PROTEIN ENGINEERING CANADA CONFERENCE

SCIENTIFIC PROGRAM Friday June 20th, 2014 – Opening Mixer and Registration Opening Mixer at The Fox & Feather Pub & Grill (283 Elgin Street, Room Fox 2, 2nd floor) A registration table will open at 6:30PM. Name badges can be picked up at the registration table.

6:30-11:00PM

Saturday June 21st, 2014 – Sessions 1-3 (FSS 2005) Registration on 1st floor of Faculty of Social Sciences (FSS) Building (120 University Private) Name badges can be picked up at the registration table.

8:00-8:20AM

Session 1 (Chair: Jean-François Couture, University of Ottawa) Welcoming remarks 8:20-8:30AM Miroslaw Cygler, University of Saskatchewan (Saskatoon, SK, Canada) 8:30-9:00AM Title to be announced. David Kwan, University of British Columbia (Vancouver, BC, Canada) 9:00-9:15AM Talk selected from abstract: Engineering a glycoside hydrolase by directed evolution for blood antigen removal

Rafael J. Najmanovich, University of Sherbrooke (Sherbrooke, QC, Canada) 9:15-9:30AM Talk selected from abstract: A coarse-grained elastic network atom contact model and its use in the simulation of protein dynamics and the prediction of the effect of mutations

Robert E. Campbell, University of Alberta (Edmonton, AB, Canada) 9:30-10:00AM Visualizing biochemistry where and when it happens with engineered fluorescent proteins

Coffee Break (FSS 4006) 10:00-10:30AM Session 2 (Chair: Joelle N. Pelletier, University of Montreal) David D. Boehr, Pennsylvania State University (University Park, PA, USA) 10:30-11:00AM Dynamic amino acid networks in enzyme catalysis Katherine Donovan, University of Canterbury (Christchurch, New Zealand) 11:00-11:15AM Talk selected from abstract: Not different, just better: The adaptive evolution of a key glycolytic enzyme

James A. Davey, University of Ottawa (Ottawa, ON, Canada) 11:15-11:30AM Talk selected from abstract: Accurate prediction of protein stability by explicit negative multistate design

Amy E. Keating, Massachusetts Institute of Technology (Cambridge, MA, USA) 11:30-12:00PM Novel peptide ligands for Bcl-2 family proteins Lunch Break (on your own) 12:00-2:00PM

Session 3 (Chair: Nicolas Doucet, INRS-Institut Armand-Frappier, University of Quebec) Rama Ranganathan, UT Southwestern Medical Center (Dallas, TX, USA) 2:00-2:30PM The evolutionary “design” of proteins Gevorg Grigoryan, Dartmouth College (Hanover, NH, USA) 2:30-3:00PM Computational design of selective targeting peptides Susan M. Aitken, Carleton University (Ottawa, ON, Canada) 3:00-3:15PM Talk selected from abstract: Characterization of tryptophan residues in yeast cystathionine β-Synthase and their potential as probes of conformational change

Aron Broom, University of Waterloo (Waterloo, ON, Canada) 3:15-3:30PM Talk selected from abstract: Design of a kinetically stable and functional protein via modular symmetry

Tanja Kortemme, University of California, San Francisco (San Francisco, CA, USA) 3:30-4:00PM Design of modular sensor/actuators and light-controlled protein machines Kimberley A. Reynolds, UT Southwestern Medical Center (Dallas, TX, USA) 4:00-4:30PM Designing and mapping protein interactions with evolutionary statistics Poster Session (FSS 4007) 4:30-6:30PM

Sunday June 22nd, 2014 – Sessions 4-5 (FSS 2005) Session 4 (Chair: Natalie K. Goto, University of Ottawa) Todd O. Yeates, University of California, Los Angeles (Los Angeles, CA, USA) 8:30-9:00AM Design and structures of giant self-assembling protein cages and nanomaterials Yi Shen, University of Alberta (Edmonton, AB, Canada) 9:00-9:15AM Talk selected from abstract: Engineering of genetically encoded pH sensors based on red fluorescent proteins

Alexander Foo, University of Ottawa (Ottawa, ON, Canada) 9:15-9:30AM Talk selected from abstract: Modulation of E. coli rhomboid protease activity by hydrophobic mismatch

G. Andrew Woolley, University of Toronto (Toronto, ON, Canada) 9:30-10:00AM Blue light induced domain swapping Coffee Break (FSS 4006) 10:00-10:30AM Session 5 (Chair: Roberto A. Chica, University of Ottawa) Ivan Korendovych, Syracuse University (Syracuse, NY, USA) 10:30-11:00AM Minimalist design of esterases Joelle N. Pelletier, University of Montreal (Montreal, QC, Canada) 11:00-11:30AM Engineering peptides and proteins for efficient biodetection using a portable surface plasmon resonance platform

Nobuhiko Tokuriki, University of British Columbia (Vancouver, BC, Canada) 11:30-11:45AM Talk selected from abstract: Evolutionary (ir)-reversibility of enzymatic activity in laboratory evolution

Stephen L. Mayo, California Institute of Technology (Pasadena, CA, USA) 11:45-12:15PM A portfolio approach to protein property optimization Closing remarks and student awards announcements 12:15-12:25PM

TABLE OF CONTENTS

KEYNOTES

Miroslaw Cygler 12Protein engineering helping to identify functional regions of proteins

Robert E. Campbell 13Visualizing biochemistry where and when it happens with engineered fluorescent proteins

Axe, Jennifer M., O’Rourke, Kathleen F., Yezdimer, Eric M., Kerstetter,Nicole, and Boehr, David D.

14

Dynamic amino acid networks in enzyme catalysis

Amy E. Keating 15Novel peptide ligands for Bcl-2 family proteins

Rama Ranganathan 16The Evolutionary “Design” of Proteins

Fan Zheng, Heather L. Jewell, Gevorg Grigoryan 17Computational Design of Selective Targeting Peptides

Tanja Kortemme 18Design of modular sensor/actuators and light-controlled protein machines

Kimberly Reynolds 19Designing and mapping protein interactions with evolutionary statistics

Yen-Ting Lai, Dan McNamara, Yuxi Liu, Neil King, Jacob Bale, David Baker,and Todd Yeates

20

Design and Structures of Giant Self-Assembling Protein Cages and Nanomaterials

Jakeb M. Reis, Darcy C. Burns & G. Andrew Woolley 21Blue light induced domain swapping

Ivan V. Korendovych 22Minimalist Design of Esterases

Joelle N. Pelletier, Jean-Francois Masson, Olivier Bolduc, Christopher M.Clouthier, Natalia Bukar, Sandy Shuo Zhao, David M. Charbonneau, AlexandraAube, Julien Breault-Turcot, Pierre Chaurand

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Engineering peptides and proteins for efficient biodetection using a portable SurfacePlasmon Resonance platform

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Stephen L. Mayo 24A Portfolio Approach to Protein Property Optimization

ORAL PRESENTATIONS

Alexander Foo, Natalie Goto 26Modulation of E. coli Rhomboid Protease Activity by Hydrophobic Mismatch

Broom A., Ma M., Jacobi Z., Berthin L., Meiering EM. 27Design of a kinetically stable and functional protein via modular symmetry

David H. Kwan, Stephen G. Withers 28Engineering a Glycoside Hydrolase by Directed Evolution for Blood Antigen Removal

James A. Davey, Christian K. Euler, Roberto A. Chica 29Accurate prediction of protein stability by explicit negative multistate design

Katherine A. Donovan, Fen Peng, Sarah A Kessans, Tim F Coper, RenwickCJ Dobson

30

Not different, just better: The adaptive evolution of a key glycolytic enzyme

Nobuhiko Tokuriki 31Evolutionary (Ir)-Reversibility of Enzymatic Activity in Laboratory Evolution

Vincent Frappier, Laurent Bruneau-Cossette & Rafael Najmanovich 32A coarse-grained elastic network atom contact model and its use in the simulation ofprotein dynamics and the prediction of the effect of mutations

Edgar Abouassaf, Duale Ahmed and Susan M. Aitken 33Characterization of Tryptophan Residues in Yeast Cystathionine β-Synthase and TheirPotential as Probes of Conformational Change

