheatham III, T.E. 70Chen, A.A. 25Chen, I.A. 8Chen, J. 48

Chen, M.C. 12Chen, X.S. 78Cheng, Y. 49Cheong, V.V. 109Cherven, J. 40Chiang, C. 76Christensen, L.A. 74, 75Chung, W.J. 29Chvatalova, B. 40Civis, S. 11Clauvelin, N. 34, 36Cobb, G. 98Colasanti, A.V. 34Collier, M.L. 8Connolly, K. 22Conway, J.W. 88Costanzo, G. 6Cui, F. 39Culver, G.M. 22

Dailidonis, V.V. 60Dalyan, Y.B. 55, 56Danilov, V.I. 60Datta, P.P. 117Davis Harris, G. 44Deamer, D. 9Del Sol Mesa, A. 98Delaney, S. 36, 50, 66, 73, 78Delavari, A. 136Demirel, E. 84DeRosa, M. 89DeRosa, M.C. 57, 90desGeorges, A. 19, 20Dewhurst, S. 95Dey, S.K. 77Dhingra, P. 67Dill, K. 110DiMaio, J.T.M. 95Diskin-Posner, Y. 38Dong, X. 15, 49Dror, I. 43, 45Duax, W.L. 13Dupureur, C.M. 44Dzhelyadin, T.R. 44Dziak, D. 13

Easterhoff, D. 95Edgell, D. 64Edwardson, T.G.W. 87

Eisenstein, M. 24Elbaum-Garfinkle, S. 98Eldar, A. 38Endo, M. 93Endutkin, A. 72Engelhart, A.E. 4Eritja, R. 12

Fabris, D. 25, 129Fan, Z. 92Farley, K.I. 12Fedorova, O.S. 72Fedoseyeva, V.B. 35Felipe-Abrio, I. 81Ferris, J.P. 5, 6Ferus, M. 11Fleming, A.M. 30Flores-Juarez, C.R. 79Fojta, M. 28Foquet, M. 134Forsburg, S.L. 78Foster, A.G. 90Fox, G.E. 2Franceschini, L. 134Frank-Kamenetskii, M.D. 57Frank, J. 19, 20Frankenstein, Z. 24Frenkel, Z.M. 13Freudenreich, C.H. 83Fu, J. 19, 20Fu, Y. 19, 78

Gabrielian, A. 82Gallego, I. 12Garcia-Diaz, M. 80Garcıa-Muse, T. 81Garcıa-Rubio, M.L. 81Garcia, A.E. 25Garipova, M.I. 118Garton, M. 112Geng, C. 91Gevorkyan, K. 82Ghosh, D. 77Ghosh, S. 108Gnanapradeepan, K. 83Gomes, J. 103Gonzalez III, G. 112Gonzalez-Jasso, E. 79Gordan, R. 45

Journal of Biomolecular Structure and Dynamics, 2013http://dx.doi.org/10.1080/07391102.2013.341943

Copyright � 2013 Taylor & Francis

Govorov, A.O. 92Grassucci, B. 20Grassucci, R.A. 19Greven, M.C. 49Grigoryan, Z.A. 60Grosberg, A.Y. 135Grossfield, A. 95Gryaznov, S.M. 3Guilbert, C. 128Gupta, A. 26Gupta, N. 22Gustilo, E.M. 71Guzev, M.A. 70

Hall, D.R. 57Hambardjieva, E. 80Hambardzumyan, L.A. 58Hamblin, G.D. 88Hambrdzumyan, L.A. 59Hanawalt, P.C. 83Hancock, M. 31Hanna, J. 139Hansma, H.G.L. 9Haran, T.E. 37Harris, K. 71Harris, K.A. 17Harris, R.S. 74Harutjunyan, A. 82Harutjunyan, G. 82Harutyunyan, S.V. 55Hashem, Y. 19, 20Hausman, D. 84Hazra, S. 115He, G. 44Heddi, B. 29, 109Heinemann, U. 139Hogele, A. 92Holahan, M.R. 57Holliger, P. 7Hong Zhou, Z. 78Honig, B. 43, 48, 116Horton, J. 45Hoshyar, R. 27, 62House, N. 83Huang, J. 50Hud, N.V. 12, 29Huo, R. 42Hur, S. 127

