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conformation in an ATP dependent manner. Chaperonin-mediated protein fold-ing is achieved on the closure and opening of a built-in lid under ATP cycle.Recent study with cryo-electron microscopy and single particle analysis sug-gested that the ring structure of chaperonin twisted to seal off the central cavity.However such dynamics could not be traced experimentally since the motionwas expected to be too small to detect by visible light technology.Here we show that ATP dependent dynamic motion of group II chaperoninfrom Thermococcus strain KS-1 at single molecular level with high accuraterotational view by diffracted X-ray tracking (DXT). DXT has been consideredas a powerful technique in biological science for detecting subtle dynamic mo-tion of the target protein at single molecular level. The dynamics of a singleprotein can be monitored through trajectory of the Laue spot from the nanocrys-tal which was labeled on the objective protein immobilized on the substrate sur-face. UV-light triggered DXT using caged ATP revealed that the chaperonin’slid closed partially within one second after ATP binding, the closed ring twistedcounter clock-wisely from top to bottom view of chaperonin with 130 m rad/secin angular speed within 2 to 6 seconds, and the twisted ring turned back to theoriginal open state with 80 m rad/sec clockwise twisting motion. Our analyseswith precise rotational and macroscopic views for chaperonin’s dynamics showthat there are distinct two modes in lid-closure process, in-cooperative closureand cooperative counter-clockwise twisting motion. Moreover, we found thatthe ring’s twisting motion correlated to the folding activity of group IIchaperonin.

302-Pos Board B88Operational Plasticity of Hsp104, a Prion DisaggregaseJames Shorter.University of Pennsylvania, Philadelphia, PA, USA.It is poorly understood how Hsp104, a hexameric AAAþ ATPase from yeast,disaggregates the diverse structures and unrelated polypeptides that encompassthe stress-induced aggregated proteome, myriad prions, and alpha-synucleinoligomers and amyloids connected to Parkinson’s disease. Here, we establishthat Hsp104 hexamers adapt fundamentally different mechanisms of intersubu-nit collaboration (with respect to substrate handling and ATP hydrolysis) to dis-aggregate stress-induced aggregates versus amyloid. Remarkably, ClpB, theE. coli homolog of Hsp104, co-ordinates ATP hydrolysis and substrate han-dling differently and cannot remodel amyloid. Thus, we reveal the operationalplasticity of Hsp104 hexamers, which empowers the disaggregation of amyloidand non-amyloid clients that impose divergent mechanical demands.

303-Pos Board B89Binding Regions in the Skp Chaperone for Client Membrane Proteins.A Site-Directed Fluorescence StudyMeenakshi Sharma, Regina Pape, Jorg H. Kleinschmidt.University of Konstanz, Konstanz, Germany.In Gram-negative bacteria, outer membrane proteins (OMPs) are synthesized inthe cytoplasm. The translocation of OMPs across the periplasm in unfoldedstate is assisted by periplasmic molecular chaperones. The Seventeen-Kilo-Dalton protein, Skp, is a homotrimeric periplasmic chaperone known to facil-itate folding and insertion of various OMPs into the membrane. To gaina better insight into the mechanism, by which Skp binds its client proteins inthe periplasm, we designed, expressed and isolated a new Skp construct,Scp, from E. coli. in this construct, the three Skp monomers were linkedtogether with two short and flexible linkers. The function of Scp was confirmedby comparison with wild-type Skp in membrane protein folding experimentswith OmpA.To examine the topology of Scp$OmpA complexes, we have used site directedmutagenesis and fluorescence spectroscopy. Our first aim was to identify bind-ing regions in the Scp chaperone at the level of individual amino acid residues.Nine single-cysteine Scp mutants were constructed. The cysteine residues inthese mutants were spectroscopically labeled with the fluorescent probe IAE-DANS. Fluorescence resonance energy transfer (FRET) was then used to probethe interaction between the Scp mutants and a set of single-tryptophan mutantsof OmpA.

304-Pos Board B90Nucleic Acid Chaperone Activity of Wild Type and Mutant FIV Nucleo-capsid ProteinsHao Wu1, Micah J. McCauley1, Robert J. Gorelick2, Ioulia Rouzina3,Karin Musier-Forsyth4, Mark C. Williams1.1Northeastern University, Boston, MA, USA, 2AIDS and Cancer VirusProgram, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD, USA,3University of Minnesota, Minneapolis, MN, USA, 4The Ohio StateUniversity, Columbus, OH, USA.