Yi Shen, Robert Campbell 34Engineering of genetically encoded pH sensors based on red fluorescent proteins

POSTER PRESENTATIONS

1. Adam M. Damry, James A. Davey, Christian K. Euler & Roberto A. Chica 36Precise Control of Amino Acid Side Chain Conformation with Multistate ComputationalProtein Design

2. Anil Kumar, Anna S. I. Jaikaran, and G. Andrew Woolley 37Towards photo-control of translation initiation using photoactive yellow protein

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3. Antonia T. Pandelieva, Guido F. Calderini, Andrew Wong, and Roberto A.Chica

38

Increasing Red Fluorescent Protein Quantum Yield through Rational Design

4. Antony D. St-Jacques and Roberto A. Chica 39Engineering Enzyme Substrate Specificity Using Multistate Computational Protein De-sign

5. Camille Henault, Peter Juranka, Julian Surujballi, John E. Baenziger 40Sequence variability leads to distinct roles for M4 as an allosteric regulator of pentamericligand-gated ion channels

6. Carlos Dulcey, Nicolas Doucet 41Molecular characterization of the LipIAF5-2 lipase for the synthesis of value-added chem-icals

7. Caroline M. Rufo�, Yurii S. Moroz�, Olesia V. Moroz�, Jan St{ohr, Tyler A.Smith, Xiaozhen Hu, William F. DeGrado and Ivan V. Korendovych

42

Short peptides self-assemble to create catalytic amyloids

8. Corey Stevens and Peter Davies 43Using multiply branched antifreeze proteins as novel ice-binding reagents

9. Curtis J.W. Walton, Frederic Thiebaut, Jean-Francois Couture, Roberto A.Chica

44

Investigating the structural determinants of substrate specificity in a stereo-invertingaminotransferase.

10. David M. Charbonneau, Alexandra Aube, Jean-Francois Masson and Joelle N.Pelletier

45

Clinical integration of a portable SPR device: Pharmacodynamics of L-asparaginase inthe treatment of childhood acute lymphoblastic leukaemia

11. Diana I. Martınez-Tobon, Jared Lehne, Anastasia Elias, and DominicSauvageau

46

Polyhydroxyalkanoate (PHA) depolymerases for the potential development of an im-proved bioplastic degrading enzyme

12. Donald Gagne & Nicolas Doucet 47Comparison of protein flexibility among human homologues of the RNase A superfamily

13. Elizabeth Raymond, Jennifer Yoon, Korrie Mack, Yurii Moroz, Ivan Koren-dovych

48

Introducing New Functions into Old Proteins

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14. Florian Baier, Nobuhiko Tokuriki 49Connectivity between catalytic landscapes of the metallo-β-lactamase superfamily

15. Gayane Machkalyan, Terry Hebert and Gregory J Miller 50Decoding signalling roles of diphosphoinositol polyphosphates

16. Guillaume Brault & Nicolas Doucet 51Intracellular protein expression as an efficient vessel for biosynthesis of flavoring esters

17. Habib Horchani, Sylvain Bussieres, Line Cantin, Mustapha Lhor, Jean-Sebastien Laliberte-Gemme, Rock Breton, Christian Salesse

52

Enzymatic activity of Lecithin:retinol acyltransferase: A thermostable and highly activeenzyme with a likely mode of interfacial activation

18. Irina Shlaifer, Joanne Turnbull 53Expression, purification and characterization of an archaeal enzyme involved in tyrosinebiosynthesis

19. J.P. Daniel Therien, Peter F. Juranka, and John E. Baenziger 54The Role of Intramembrane Chemistry in the Gating of Pentameric Ligand-Gated IonChannels

20. Jakeb M. Reis, Darcy C. Burns, G. Andrew Woolley 55Blue light induced domain swapping

21. Janet E.B. Barber, Adam M. Damry, Guido F. Calderini, Curtis J.W. Walton& Roberto A. Chica

56

Engineering D-amino acid aminotransferase mutants displaying increased activity towardsaromatic D-amino acids

22. Jessica A. Rumfeldt, Elizabeth M. Meiering 57Structural dynamics of CuZn Superoxide dismutase monitored by hydrogen-deuteriumexchange

23. Jessica Kelly Moisan, Fatma Meddeb-Mouelhi, and Marc Beauregard 58Biophysical characterization of recombinant carboxylesterase EstGtA2 from Geobacillusthermodenitrificans

24. Jiayin Sun, Casey L. Carswell, John E. Baenziger 59Engineering Lipid Sensitivity in Prokaryotic Ligand-Gated Ion Channels

25. Kritica Arora, Parker Anderson, Ana Asenjo, Lama Talje, Monika Joshi, Her-nando Sosa, Benjamin Kwok, and John Allingham

60

Mitotic kinesin structure displays elusive near rigor state of the motor domain

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26. Joshua Pottel, Anna Tomberg, Calem Bendell, Nicolas Moitessier 61Development of Computational Tools towards the Virtual Engineering of Biocatalysts

27. Justin Kollman 62Filament assembly by CTP synthase: A novel mechanism for allosteric regulation ofenzyme activity

28. Katherine Brechun, Vitali Borisenko, Lori Yin, Asim Rashid, Huixin Lu, AndrewWoolley

63

Fluorescence-based monitoring of PYP photo-switching in vivo

29. Landon Zarowny and Robert E. Campbell 64Towards Integrating Calcium Sensors for In vivo Neuronal Activity Imaging

30. Li Cui, Fatma Meddeb, Marc Beauregard 65Use of enzymes to improve the refining and physical properties of kraft pulp

31. Loretta Au, and David F. Green 66Decoupling epistatic relationships that maintain structural stability in a β-propeller pro-tein

32. Manel Ghribi, Fatma Meddeb- Mouelhi, and Marc Beauregard 67Characterization of hydrolytic enzyme-producing bacteria isolated from paper mill

33. Mark Horsman, Christopher Boddy 68Examining the substrate and carrier protein specificity of the Bacillaene thioesterase

34. Matthew Wiens, Robert Cambell 69Tandem dimer fluorescent proteins

35. Maximilian Ebert, Brahm Yachnin, Guillaume Lamoureux, Albert Berghuis,Joelle Pelletier

70

Structural analysis and molecular dynamics of the self-sufficient P450 CYP102A5 andCYP102A1: A a combined computational/experimental approach to increase the effi-ciency of biocatalyst engineering

36. Miroslava Strmiskova, Natalie K. Goto, Jeffrey W. Keillor 71Optimization of a fluorogenic labelling technique for in cellulo protein visualization

37. Muhammad Hussein Alu’datt 72Effect of removal of free and bound phenolic compounds on molecular, chemical andbiological properties of separated peptides from hydrolyzed protein fractions from Blackcumin.

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38. Mustapha Lhor, Habib Horchani, Mario Methot and Christian Salesse 73Retinol dehydrogenase 11 membrane anchoring is mediated by its N-terminal α-helixwith a preferential binding to phosphoethanolamine lipids

39. Nancy Hom, Ertan Ozyamak, Arash Komeili, Justin Kollman 74Structural investigations of bacterial magnetotactic organelle formation and positioning

40. Nhung Nguyen Thi & Nicolas Doucet 75Probing the clamping movement of xylanase B by NMR spectroscopy

41. Christopher Corbeil, Alain Ajamian, Paul Labute 76A Unified Framework for Computer Aided Biologics Design

42. Philippe Egesborg, Helene Carlettini, Jordan P. Volpato, Nicolas Doucet 77Combinatorial Active-Site Variants Confer Sustained Clavulanate Resistance in BlaC β-Lactamase from Mycobacterium tuberculosis

43. Sabina Sarvan, William Lam and Jean-Francois Couture 78Structural insights into the interaction between the Fur family of metalloregulators andDNA

44. Shane J. Caldwell and Albert M. Berghuis 79Unusual crystal packing highlights dynamic flexibility of an enzymatic kinase domain

45. Shen Wan, Illimar Altosaar, and Joann K. Whalen 80Phytoremediation of nitrous oxide: Expression of nitrous oxide reductase from pseu-domonas stutzeri in transgenic plants

46. Shuaiqi Guo, Christopher Garnham, and Peter Davies 81Making an ice-binding protein de novo

47. Sophie Gobeil, Jaeok Park, Christopher Clouthier, Donald Gagne, NicolasDoucet, Albert Berghuis, and Joelle Pelletier

82

Structure-based recombination of β-lactamases: functional, structural and dynamic in-vestigation of artificially-evolved enzymes

48. Lanouette S., Davey J., Chica R., Figeys D., Couture J.-F. 83HIGH-THROUGHPUT ASSAYS AND IN SILICO DESIGN ACCURATELY PREDICTNOVEL SMYD2 SUBSTRATES

49. Vincent Frappier, Laurent Bruneau-Cossette, Joe DeBartolo, Rafael Naj-manovich and Amy E. Keating

84

LeSTATIUM: Incorporating a treatment of protein conformational space in the STATIUMstatistical potential

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50. Yachnin, Brahm J., McEvoy, Michelle B., MacCuish, Roderick J. D., Morley,Krista L., Mittermaier, Anthony K., Lau, Peter C. K., Berghuis, Albert M.