Ilkhani, H. 27Israeloff, N.E. 42Ivanov, A.A. 52Iyer, S. 49

James, T.L. 128Jasper, K.M. 32Jayaram, B. 67, 103Jernigan, R.L. 106Jia, K. 106Joan Curcio, S.M. 15Jobe, A. 19, 20Johnson, S.E. 53, 54

Johnston, W. 35Jordan, J.J. 37Joshi, P.C. 5, 6Joshi, R. 41Jossinet, F. 19, 20Jurecka, P. 67, 70

Kabanov, A.V. 10Kaiser, R. 71Kamzolova, S.V. 122Kamzolova, S.G. 44Karamychev, V.N. 41Karapetian, A.T. 58, 59, 60Karthick, R. 112Kashyn, I.A. 124, 126Kaushal, S. 83Keller, S. 24Kerem, B. 75Khechinashvili, N.N. 10Khutshivilli, I. 54Kiran Kumar, M. 118Kirsanov, K.I. 52Kleinstiver, B. 64Koeller, K.J. 44Kolaczyk, T. 64Komarov, V.M. 10Kompanichenko, V.N. 16Kondratyev, M.S. 10Korlach, J. 134Kornoushenko, Y.V. 126Koulgi, S. 41Kozakov, D. 57Krupkin, M. 1Kuhrova, P. 25Kundaje, A. 49Kurouski, D. 94Kuznetsov, N.A. 72Kuzyk, A. 92

Lackey, L. 74Lafuente-Barquero, J.F. 81Land, A.M. 74Lannan, F.M. 29Larkin, J. 134Law, E.K. 74Lazarovici, A. 65Lech, C.J. 29Lednev, I.K. 94Lee, H.-T. 53Lee, H. 48Lee, S.-M.A. 83Lehmann, M. 84Lekontseva, N.V. 51Leonard, B. 74Leszcyska, G. 71Li, L. 4, 5, 8Li, M. 74Li, P.T.X. 24,25Liao, H.Y. 19, 20Liedl, T. 92Liman, J. 35Lin, X. 49

Lippens, J. 129Liu, F. 32Liu, J. 106Liu, P. 48Liu, S.-Q. 98, 100Liu, Y. 90,96Liu, Z. 31Lomzov, A. 46Lott, B.B. 22Lovett, S.T. 81Lu, Y. 65Lu, Z.J. 19Luque, F.J. 67, 70

MacCallum, J. 110Macdonald, M. 95Machado, A.C.D. 46, 65Madison-Antenucci, S. 19, 20Madularu, D. 57Maglia, G. 134Maher III, L.J. 42Maie, K. 61Malathi, R. 112Malelahi, S. 27Malevanets, A. 112Malkiewicz, A. 71Mamajanov, I. 29Mamasakhlisov, Y.Sh. 60Mandel-Gutfreund, Y. 43, 47Manjari, S.R. 121Mann, R. 48Mann, R.S. 43Markarian, S.A. 59Marky, L.A. 53, 54Masterson, P. 95Mastronardi, E. 89Mathews, D.H. 17, 18, 19Matthews, K.S. 96Mauro, E.D. 6Mazur, S. 41McCauley, M.J. 42, 58McConnell, E.M. 57McDowell, B. 64McGinty, R. 84McGown, L.B. 26McGuinness, K.N. 109Mckeague, M. 89McKinnon, S. 112McLaughlin, C.K. 87, 88Mecia, L.B. 50Medrano, V. 36Mejia, E. 80Melkonyan, L.V. 110Meluzzi, D. 33Menendez, D. 37Merwin, S. 15Miao, H. 95Minasyants, M.V. 59Minsky, A. 132Mirkin, S. 84Mirkin, S.M. 83Mishra, A. 67