The feline immunodeficiency virus (FIV) nucleocapsid (NC) protein contains66 amino acids. It has similar composition to HIV-1 NC, with a high numberof positively charged amino acids and two zinc fingers, each having a singlearomatic residue (F12 and W44). However, there are some differences betweenthe structures of these two proteins, which likely contribute to their differentnucleic acid chaperone properties. In addition to a short N-terminal basicdomain, FIV NC has a C-terminal positively charged tail. Moreover, whereasHIV-1 NC’s two aromatic residues are located on the same side of each zincfinger domain, FIV NC’s aromatic residues are located on opposite sides ofthe zinc fingers. It is known that HIV-1 NC has optimal chaperone activity, in-cluding the ability to strongly aggregate nucleic acids, destabilize nucleic acidsecondary structure and facilitate rapid protein-nucleic acid interaction kinet-ics. We use single molecule experiments to measure the characteristics ofwild type and mutant FIV NC. By stretching single DNAmolecules in the pres-ence of these proteins, we measure their ability to induce aggregation, stabilizeor destabilize dsDNA, and facilitate nucleic acid annealing. Our results showthat wild type FIV NC induces significant DNA aggregation, but instead ofdestabilizing double-stranded DNA, it appears to stabilize the DNA. FIV NCvariants containing mutations in the aromatic residues, basic residues, andzinc finger residues possess strongly altered nucleic acid chaperone properties.This work allows us to directly relate FIV NC structure with its function.The single molecule experiments are also compared to ensemble nucleic acidbinding and chaperone studies. Taken together, this study sheds new light onthe mechanism by which specific types and locations of residues contributeto NC’s nucleic acid chaperone activity and retroviral replication in thesesystems.

305-Pos Board B91Elucidating Molecular Constraints that Effect Alpha Crystallin Oligomer-ization, Stability, and Chaperone FunctionJean Santos, Raysa Cabrejo, Patricia O’Hara, James Hebda.Amherst College, Amherst, MA, USA.Alpha Crystallin is the major protein component of the human lens and playsan important role in the prevention of cataracts. a-Crystallin (aX) oligomersconsist of two isoforms, aX-A and aX-B which share high sequence similarityand define the common a-Crystallin fold found in many small heat shock pro-teins (sHSPs). aX-A and aX-B are hypothesized to play two important roleswithin the lens. First, aX-A and aX-B belong to a group of proteins calledCrystallins (a, b, and g) that are very stable proteins that play a role in pre-serving a uniform density within the lens, which allows it to focus light.The Crystallin proteins’ ability to form diverse and stable oligomers resultsin a glass-like rather than crystalline organization to the lens protein material,which also aids in the long-term stability of this high-density protein organ.Second, aX-A and aX-B both function as sHSPs that bind to misfolded pro-teins, preventing formation of large, insoluble protein aggregates (the begin-ning of cataracts). Our lab is investigating the molecular interactionsbetween aX-A and aX-B that result in its stability, diverse oligomerization,and chaperone function. To this end we are using a model, inducible misfold-ing protein (insulin B-chain) to study chaperone function by light scatter undervarious conditions. We are also using random and targeted modification ofaX-A and aX-B to simulate long-term protein damage and degradation ob-served in aged lenses. Focus on the C-terminal strand exchange observed inrecent crystal structures and proposed to aid in aX-A and aX-B polydisperseoligomerization is additionally aiding experimental design. We hope to iden-tify specific molecular interactions that result in aX-A and aX-B’s chaperonefunction, and determine how those interactions relate to stability and self-oligomerization.

Protein-Ligand Interactions I

306-Pos Board B92Virtual Screening and Docking of Potential Protein Kinase B InhibitorsSefika Kutlu Ulgen, Seval Aladag, Elif Ozkirimli.Bogazici University, Istanbul-Turkey, Turkey.This study aims to carry out a molecular docking process of novel inhibitors forProtein kinase B-beta (PKBb / AKT-2), activation of which has been observedin 30-40% of ovarian and pancreatic cancers. In this project, it is aimed to findnovel potential ATP competitive inhibitors. 3D pharmacophore filtering byPhase and multistep docking and scoring by Glide are applied to two crystalstructures of Akt-2 to take into account the conformational change upon thebinding of different inhibitors. The molecular library CoCoCo is screened tofind hits. This procedure combines the pharmacophore perception and database

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screening methods, considers the protein conformation change upon binding byusing two different protein conformations and also takes into account the sol-vation effect by including conserved water molecules in the active site. The topranked molecules from each pharmacophore hypothesis are further analyzedaccording to their interactions with AKT-2 and the ultimate docking resultsfrom Glide are compared with previously identified inhibitors based on struc-ture and chemistry.