85

The Structural Basis for BVMO Substrate Profile and Stereospecificity

51. Yannick Hebert-Ouellet , Vinay Khatri , Fatma Meddeb-Mouelhi, and MarcBeauregard

86

Tracking wood fibers decrystallization with carbohydrate binding module

52. Yazan M. Abbas, Irene Xie, Bhushan Nagar 87Structural insight into IFIT3 domain swapping

53. David Bernard, Donald Gagne & Nicolas Doucet 88Comparison of predicted structures and dynamics of various mammalian EDN and ECPorthologues

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KEYNOTES

11

Protein engineering helping to identify functional regions of proteins

Miroslaw Cygler

Dept. Biochemistry, University of Saskatchewan, Saskatoon, SK S7N 5E5

We have used protein engineering in combination with functional assays to identify region of pro-teins that are associated with their function. I will provide two examples. The first is the mappingthe surface of a bacterial cysteine desulfurase with several proteins that were know to interact withit. The second example is the identifying regions on a surface of the oligomeric protein, bacterialpolysaccharide co-polymerase, that are defining the length of the synthesized polysaccharide.

12

Visualizing biochemistry where and when it happens with engineered fluorescentproteins

Robert E. Campbell

Department of Chemistry, University of Alberta

The Campbell research group is focused on the use of protein engineering for the development offluorescent proteins and fluorescent protein-based reporters for live cell imaging. The key to muchof this work is establishing directed evolution work flows that enable the screening of many thou-sands of variants to identify the select few with improved photophysical properties. Over the lastdecade, this approach has proven to be both effective and versatile and has led to the developmentof a variety of new genetically encoded fluorescent probes. Indeed, by exploiting iterative cycles offluorescence image-based screening, combined with genetic, optical, or chemical manipulations ofbacterial colonies, the Campbell group has developed a growing selection of fluorescent protein-basedtools with improved brightness, photostability, and biosensing or photoconversion properties. In thisseminar I will present some of our most recent efforts to engineer an improved generation of reporters.Specifically, I will provide an update on the expanding palette of Ca2+ reporters our lab has beendeveloping, and describe how we are using similar engineering efforts to make reporters for membranepotential and neurotransmitters.

13

Dynamic amino acid networks in enzyme catalysis

Axe, Jennifer M., O’Rourke, Kathleen F., Yezdimer, Eric M., Kerstetter, Nicole, and Boehr, David D.

The Pennsylvania State University, Department of Chemistry

Our previous studies (1) have demonstrated that enzymes fluctuate into lowly populated conforma-tions that correspond to their next structural state in the catalytic cycle. These fluctuations appear toprepare the enzyme for the next structural transition, and may be rate-limiting bottlenecks in enzymecatalysis. These findings have also suggested that the evolution of these internal protein motionsmay be critical for efficient enzyme catalysis. However, it is poorly understood how these differentmotions and structural transitions are effectively coordinated. One proposal is that there are inter-action networks of amino acids operating throughout the protein that synchronize these structuraltransitions. We have now used an NMR method, CHESCA (2) (chemical shift covariance analysis),to delineate amino acid network(s) in the alpha subunit of tryptophan synthase (TS) (3). Strikingly,the amino acid networks defined by this method are different in the free protein compared to whenTS is actively turning over substrate/product. In particular, there are long-range connections in the“active” state between regions directly involved in chemical catalysis, which are missing in the “free”state. In the “free” state, there are long-range interactions between dynamic loops that gate accessto the active-site, which are missing in the “active” state. Amino acid networks in enzymes are thusdynamic, and change to regulate the various stages of catalysis, from substrate binding and productrelease to chemical catalysis itself. Wiring these dynamic amino acid networks into enzymes will becritical in coordinating functionally relevant motions and structural transitions in engineered enzymes.

References1. D.D. Boehr, D. McElheny, H.J. Dyson and P.E. Wright, Science, 2006, 313, 1638.2. R. Selvaratnam, S. Chowdhury, B. VanSchouwen and G. Melacini, Proc. Natl. Acad. Sci. USA,2011, 108, 6133.3. J.M. Axe and D.D. Boehr, J. Mol. Biol., 2013, 425, 1527.

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Novel peptide ligands for Bcl-2 family proteins

Amy E. Keating

MIT Department of Biology

Specific interactions among Bcl-2 family proteins are critical for regulating programmed cell death.Five mammalian anti-apoptotic proteins counteract pro-death signals by binding to the BH3 motifsof diverse pro-apoptotic proteins. In many cancer cells, up-regulation of anti-apoptotic Bcl-2 fam-ily proteins neutralizes pro-death signaling molecules and provides resistance to chemotherapeuticagents. The structures of anti-apoptotic Bcl-2 family proteins bound to helical BH3-motif peptidesfrom pro-apoptotic proteins are highly conserved. Yet the interactions among family members arehighly selective, and only some combinations of pro- and anti-apoptotic proteins form high affinitycomplexes. To better understand the molecular recognition properties of these proteins, we have beenstudying sequence-level determinants of binding using a variety of experimental and computationalmethods. Our goal is to develop models that can be used for interaction specificity prediction anddesign. I will discuss the types of experimental interaction data we have collected to define interac-tion specificity, the types of predictive computational models we have constructed, and some of oursuccesses in predicting and re-engineering specific Bcl-2 family interactions.

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The Evolutionary “Design” of Proteins

Rama Ranganathan

The Green Center for Systems Biology, UT Southwestern Medical Center, Dallas TX, 75390

Natural proteins can fold spontaneously into well-defined three-dimensional structures, and can displaycomplex biochemical properties such as signal transmission, efficient catalysis of chemical reactions,specificity in molecular recognition, and allosteric conformational change. All of this is achieved whilealso preserving the capacity for rapid adaptive variation in response to fluctuating selection pressures,a central feature of evolving systems. What are the basic principles in the “design” of natural pro-teins that underlie all of these properties? To address this, we developed an approach (the statisticalcoupling analysis or SCA) for globally estimating the pattern of functional interactions between siteson proteins through statistical analysis of the evolutionary divergence of a protein family1,2. Thisanalysis indicates a novel decomposition of proteins into sparse groups of co-evolving amino acidsthat we term “protein sectors”9. The sectors comprise physically connected networks in the tertiarystructure and can be modular – with different sectors representing different functional properties.Experiments in several protein systems demonstrate the functional and adaptive importance of thesectors 1,3,4,7,8,10,11,12 and recently, the SCA information was shown to the necessary and suffi-cient to design functional artificial members of two protein families in the absence of any structuralor chemical information5,6. These results support the hypothesis that sectors represent the basicarchitecture underlying folding, function, and adaptive variation in proteins. We are now working ontwo key problems: (1) understanding the physical mechanisms underlying sectors, and (2) defininghow the dynamics of the evolutionary process controls the emergence and structural architecture ofsectors in proteins.[1] Lockless, R. Ranganathan, Science, 286, 295-9 (1999).[2] Suel et al., Nature Struct. Biol., 10., 59-69 (2003).[3] Hatley, et al., PNAS, 100: 14445-14450 (2003).[4] Shulman et al., Cell, 116: 417-429 (2004).[5] Socolich et al., Nature, 437: 512-518 (2005).[6] Russ et al., Nature, 437: 579-583 (2005).[7] Mishra et al., Cell, 131: 80-92 (2007).[8] Lee et al., Science 322: 438-442 (2008).[9] Halabi et al., Cell 138: 774-785 (2009).[10] Smock et al., Mol. Sys. Biol.: 6: 414 (2010).[11] Reynolds et al., Cell, 147: 1564-1575 (2011).[12] McLaughlin et al., Nature 491: 138-142 (2012).