Mishra, B.N. 101Mishra, D.K. 111Mishra, S. 116Mittermaier, A. 31Mladek, A. 11Moitessier, N. 31Monreal, C. 89, 90Morgunov, I.G. 122Morii, T. 86Morozov, V.F. 110Morrow, J.R. 62Mousavi, M.F. 27Movileanu, L. 136Mukhaelyan, Z.H. 58Mukherjee, G. 67Mukhopadhyay, B.P. 111Mungi, C. 137Munikumar, M. 105, 113, 114Murphy IV, F.V. 71Murugesapillai, D. 42Muser, S.E. 91Muthukumar, M. 133Mutsafi, Y. 132Myers, R.M. 49

Nakamura, M. 61Nakata, E. 86Nakazato, T. 22Nanaware-Kharade, N. 112Nanda, V. 109Nath, A. 98Nathan, J.R. 119Negureanu, L. 138Neil, A.J. 83Nelson Holte, M.H. 42Neumann, R.D. 41Ngan, C.H. 57Nilsson, B.L. 95Niranj Chandrasekaran, S. 8Noble, W.S. 49Noel, J.K. 21Norouzi, D. 33Novikova, O. 15

Obbad, S.I. 84Ohayon, Y.P. 84, 85Olmon, E.D. 73Olson, C.M. 54Olson, W.K. 34, 36, 104Ortiz, J.F. 95Ostermeir, K. 106Otsuka, Y. 61Otyepka, M. 25, 67, 70Ozeri-Galai, E. 75

Parker, S.C.J. 76Padhi, A.K. 103Panchenko, A. 115Panyutin, I.G. 41Pardatscher, G. 92Parsadanyan, M.A. 59Paukstelis, P.J. 91

Author Index 141

Pearson, S. 102Pecinka, P. 40Peisley, A. 127Perez, A. 110Perez, J.J. 136Peterson, E. 112Petrey, D. 116Petrov, V.V. 123Phan, A.T. 29, 109Phillips, S.E.V. 50Pierce, B.G. 49Pino, S. 6Pless, R.C. 79Poghosyan, N.L. 110Popov, A.V. 68Porter, L.L. 107Powner, M.W. 4Pradhan, D. 105, 113, 114Prislan, I. 54Priyadarshini, V. 105, 113, 114Pyshnyi, D. 46

Qu, G. 15

Raghunath, G. 112Ragunathan, M. 119Rai, S. 101Rajamani, S. 137Ramasree, D. 102Ranatunga, N. 78Rando, O.J. 49Rasmussen, K.O. 49Rathore, A. 74Reiling, C. 53, 54Ren, G. 68Resnick, M.A. 37Rhoades, E. 98Rivas, M. 2Rivera, K.G. 8Robbins, T.J. 56, 126Rohs, R. 43, 45, 46, 63, 65, 66Rojas, A.V. 95Roller, E.-M. 92Rose, G.D. 107Rosenthal, K. 37Roske, Y. 139Rouzina, I. 58Rozenberg, H. 38Rusling, D.A. 85

Sabo, P.J. 65Saeki, T. 61Sagi, J. 28Saha, C. 77Saladino, R. 6Saleh, S.S. 83Salsbury, Jr., F. 138Salyanov, V.I. 52Samchenko, A.A. 10Sanbonmatsu, K.Y. 21Sander, S.A. 62

Sandstrom, R. 65Sang, P. 100Sankar, K. 106Santiago, R. 24Santos-Pereira, J. 81Sanyakamdhorn, S. 85Sargsyan, S.A. 110Sarita Rajender, P. 102Scalabrin, M. 129Schermerhorn, K. 66Schermerhorn, K.M. 78Schiffer, C.A. 74Schimmel, P. 1Schreiber, R. 92Schutz, A. 139Seeman, N.C. 84, 85Selvaraj, C. 119, 127Sengupta, J. 21Senitzki, A. 37Serdyuk, I.N. 51Sergeev, A.V. 51Serpell, C.J. 87Sha, R. 84, 85Shafer, A. 65Shah, R.C. 84Shahhamzeie, N. 27Shahinyan, M.A. 59Shakked, Z. 38Shandilya, S.M.D. 74Sharav, J. 37Sharma, N. 41Shasmal, M. 21Shaw, D.E. 108Shazman, S. 48Shen, N. 45Sheng, J. 4Shika, S. 119Shin, J.H.S. 83Siltberg-Liberles, J. 95Simmel, F.C. 92Sinai, M.I.-T. 75Singh, G. 34Singh, P. 127Singh, R.K. 127Singh, S.K. 119, 127Singh, T. 67Sinha, S. 130Skurichin, E.E. 70Slattery, M. 43Slaymaker, I.M. 78Sleiman, H. 88Sleiman, H.F. 31, 87, 88Smirnova Alexander, E.A. 82Snider, A. 83Snyder, M. 49Solow, J. 35Somvanshi, P. 101Sonavane, U. 41Soni, A. 67Sorokin, A.A. 44Soskine, M. 134