307-Pos Board B93Stable Complexes of LYN Kinase and Druglike Small MoleculesD.S. Dalafave.The College of New Jersey, Newtown, PA, USA.Glioblastoma multiforme (GBM) is a very aggressive brain cancer in humans.Even with improvements in surgical, radiation, and chemotherapy treatments,current prognosis is bleak, with only a 14-month median survival time. LYN,an important kinase involved in cell regulation, is often overexpressed inGBM. This work addresses computational design of druglike small moleculesthat could potentially inhibit LYN and thus hinder GBM progression. New pu-tative LYN inhibitors were obtained by atomic substitutions and structuralalterations of bafetinib, a small molecule previously found to bind LYN. Drug-like properties and toxicities of the designed molecules were evaluated usingthe Osiris Property Explorer program. Molecules with no implied toxicitiesand most favorable druglike properties were used for docking studies in theArgusLab program. Designed molecules that made the most stable dockingconfigurations with LYN, but no stable configurations with other kinases,were identified as LYN-specific. Binding energies of the stable complexesformed by these molecules and LYN were calculated. Possible utilization ofthe designed molecules in drug research against GBM is discussed.

308-Pos Board B94Crystal Structure of the Kinase Domain of ABL in Complex with a PotentRationally-Designed Derivative of the PP1 Inhibitor, AB129M. Nidanie Henderson1, Kathryn E. Malecek1, Deborah L. Makino2,Xiaoxian X. Cao2, Kevan M. Shokat3, John Kuriyan2.1Carleton College, Northfield, MN, USA, 2University of California,Berkeley, CA, USA, 3University of California, San Francisco, CA, USA.As part of our effort to understand the determinants of drug binding to the Abltyrosine kinase, we determined the crystal structure of the Abl kinase domainbound to a derivative of the PP1 nucleoside analog. Just as the cancer drug im-atinib selectively inhibits the tyrosine kinase activity of c-Abl, c-Kit, and thePDGF receptor, but is a poor inhibitor of the closely related Src kinases (1),PP1 is a selective inhibitor of the Src family (2), c-Kit (3), and PDGFR kinasesbut not of Abl tyrosine kinase activity (3, 4). In fact, PP1 is 1000X less effectiveat inhibiting Abl and Bcr-Abl kinases than at inhibiting the Src kinases (3, 4).We rationally designed a derivative of the PP1 compound to introduce hydro-gen bonding between it and a strictly conserved lysine residue that coordinatesthe alpha and beta phosphate groups of ATP in protein kinases (5). By expand-ing the interactions between the inhibitor and Abl kinase, we sought to makethe PP1 derivative, AB129, less selective. Our crystal structure of the Abl ki-nase domain bound to the AB129 inhibitor shows three important features.First, the compound binds to the kinase domain in the active conformation: al-pha C helix is rotated inward facilitating a salt bridge between Lys271 andGlu286 and the conserved Asp-Phe-Gly (DFG) motif is oriented Asp-In. Incontrast, the structure of PP1 bound to the Src family kinase Hck is in the in-active conformation (6). Second, the strictly conserved Glu286 residue, notLys271, hydrogen bonds to the hydroxyl group of AB129. Third, the conforma-tion of the phosphate binding loop (P-loop) is extended. We provide kinetic andcalorimetric data to support our rationale for the observed conformationalchanges.