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Computational Design of Selective Targeting Peptides

Fan Zheng, Heather L. Jewell, Gevorg Grigoryan

Dartmouth College

Reagents that target protein-interaction domains to rewire signaling pathways are of great relevancein biomedical research. Computational protein design may offer a means of creating such reagentson demand, but methods for encoding selectivity are sorely needed. This is particularly relevant whentargeting a member of a ubiquitous domain family, e.g. PDZ domains—modules that bind C-terminalsequences of partner proteins. Compounding the difficulty of this scenario for protein design is theneed to consider a multitude of potential binding modes with any off-target domains in order toestablish target selectivity. We were motivated to address this problem by a colon cancer signalingpathway in which recognition of a receptor C-terminus by one PDZ domain (N2P2) increases tu-morigenicity, while binding of another (M3P6) to the same site has the opposite effect. Peptidesthat inhibit N2P2 without affecting M3P6 would thus offer a potential means of down-regulatingoncogenicity. We developed a computational design procedure that accounts for peptide flexibility inbinding, yet is highly efficient enabling rapid analysis of tradeoffs between N2P2 affinity and selec-tivity against M3P6. Designed peptides bound N2P2 as intended (verified with NMR footprinting)with affinities in the low-micromolar range and no detectable M3P6 binding. Peptides designed forreverse discrimination bound M3P6 tighter than N2P2, further testing our framework. We showthat significant domain-specific energetic coupling exists between peptide residues, so that positionalpreferences alone are not sufficient to describe the landscape of affinity and specificity. Our success indiscriminating between M3P6 and N2P2, despite their overlapping binding preferences, is particularlyexciting given that a homology model of M3P6 was used in design, as no experimental structure wasavailable. Our design framework is general and can be applied in a wide range of scenarios to engineerselective targeting.

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Design of modular sensor/actuators and light-controlled protein machines

Tanja Kortemme

UCSF

Engineered biological systems, ranging from molecules with novel functions to entire organisms, havetremendous practical importance; they can also fundamentally change how we ask questions aboutthe biological design principles of function and fitness. Emerging advances in computational methodsto design proteins are beginning to enable exciting applications of engineered functions not seen innature. I will present our recent progress on computational design of sensor/actuators that can detectand respond to small molecules in living cells, and on reengineering protein machines that can becontrolled by specific external inputs such as light.

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Designing and mapping protein interactions with evolutionary statistics

Kimberly Reynolds

University of Texas, Southwestern Medical Center

Cellular function and fitness depends on the cooperative action of many proteins. Allosteric regula-tion, the formation of physical complexes, and the organization of enzymes into metabolic cascadesrepresent several ways in which proteins can be functionally coupled to one another to produce usefulcellular behaviors. How are such functional interactions between proteins encoded at the amino acidlevel, and how do they evolve? Statistical analysis of amino acid co-evolution in individual proteindomains reveals a general architecture for natural proteins in which sparse networks of amino acidsunderlie basic aspects of structure and function. These networks, now termed sectors, form physicallycontiguous pathways embedded in the tertiary structure. Using the metabolic enzyme dihydrofolatereductase (DHFR) as a model system, we show that (1) sector-connected residues on the proteinsurface are hot spots for the gain of allosteric regulation and (2) the sector of DHFR co-evolveswith a second, epistatically coupled metabolic protein. These findings suggest practical guidelines forthe engineering of new allosteric systems, permit description of a plausible model for the evolutionof intermolecular communication, and motivate a global analysis of evolutionary couplings betweenproteins in cellular systems.

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Design and Structures of Giant Self-Assembling Protein Cages and Nanomaterials

Yen-Ting Lai (1), Dan McNamara (1), Yuxi Liu (1), Neil King (2), Jacob Bale (2), David Baker (2),and Todd Yeates (1)

(1) Dept. of Chemistry and Biochemistry, Univ. of California, Los Angeles, CA, USA (2) Dept.of Biochemistry, Univ. of Washington, Seattle, WA, USA

Nature is replete with self-assembling molecular structures having diverse cellular functions. Some ofthe largest and most sophisticated types are built from many copies of the same or similar proteinmolecules arranged following principles of symmetry. A long-standing engineering goal has been todesign novel protein molecules to self-assemble into geometrically specific structures similar to theextraordinary structures evolved in Nature. Practical routes to this goal have been developed byusing ideas in symmetry to articulate the minimum design requirements for achieving various typesof symmetric architectures, including cages, extended two-dimensional layers, and three-dimensionalcrystalline materials [1]. The key requirement is generally that two distinct self-associating interfaceshave to be designed into the protein molecule, following specific geometric specifications. Recentexperiments have demonstrated success using two alternate strategies, one based on fusing togethertwo simple oligomers (e.g. a dimer and a trimer) in a geometrically specific orientation [1-3], and onebased on designing a new protein-protein interface into a natural oligomer (which already bears oneinterface)[4]. The success of these strategies has been proven by determining crystal structures ofseveral giant, self-assembling protein cages and clusters (100-230 A in diameter), created by design[2-6]. The ability to create sophisticated supramolecular structures from designed protein subunitsopens the way to broad applications in synthetic biology. Design principles and strategies will bediscussed, along with new results.

[1] Padilla, et al. (2001). PNAS 98, 2217-21.[2] Lai, et al. (2012). Science 336, 1129.[3] Lai, et al. (2013). JACS 135, 7738-43.[4] King, et al. (2012). Science 336, 1171-4.[5] King, et al. (2014) Nature (in press).[6] Lai, et al. “Structure of a Designed Protein that Self-Assembles into a Highly Porous, 230 ACube” (under review).

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Blue light induced domain swapping

Jakeb M. Reis, Darcy C. Burns & G. Andrew Woolley

University of Toronto

AbstractThe design of new optogenetic tools would be facilitated by the development of protein scaffolds thatundergo large, well defined structural changes upon exposure to light. We describe here a variant ofthe blue light photoreceptor photoactive yellow protein (PYP), in which a surface loop is replaced bya heterodimeric coiled-coil forming sequence (E-helix). The protein forms domain swapped dimerswith a dimerization affinity of Kd = 10 µM in the dark. These interconvert with monomers on thetimeframe of weeks. Blue light irradiation decreases the dimerization affinity (Kd = 300 µM) anddramatically enhances the rate of domain swapping, leading to the production of monomers on atime frame of >1 min. Whereas the dimer form of the protein specifically binds a partner K-helixsequence in a coiled-coil motif, the monomeric form is unable to do so. Blue light induced domainswapping thus provides a mechanism for control of protein activity with very low thermal backgroundactivation.

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Minimalist Design of Esterases

Ivan V. Korendovych

Syracuse University

Design of a new catalytic function in proteins, apart from its inherent practical value, is important forfundamental understanding of enzymatic activity. We will present applications of a computationallyinexpensive, minimalistic approach to design of esterases. Two representative cases will be discussed.1. Introducing a single histidine residue into a 74-residue-long C-terminal domain of calmodulinconfers esterase activity on this non-enzymatic protein. The catalytic efficiency of the resultingallosterically regulated catalyst, named AlleyCatE, is on par with those of the best examples ofcomputationally designed esterases. Directed evolution allowed for further improvement of protein’scatalytic efficiency.2. We designed a series of 7-residue peptides that self-assemble into amyloid-like fibrils to act asZn2+-dependent esterases. Zn2+ helps stabilize the fibril formation, while also acting as a cofactorto catalyze acyl ester hydrolysis. These results indicate that amyloid fibrils are able to not only cat-alyze their own formation – they also can catalyze chemical reactions.