Sperling, J. 23, 24Sperling, R. 23, 24Spitser, S.A. 74Sponer, J. 11, 25, 67, 70Sponer, J.E. 11Sproat, B. 71Srivastava, A. 130Stamatoyannopoulos, J.A. 65Stelfox, Alice, J. 50Stepanov, V. 2Stephenson, W. 24, 25Su, X.A. 83Subramaniam, V. 97Subramanian, H.K.K. 76Sugiyama, H. 93Sultan, Y. 90Sundquist, W.I. 131Susova, O.Yu. 52Swargam, S. 105, 113, 114Szostak, J.W. 2, 3, 4, 5

Tajmir-Riahi, H.A. 85Takada, T. 61Takamatsu, Y. 61Tao, Y. 98, 100Temlyakova, E.A. 44Tenenbaum, S. 24, 25Thakkar, S. 112Theimer, C.A. 32Thomas, C.D. 50Tick, J. 13Tien, N. 86Timchenko, A.A. 51Tjhen, R. 128Todolli, S. 104Tomashevski, A.A. 123Tomasko, M. 28Tonoyan, S.A. 110Topilina, N.A. 15Toso, D.B. 78Tran, Q. 2Trifonov, E.N. 13Tullius, T.D. 76Tuzikov, A.V. 124, 126

Ulyanov, N.B. 128Uma, V. 102, 118Umamaheswari, A. 105, 113,114Usheva, A. 49Usmanova, Rita R. 118Uversky, V.N. 95

Vajda, S. 57Varani, G. 131Vardevanyan, P.O. 58, 59Varughese, K.I. 112Vasavi, M. 118Vasilieva, E. 44Vasquez, K.M. 74, 75Vendeix, F.A.P. 71

Venkataramana Reddy, C. 118Vishveshwara, S. 108Volodin, A. 82Vorlıckova, M. 28Vorobjev, Y.N. 68Vorobjev, Y. 46Vuruputuri, U. 103

Wachtershauser, G. 9Walker, R. 17Walsh, R. 57Wang, D. 41Wang, E. 84Wang, G. 74, 75Wang, J. 49Wang, L. 64Wang, Y. 22, 56, 126Wanker, E.E. 139Wanunu, M. 133, 134Warshel, A. 121Weng, Z. 49Westhof, E. 19, 20Whitakera, N.A. 126Whitfield, T.W. 49Whitford, P.C. 21Williams, M.C. 42, 58Wochner, A. 7Wodak, S.J. 112Wolfs, J.M. 64Wu, B. 127

Xie, Y.-H. 98, 100Xu, Z. 19

Yakubovskaya, E. 80Yamana, K. 61Yang, L. 63Yonath, A. 1

Zacharias, M. 106Zerbe, B. 57Zgarbova, M. 67, 70Zhang, L. 68Zhang, Q.C. 116Zhang, Q. 19, 20Zhang, S. 3Zhang, X. 17Zhao, S. 19Zharkov, D. 72Zharkov, Dmitry O. 68Zhou, T. 43, 45, 46, 63, 65,66Zhuang, J. 49Zhuravlev, Yu.N. 70Zhurkin, Victor B. 39, 41Zhuze, Alexei L. 52Zimmerman, E. 1Zimmermann, Michael T. 106Zuker, M. 24

142 Author Index

Documents

ABSTRACTS New biology from ancient enzymes 2 A prebiotic RNA … · 2013-05-30 · the last common ancestor and hence its study can provide insight to early events in the origin of