309-Pos Board B95Crystallization of the Abl Kinase Domain with Combi-Inhibitors, ZRF1and ZRF2Steve C.E. Amaefuna1, Angela M. Harrington1, Bertrand Jean-Claude2,Bhushan Nagar2, M. Nidanie Henderson1.1Carleton College, Northfield, MN, USA, 2McGill University, Montreal,QC, Canada.The uncontrolled kinase activity of the oncogenic protein Bcr-Abl is a hall-mark of Chronic myelogenous leukemia (CML). Although the drug imtanibis highly effective at shutting down aberrant Abl kinase activity, someCML patients harbor additional mutations in their bcr-abl gene that renderstheir gene product, the Bcr-Abl protein, resistant to this drug. Since second

generation Abl kinase inhibitors such as dasatinib are effective only againstparticular resistant mutant proteins, new targeted agents are needed to combatdrug resistance. Two combi-inhibitors ZRF1 and ZRF2, which block Bcr-Ablactivity and damage DNA in the cell, have been shown to be more potent thanimatinib in cellular assays. Since both ZRF1 is hydrolyzed in the cell to ZRF0,an imatinib analog in which only the mono-substituted benzamide piperazinylmoiety is replaced with bi-substituted amino and trifluoromethyl groups, wewondered why these two changes had such a profound effect on the potencyof the drug. To determine whether ZRF1/2’s increased potency was due to theDNA damaging agent attached to the parent compounds, we sought to co-crystallize the Abl kinase domain with these two inhibitors at room tempera-ture and 4�C using commercially available sparse-matrix screens. Wescreened over 400 crystallization conditions, with Abl and ZRF1, ZRF2, ordasatinib inhibitors but were unable to obtain crystals of either the Abl-ZRF1 or Abl-ZRF2 drug complex. Protein crystals in wells containing theAbl kinase domain and dasatinib, however, were evident in every tray. SinceZRF1 and ZRF2 are hydrolyzed in the cell, future co-crystallization studies ofAbl and its mutant proteins will employ the hydrolyzed inhibitor analogs asthe ligand.

310-Pos Board B96BindingDB: A Protein-Ligand Database for Drug DiscoveryGeorge Nicola, Tiqing Liu, Linda Hwang, Michael Gilson.University of California San Diego, La Jolla, CA, USA.The large and growing body of experimental data on biomolecular binding is ofenormous value in developing a deeper understanding of molecular biology, indeveloping new therapeutics, and in various molecular design applications.However, most of these data are found only in the published literature andare therefore difficult to access and use. BindingDB is a public web-accessible database of measured binding affinities for various molecular types.The BindingDB allows queries based upon a range of criteria, including chem-ical similarity or substructure, sequence homology, numerical criteria and reac-tant names. The data specification includes significant experimental detail. Thetime and expense required to extracting data from the literature, for this andmany other databases, highlight the importance of moving toward machine-readable components of publications.

311-Pos Board B97GALAXYDock: Docking Ligands to Receptor Proteins with Selected Flex-ible Side-ChainsWoong-Hee Shin, Chaok Seok.Seoul National University, Seoul, Korea, Republic of.Protein-ligand docking techniques are one of the essential tools for structure-based drug design. Although conformational changes of protein receptor fre-quently occur in the binding process, a large fraction of contemporary dockingprograms ignore receptor flexibility. Consideration of protein flexibility effi-ciently and accurately in docking studies is challenging due to difficulties infinding accurate scoring function and in sampling conformations efficiently.Previously we developed a docking program called LigDockCSA which incor-porates LigDock scoring function that combines AutoDock3 and PLP scoringfunction and conformational space annealing (CSA) as a sampling method.However, the program treats protein rigid. Here, we present GALAXYDock,an extension of LigDockCSA that accounts flexibility of pre-selected side-chains of receptor protein in the binding site. To test the performance of thenew docking method, 3 sets of protein-ligand complexes that share similarbackbone structures but have different side chain conformations were selectedas test sets: HIV-PR, LXRb and cAPK. The cross-docking results show that theaccuracy of binding pose prediction is increased about 20% when comparedwith our previous rigid-receptor results.

312-Pos Board B98Docking Simulations of Perylene-HSA BindingMohammed J. Farooqi1, Mark A. Penick1, George Negrete1,Lorenzo Brancaleon2.1University of Texas at San Antonio, San antonio, TX, USA,2University of Texas at San Antonio, San antonio, TX, USA.The study of the binding and effects of polyaromatic hydrocarbons (PAH) toproteins remains one of the fundamental aspects of research in biophysics.Among other processes, ligand binding can regulate the function of proteins in-cluding inhibiting their action. Binding to small ligands remains a very im-portant aspect in the study of the function of many proteins. We studieda new class of 3,9-substituted perylene derivatives designed to optimize