22

Engineering peptides and proteins for efficient biodetection using a portableSurface Plasmon Resonance platform

Joelle N. Pelletier, Jean-Francois Masson, Olivier Bolduc, Christopher M. Clouthier, Natalia Bukar,Sandy Shuo Zhao, David M. Charbonneau, Alexandra Aube, Julien Breault-Turcot, Pierre Chaurand

Departement de chimie, Universite de Montreal

Surface plasmon resonance (SPR) is a method of choice for rapid, label-free detection of molecularinteractions. Nonetheless, the high cost of SPR instrumentation and the inability to apply SPR de-tection to crude samples prevent its general use. Non-specific adsorption in biofluids severely limitsthe use of biosensors in real-world applications; crude cell lysate also poses a significant challengebecause of a large concentration of highly adherent lipids. Working with a low-cost, portable SPRprototype designed by the team, we have developed peptide-based surface chemistry tuned to effi-ciently suppress surface fouling when using complex samples such as cell lysate and human serum. Inparallel, we have developed assays for the detection of diverse proteins and therapeutic drugs. Thequantification of the chemotherapy agent methotrexate in clinical samples will be presented. Thismethod is intended for therapeutic drug monitoring in patients undergoing cancer chemotherapy, toensure safety and efficacy of the treatment. To this effect, a competitive binding assay based onSPR using folic acid-functionalized gold nanoparticles and the enzyme human dihydrofolate reduc-tase allowed detection of nanomolar to micromolar concentrations of methotrexate. Application of anengineered, methotrexate-resistant variant of dihydrofolate reductase doubled the working range ofthe assay. Potential interferents in the patients’ blood, such as folic acid, trimethoprim and metabo-lites of methotrexate, did not affect the signal. The method was successfully validated relative to afluorescence polarization immunoassay, commonly used in clinical settings, and the reference methodLC-MS/MS. This example illustrates the potential for this portable SPR platform to serve as a flexibletool for detection of molecular interactions in biological samples.

23

A Portfolio Approach to Protein Property Optimization

Stephen L. Mayo

Caltech Division of Biology and Biological Engineering

Research Interests: Computational protein design, enzyme design, protein sequence evolution, protein-protein recognition.

24

ORAL PRESENTATIONS

25

Modulation of E. coli Rhomboid Protease Activity by Hydrophobic Mismatch

Alexander Foo, Natalie Goto

University of Ottawa

Rhomboids comprise a broad family of intramembrane serine proteases that are found in a widerange of organisms, and participate in a diverse array of biological processes such as homeostasis,apoptosis, and quorum sensing. The E. coli GlpG rhomboid, being the best-characterized memberof the family, is a convenient model for the study of rhomboid structure and function. Despite thewealth of data available for GlpG, little is known about how its activity is regulated, with most stud-ies suggesting that it is constitutively active. However, it has been shown that changes in the lipidenvironment can alter activity of many other integral membrane proteins. To explore the possibilitythat GlpG can also be regulated by its local environment, we assessed its catalytic activity in a varietyof detergent micelle systems. We found that detergent micelles containing alkyl chains with 10 to 12carbon atoms supported the highest rates of proteolysis, while the use of longer alkyl chains decreasedactivity. The activity of GlpG purified into mixed micelles comprised of phosphocholine detergents ofdiffering alkyl chain lengths also followed this trend. This dependence on alkyl chain length suggeststhat the size of the detergent micelle hydrophobic phase is an important factor in activity, with theability of C12-detergents to match the 25 A wide hydrophobic region of GlpG playing a key role inmaintaining high levels of proteolysis. Thermal denaturation studies show that this effect is indepen-dent of protein stability, while Km measurements do not provide evidence for competitive inhibitionby longer-chain detergents. Instead other factors, such as changes in GlpG conformational flexibility,may be involved in this change in activity. Similar mechanisms may also modulate GlpG functionin a more structured lipid bilayer environment, a possibility that is currently being investigated as amechanism through which rhomboids might generally be regulated in biological systems.

26

Design of a kinetically stable and functional protein via modular symmetry

Broom A., Ma M., Jacobi Z., Berthin L., Meiering EM.

University of Waterloo

How can we rationally or computationally design well-behaved, functional, and stable proteins? Weshow one possible solution by mimicking the modular nature of evolution to design a moderatelysized, tri-symmetric, globular protein with immense kinetic stability. The protein, ThreeFoil, is highlyresistant to proteases, has a melting temperature near water’s boiling point, and possesses multivalentsugar binding functionality.

The kinetic stability is exemplified by an unfolding half-life in water of more than a decade. While thebinding constant for a monosaccharide is very weak, being in mM range, the tri-symmetric structureallows for branched glycans to be bound with much greater affinity. Despite these attractive features,some drawbacks exist. With a folding half-life in water of about an hour, ThreeFoil folds slower thanthe ”biological folding limit”, which can make experimentation and purification by refolding from in-clusion bodies difficult. Additionally, despite the immense kinetic stability, it possesses only moderatethermodynamic stability with a delta-G of approximately 7 kcal/mol. This modest thermodynamicstability may limit its ability to act as a scaffold for designing new multivalent sugar binding functions.

What are the molecular origins of the high folding and unfolding energy barriers? How can thefolding rate - and consequently thermodynamic stability - be improved without sacrificing the ideallyslow unfolding? Symmetry, topological frustration and complexity, along with residual structure inthe ”unfolded” state may shed light on these questions and lead to a protein with the desired, well-designed characteristics.

27

Engineering a Glycoside Hydrolase by Directed Evolution for Blood AntigenRemoval

David H. Kwan, Stephen G. Withers

University of British Columbia

The major A and B carbohydrate antigens are clinically the most important blood antigens for bloodtransfusion. We are currently investigating enzymatic methods of removing these antigens from thesurface of red blood cells. The principal antigenic determinants of A and B blood groups are theterminal trisaccharide components; GalNAc-α-1,3-(Fuc-α-1,2)Gal in the A-antigen, and Gal-α-1,3-(Fuc-α-1,2)Gal in the B-antigen. Recent discovery of blood antigen-cleaving bacterial enzymes fromthe GH98 family of glycosidases (Carbohydrate Active Enzyme database: www.CAZy.org) has openedup a new approach for enzymatic removal of both A and B antigens with a single enzyme. These en-zymes, found in Clostridium perfringens and Streptococcus pneumoniae, have been dubbed EABasesfor their endoglycosidase activity towards both A and B antigens, cleaving the full trisaccharide fromthe core chain by hydrolyzing the Gal-β-1,4-GlcNAc-linkage. Under physiological conditions, EABasecan efficiently cleave the Gal-β-1,4-GlcNAc-linked B-trisaccharide. However, the A-trisaccharide ismore problematic since it is attached to RBC glycans by either a Gal-β-1,4- or Gal-β-1,3-linkage,with the latter being poorly cleaved by EABase. We have used structure-guided directed evolution toengineer the EABase enzyme in order to broaden its substrate specificity for both Gal-β-1,4- and Gal-β-1,3-linked blood antigens. After iterative mutagenesis and screening of several libraries, we haveidentified a mutant that can catalyze the cleavage of the A-trisaccharide from a Gal-β-1,3-GlcNAclinkage in an oligosaccharide substrate 170-fold more efficiently than the wild-type enzyme.

28

Accurate prediction of protein stability by explicit negative multistate design

James A. Davey, Christian K. Euler, Roberto A. Chica

Department of Chemistry, University of Ottawa

Current computational protein design (CPD) methodologies allow for the in silico screening of aminoacid sequences on a scale that is experimentally impossible to achieve. While CPD can reliably enrichpredictions of stable protein sequences, the methodology is currently unable to accurately correlatepredicted and experimentally-validated stabilities of protein sequences. Traditionally, CPD is per-formed using a positive design approach whereby sequences are scored and ranked on a model ofthe folded protein structure, approximated by either a single fixed protein backbone or a backboneensemble. In an attempt to reconcile calculation and experiment, we present the first negative de-sign approach that utilizes a backbone ensemble as an explicit negative state model. Our negativestate model, intended to approximate the unfolded protein, was created from an off-target backboneensemble shown to poorly predict and enrich stable protein sequences. Conversely, our model for thefolded protein was created from an on-target ensemble shown to correctly predict and enrich stableprotein sequences. A correlative (R2 = 0.82) quantitative structure-activity relationship (QSAR)was generated using a training set of 18 known stable Streptococcal protein G domain β1 (Gβ1)sequences. Our QSAR was then applied to the prediction of stability for 10 new Gβ1 sequences. Ex-perimental validation of the stabilities of these 10 new sequences shows excellent agreement betweenprediction and experiment (R2 = 0.77) providing a proof-of-concept for the application of off-targetbackbone ensembles in explicit negative multistate design for the prediction of protein stability.

29

Not different, just better: The adaptive evolution of a key glycolytic enzyme

Katherine A. Donovan, Fen Peng, Sarah A Kessans, Tim F Coper, Renwick CJ Dobson

University of Canterbury

Although we have a good understanding of adaptation at the organismal level, there is a paucityof data addressing how organisms adapt at the molecular level. Our study builds on a laboratory ex-periment carried out by Richard Lenski and colleagues in which 12 replicate bacterial populations wereevolved from a common ancestor in an identical glucose-limited environment for <60,000 generations.The fitness of each population increased relative to the ancestor. Whole genome and candidate genesequencing has found that the fixed mutations are concentrated in relatively few genes. One of thesetarget genes is pykF, which encodes for the glycolytic enzyme pyruvate kinase, which is central to theregulation of energy metabolism. There are eight different mutations found scattered across pyruvatekinase. How are the mutations in pykF causing the increased fitness found in the bacterial phenotype?

Interestingly, the fitness, functional and stability data for the adaptively evolved pyruvate kinaseenzymes demonstrate different fitness effects and dynamics. However, the crystal structures andsolution structural profiles (SAXS) are surprisingly similar. In conclusion, although the long-termevolution experiment demonstrates a high degree of parallelism with respect to fitness and muta-tional patterns, our data suggest much less parallelism with respect to protein function. Moreover,our results point to protein dynamics as an important mode for adaptive evolution in proteins.

30

Evolutionary (Ir)-Reversibility of Enzymatic Activity in Laboratory Evolution

Nobuhiko Tokuriki

Michael Smith Laboratories, University of British Columbia

The extent to which mutations interact each other or epistasis dictates how protein evolves to newfunctions. Highly constrained fitness landscapes indicate that protein evolution is deterministic andpredictable. Understanding molecular basis underlying epistasis is important to enhance our abilityto engineer proteins and enzymes in the laboratory.

To explore ruggedness of fitness landscapes, we performed extensive laboratory enzyme evolutionbetween two enzymatic activities, phosphotriesterase (PTE) and arylesterase (AE). First, we evolvedPTE into AE with 22 rounds of directed evolution, resulting a complete function transition betweenPTE and AE with <109 specificity switch. Then, starting the newly evolved AE, we evolved AE backto PTE activity. The phenotype of enzyme (PTE activity) was highly reversible; with 12 rounds ofdirected evolution, we obtained a new efficient PTE on par with the wild-type PTE. However, on thesequence level, the evolution followed only partially the reversal trajectory; the newly evolved PTE(neoPTE) differ 28 mutations from the wild-type PTE (ancPTE). Intriguingly, many mutations differbetween neoPTE and ancPTE were deleterious when we tested on top of each background, neoPTEare located on a distinct, incompatible peak from the original one on the fitness landscape.

I discuss molecular basis underpinning the incompatibility and irreversibility of the enzyme evolution.Detailed characterization of crystal structures of various enzymes revealed that the incompatibilityis due to rewiring intra-molecular interaction networks to displace and replace key components inthe active sites. The active site configuration that degenerated for PTE activity during the forwardevolution was reconciled by a different subset of mutations in the reverse evolution. I also discusshow remote mutations optimize key components in the active site in sub-angstrom level, and the roleof protein dynamics during the enzyme evolution.

31

A coarse-grained elastic network atom contact model and its use in the simulationof protein dynamics and the prediction of the effect of mutations

Vincent Frappier, Laurent Bruneau-Cossette & Rafael Najmanovich

Universite de Sherbrooke

Simplified normal mode analysis (NMA) methods are widely used to study dynamic aspects of proteinstructures but based on geometry alone. We present ENCoM, an Elastic Network Contact Model thatemploys a potential energy function that includes a pairwise atom-type non-bonded interaction termand thus makes it possible to consider the effect of the specific nature of amino-acids, and thus ofmutations, on dynamics within the context of NMA. We compare ENCoM to existing methods for theprediction of the effect of mutations on protein stability, showing than unlike other methods, ENCoMis particularly apt at predicting stabilizing mutations. We also demonstrate the use of ENCoM for theprediction of the effect of mutations on protein function with the prediction of NMR S2 differences fora G121V mutant of the dihydrofolate reductase (DHFR) enzyme in E. coli. We also show preliminaryresults on the prediction of constitutively active and inactive mutations in GPCRs and the predictionof allosteric binding-sites in proteins. Lastly we discuss role of ENCoM within other research areas inthe Najmanovich Research Group, notably in the detection of binding-site similarities as well as theinclusion of full-protein flexibility in docking simulations.

32

Characterization of Tryptophan Residues in Yeast Cystathionine β-Synthase andTheir Potential as Probes of Conformational Change

Edgar Abouassaf, Duale Ahmed and Susan M. Aitken

Carleton University

Cystathionine β-synthase (CBS; E.C. 4.2.1.22) is a pyridoxal 5’-phosphate (PLP) dependent en-zyme that catalyzes the condensation of serine with homocysteine to produce cystathionine. YeastCBS (yCBS) possesses 4 tryptophan residues W132, W263, W333 and W340, each playing varyingroles in function based on their location in the protein structure. In this study, single and triplevariants of yCBS, created by the substitution of W (trp)–< F (Phe), were used to study the effectof each trp residue on the activity, preliminary stability and energy transfer (FRET) with respect tothe PLP cofactor. The order of importance of these residues on activity and stability is W263 <W333 < W132 < W340. The accessibility of each trp residue was determined via quenching withIodide, in the open and closed conformation. Our results show that W263 is the main contributor ofFRET to PLP and the W263F variant had a substantial effect on activity and stability. The W132Fand W340F variants showed no changes in activity or stability, while W333F variant had moderatelyaffected stability.

33

Engineering of genetically encoded pH sensors based on red fluorescent proteins

Yi Shen, Robert Campbell

University of Alberta

Fluorescent proteins (FPs) have been extensively used as templates for engineering of geneticallyencoded biosensors that can report biochemical changes in live cells. FPs with pH-dependent spec-troscopic features can be used as indicators of cellular pH, and have become indispensible tools forstudies of pH homeostasis and cell physiology. Using strategies including direct mutation of the chro-mophore, modulation of the chromophore environment by site directed mutagenesis of residues inclose proximity, and random mutagenesis, we engineered red FP based pH sensors with intensiomet-ric, ratiometric and inversed intensiometric fluorescence response. Here we describe and discuss thedesign and development of these pH sensitive red FPs, their spectral properties and pH-dependency,as well as the applications in fluorescence live cell imaging. In addition, we report the first exampleof a thermochromic protein, which is discovered during the course of the development.

34

POSTER PRESENTATIONS

35

No. 1

Precise Control of Amino Acid Side Chain Conformation with MultistateComputational Protein Design

Adam M. Damry, James A. Davey, Christian K. Euler & Roberto A. Chica

Department of Chemistry, University of Ottawa, Ottawa, ON K1N 6N5

Precise control of amino acid side-chain conformation is an important objective of protein designas conformation determines function. In this study, we computationally designed and experi-mentally validated mutations in streptococcal protein G domain β1 (Gβ1) that lead to desiredconformations in a specific residue, Trp43, while having minimal impact on the overall proteinstructure. To do this, multistate design was performed across a computationally generated en-semble of Gβ1 backbones intended to mimic protein conformational flexibility, allowing Trp43 toadopt non-native conformational states. The resulting ensemble was threaded with amino acidsequences which were then ranked by their predicted stability and binned according to predictedtryptophan conformations. Representative sequences where the Trp43 side chain was predictedto be either in core or solvent exposed were expressed and purified for characterization. Usingcircular dichroism, we determined that all of these Gβ1 sequences were stable and adopted thenative Gβ1 fold. Additionally, we used fluorescence spectroscopy to study the environment of theTrp43 side chain. We observed that all Gβ1 mutants predicted to have a solvent exposed Trp43side chain display lower quantum yields as well as bathochromic shifts in emission wavelengthrelative to those where the Trp43 side chain is predicted to be in core. These results suggestthat the environment of the Trp43 side chain in these mutants is more exposed to solvent, inagreement with our predictions. Our long-term goal is to predict Gβ1 sequences in which theTrp43 side chain is in equilibrium between the core and solvent exposed confirmations, an ob-jective that can only be achieved by the explicit consideration of multiple conformational statesin multistate design.

36

No. 2

Towards photo-control of translation initiation using photoactive yellow protein

Anil Kumar, Anna S. I. Jaikaran, and G. Andrew Woolley*

Chemistry, University of Toronto, 80 St. George Street, Toronto, M5S 3H6, Canada

Photo-control of translation initiation could be a powerful tool for probing the role of trans-lational processes in cellular biology. Cap-dependent translation initiation requires the for-mation of the eukaryotic initiation factor-4F (eIF4F) complex that is composed of three sub-units eIF4E, eIF4A and eIF4G. This is regulated by association/dissociation of hypophosphory-lated/phosphorylated 4E-BP (eIF4E-binding protein) via competitive binding with eIF4G.Here, we report designs and initial characterization of photoswitchable 4E-BP chimeras. Theflexible peptide linker GGSGGSGG of our previously designed circularly permuted PYP (cPYP)(Biochemistry, 2013) was replaced with active segments of 4E-BP. Three constructs were pre-pared, one with only the primary canonical site (54YXXXXL60: cPYP-4E-BP-C0) and twolonger constructs containing the secondary canonical site (78IPGVT82, cPYP-4E-BP-C1, cPYP-4E-BP-C1N1).All designed chimeras were expressed in E. coli and are highly soluble. UV-vis spectra of thedark adapted and irradiated state confirm the photoswitchable properties of all designed con-structs. The recovery of the dark-state structure as monitored by UV-vis spectroscopy afterremoval of blue light irradiation indicates that cPYP-4E-BP chimeras recover slowly in minutes.Importantly, recovery rates of all of the constructs (cPYP-4E-BP-C0, cPYP-4E-BP-C1, cPYP-4E-BP-C1N1) were increased in the presence of 4E, consistent with a light dependent interactionof the chimeras with 4E. Initial activity assays using an in vitro translation systems based onHeLa cell extracts shows inhibition of translation by cPYP-4E-BP-C1 and cPYP-4E-BP-C1N1.Further investigation using fluorescence polarization is in progress to directly characterize thebinding ability of chimeras to 4E.

37

No. 3

Increasing Red Fluorescent Protein Quantum Yield through Rational Design

Antonia T. Pandelieva, Guido F. Calderini, Andrew Wong, and Roberto A. Chica

Department of Chemistry, University of Ottawa, Ottawa, Canada

Red fluorescent proteins (RFPs) are used extensively as fusion tags to track proteins withincells, as gene expression markers, and as partners in FRET experiments. In addition, RFPsare utilized for whole-body imaging of research model animals because red light leads to re-duced phototoxicity and results in lower background autofluorescence. To improve whole-bodyimaging with RFPs, brighter variants are desired. To achieve this, we propose a rational ap-proach that involves stacking the RFP chromophore between two aromatic amino acids in orderto decrease its conformational freedom and reduce radiationless decay, thereby resulting in in-creased quantum yield. Computational protein design (CPD) was used to predict mutationsthat would allow the introduction of a Π-stacked aromatic residue in mRojoA, an RFP thatalready contains a single tyrosine residue that is stacked on the chromophore. A library of mu-tants predicted by CPD was prepared and screened for increased fluorescence intensity. Severalimproved mutants containing a histidine or tyrosine at position 63, which is located directlyabove the chromophore, were identified. The best of these mutants, containing a tyrosine atposition 63, exhibited a two-fold increase in quantum yield compared to mRojoA while having a620 nm emission wavelength. Crystallography experiments are underway to confirm the presenceof the designed Π-stacking interaction with the chromophore. In addition, we are introducingthese mutations into mCherry, a widely used RFP, in order to further improve its quantum yield.

38

No. 4

Engineering Enzyme Substrate Specificity Using Multistate Computational ProteinDesign

Antony D. St-Jacques and Roberto A. Chica

Department of Chemistry & Centre for Catalysis Research and Innovation, University of Ottawa,Ottawa, Ontario

The design of enzymes displaying desired substrate specificity is an important objective in en-zyme engineering. To help achieve this goal, computational protein design (CPD) can be used toidentify sequences that are likely to stabilize designed interactions required for efficient substratebinding. Standard CPD protocols find optimal sequences in the context of a single state, for ex-ample an enzyme with a single desired substrate bound at its active site. However, certain designobjectives such as the creation of broad specificity enzymes or the alteration of substrate speci-ficity for multisubstrate enzymes require the explicit consideration of multiple chemical states.These design objectives can be efficiently tackled using multistate design (MSD), an emergingCPD methodology that allows sequence selection to be driven by the energetic contributionsof multiple states simultaneously. Herein, we report the development of a MSD procedure toenable the identification of aminotransferase mutants displaying altered substrate specificity.Aminotransferases are used as test cases since these multisubstrate enzymes already bind atleast two substrates with side chains of different shapes and properties. We demonstrate thatmultiple chemical states are required to identify branched-chain amino acid aminotransferasemutants predicted to bind both a desired alternate substrate as well as the co-substrate requiredfor transamination, as stabilizing mutations predicted by single-state design in the context ofthe single desired substrate may be detrimental to the binding of the unconsidered co-substrate.

39

No. 5

Sequence variability leads to distinct roles for M4 as an allosteric regulator ofpentameric ligand-gated ion channels

Camille Henault , Peter Juranka , Julian Surujballi , John E. Baenziger

University of Ottawa

Pentameric ligand-gated ion channels (pLGICs) play a central role in rapid communicationbetween neurons in the central and peripheral nervous systems. pLGICs are also the sites ofaction of numerous pharmaceuticals, which alter channel gating to influence synaptic commu-nication. The prototypic pLGIC, the nicotinic acetylcholine receptor (nAChR), is influenced byendogenous and exogenous compounds that interact with the transmembrane domain. M4, themost lipid-exposed of the transmembrane α-helices, likely plays a key role in lipid sensing, pro-tein folding, and the action of many transmembrane domain allosteric modulators. Surprisingly,M4 exhibits a high degree of variability from one nAChR subunit to another. Here, we use twoprokaryotic homologs of the nAChR, GLIC and ELIC, to explore the functional consequencesof M4 sequence variability. We show that mutations in GLIC M4 invariably lead to a reductionin channel gating, suggesting that interactions with the adjacent transmembrane α-helices, M1& M3, are optimized to promote channel gating. In contrast, ELIC retains function even in theabsence of M4, while mutations in ELIC M4 typically result in a gain of function, suggesting thatinteractions are poorly optimized, with intrinsically weak coupling of agonist binding to gating.Our data suggest that the importance of M4 as an allosteric regulatory element may vary fromone nAChR subunit type to another. This variability may influence the susceptibility of differentnAChRs to allosteric modulation by compounds that interact with the transmembrane domain.

40

No. 6

Molecular characterization of the LipIAF5-2 lipase for the synthesis of value-addedchemicals

Carlos Dulcey, Nicolas Doucet

INRS-Institut Armand-Frappier

Esterification reactions catalyzed by lipolytic enzymes are of utmost importance in the con-text of industrial chemical and biochemical processes. Lipases catalyze esterification reactionsthat have gained considerable attention due to the importance of the organic esters of biologi-cal origin involved in the formulation of a wide variety of consumer products. Our laboratoryrecently extracted and characterized a lipase of unknown origin, LipIAF5-2, which was discov-ered in a previous local metagenomic study. The potential of this enzyme in organic synthesishas been investigated for the synthesis of isoamyl acetate (a short chain banana flavor ester) ina fluidized bed bioreactor, showing 100% conversion after 24 hours of reaction at 40°C. SinceLipIAF5-2 also contributes to the hydrolysis of long chain carboxylic esters, we believe it mayalso be useful for the synthesis of these molecules. Long chain fatty acid methyl esters (FAME)are important intermediates of high value-added consumer products. These intermediates areproduced in the oleochemical industry for the formulation of biosolvants, biolubricants and bio-fuels. The main objective of the current project is to explore the potential of LipIAF5-2 forthe synthesis of FAME, and increase the efficiency of the enzyme by protein engineering using asemi-rational approach. Preliminary data on the expression and purification of LipIAF5-2 will bepresented, in addition to the strategy employed for the screening of the desired catalytic activity.

41

No. 7

Short peptides self-assemble to create catalytic amyloids

Caroline M. Rufo1�, Yurii S. Moroz1�, Olesia V. Moroz1�, Jan Stohr2, Tyler A. Smith1, XiaozhenHu3, William F. DeGrado3* and Ivan V. Korendovych1*

1 Department of Chemistry, Syracuse University, Syracuse, New York 13244, USA, 2 Insti-tute for Neurodegenerative Diseases and Department of Neurology, University of California –San Francisco, San Francisco, California 94143, USA, 3 Department of Pha

Enzymes fold into unique three-dimensional structures, which underlie their remarkable catalyticproperties. The requirement to adopt a stable, folded conformation is likely to contribute totheir relatively large size (<10,000 Da). However, much shorter peptides can achieve well-definedconformations through the formation of amyloid fibrils. To test whether short amyloid-formingpeptides might in fact be capable of enzyme-like catalysis, we designed a series of seven- residuepeptides that act as Zn21-dependent esterases. Zn21 helps stabilize the fibril formation, whilealso acting as a cofactor to catalyse acyl ester hydrolysis. These results indicate that prion-likefibrils are able to not only catalyse their own formation, but they can also catalyse chemical re-actions. Thus, they might have served as intermediates in the evolution of modern-day enzymes.These results also have implications for the design of self-assembling nanostructured catalystsincluding ones containing a variety of biological and non-biological metal ions.

42

No. 8

Using multiply branched antifreeze proteins as novel ice-binding reagents

Corey Stevens and Peter Davies

Queen’s University

Antifreeze proteins (AFPs) are ice-binding proteins produced by various overwintering organismssuch as fish, plants, microorganisms and terrestrial insects, as an adaptation to sub-freezing tem-peratures. AFPs function by binding to seed ice crystals and preventing the ice’s further growth,thereby protecting the organism from damage caused by freezing. The attachment of AFPs toice depresses the freezing temperature below the melting temperature — a phenomenon termedthermal hysteresis (TH). Previous work has shown the TH activity of AFPs can be enhancedsimply by increasing the size of the AFP through the addition of a fusion protein. In attemptingto further increase the TH activity of AFPs we have linked multiple type III AFPs togethervia a dendrimer. Using a heterobifunctional crosslinker we have been able to attach a range(8-13) of type III AFPs to a second-generation polyamidoamine (G2-PAMAM) dendrimer. Theheterogeneous sample of dendrimer-linked type III constructs has shown a four-fold increase inTH activity over monomeric type III AFP. Linking AFPs together via a dendrimer will generatenovel reagents for the control ice growth.

Supported by CIHR

43

No. 9

Investigating the structural determinants of substrate specificity in astereo-inverting aminotransferase.

Curtis J.W. Walton, Frederic Thiebaut, Jean-Francois Couture, & Roberto A. Chica

Department of Chemistry, Department of Biochemistry, Microbiology, and Immunology, andthe Centre for Catalysis Research and Innovation, University of Ottawa, Ottawa, Ontario.

Aminotransferases are pyridoxal-phosphate-dependent enzymes that catalyze the synthesis ofenantiopure amino acids. Because of this, there is a growing interest in engineering them toalter their substrate specificity and to increase their catalytic activity. D-phenylglycine amino-transferase (DPAT), which produces D-phenylglycine and α-ketoglutarate from L-glutamate andbenzoylformate, is the only known stereo-inverting aminotransferase, making it an attractive tar-get for enzyme engineering. To help engineer the substrate specificity of DPAT for the productionof unnatural D-amino acids, a better understanding of its structural determinants involved insubstrate specificity is required. To achieve this, we are using a combined approach that includesx-ray crystallography, mutagenesis, and enzyme kinetics. We have solved the crystal structureof the apo form of DPAT, which enabled us to identify active site residues hypothesized to beinvolved in efficient substrate binding. These residues were subjected to saturation mutagene-sis, resulting in mutant libraries that were screened using a continuous high-throughput assaythat we previously developed. Screening of these libraries allowed us to identify key active sitearginine residues that are essential for catalytic activity, presumably because they allow efficientsubstrate binding. To better understand the role of these arginine residues, we are currentlyperforming soaking experiments to obtain crystal structures of DPAT with bound substrates.Obtaining these structures will open the door to the engineering of DPAT substrate specificityusing a rational design approach, leading to improved biocatalysts for the production of valuableD-amino acids.

44

No. 10

Clinical integration of a portable SPR device: Pharmacodynamics of L-asparaginasein the treatment of childhood acute lymphoblastic leukaemia

David M. Charbonneau, Alexandra Aube, Jean-Francois Masson and Joelle N. Pelletier

Departement de chimie, Universite de Montreal, C.P. 6128 Succ. Centre-Ville, Montreal, Qc,Canada H3C 3J7

Acute lymphoblastic leukaemia (ALL) is the most prevalent pediatric cancer diagnosed in devel-oped countries. Precursor B-cell lymphoblastic leukaemia (B-ALL) constitutes the most commonphenotype, affecting more than 70% of newly diagnosed children. L-asparaginase (ASNase) isused as a therapeutic drug in the induction phase of the treatment. Because this enzyme isof bacterial source, many children develop an allergic reaction against the therapeutic drug.Monitoring the level of anti-L-asparaginase antibodies is crucial for accurate diagnosis and foradjustment of the therapeutic drug used for treatment. However, the common technique usedto monitor anti-asparaginase, ELISA, fails to give a clear correlation between the antibody titreand the clinical picture, requires secondary detection and is not suitable for on-site analysis. Weare developing new protocols adapted to a portable surface plasmon resonance (SPR) instru-ment for direct in situ monitoring of anti-ASNase in clinical samples from treated ALL patients.We are combining surface chemistry and protein engineering to improve the sensitivity of theSPR response leading to early detection of allergic reaction. Our robust and portable biosensorwill contribute to improve the accessibility to screening in order to adjust treatment of ALLrigorously and in a timely manner.

45

No. 11

Polyhydroxyalkanoate (PHA) depolymerases for the potential development of animproved bioplastic degrading enzyme

Diana I. Martınez-Tobon 1, Jared Lehne 2, Anastasia Elias 1, and Dominic Sauvageau 1

1 Department of Chemical and Materials Engineering, University of Alberta. 2 Departmentof Biological Sciences, University of Alberta.

Polyhydroxyalkanoates (PHA) – a class of natural, biodegradable polyesters – have recentlybeen the focus of significant commercial interest. These polymers can be hydrolyzed by PHAdepolymerases - a class of enzymes that includes endo-PHA-depolymerases. Reported studieson the enzymatic activity of these proteins generally focus on isolating and characterizing singlenative enzymes. Moreover, kinetic assessments are not standardized, often do not correlate anddo not account for substrate binding kinetics. The aim of this project is to compare differ-ent PHA depolymerases expressed from wild type bacteria and heterologously, and characterizetheir degradation behaviour and stability with standardized assays by varying conditions suchas concentration, temperature, pH, and polymer substrate architecture. This serves as the basisfor the design of engineered PHA depolymerases that can degrade plastic faster under harshindustrial or environmental conditions.Initial activity assessments were made using PHA pellets or films placed in whole cell broths orin extracellular fractions of wild type organisms excreting the enzyme of interest. Results showedthat the extracellular PHA depolymerase PhaZCte, produced by Comamonas testosteroni, hadthe highest enzymatic activity of all strains tested. In addition, in order to decouple the effectof the enzyme of interest from that of other C. testosteroni enzymes, the PhaZCte gene wasintroduced in the bacterium E. coli. A purified recombinant version of PhaZCte is thus obtainedto better assess its degradation activity and stability. By repeating this process for differentPHA depolymerases displaying advantageous properties, it will be possible to determine thebest structure and active site conformation to maintain high activity under a broader range ofconditions. Furthermore, directed evolution will be used on the resulting enzyme to obtain amore robust PHA depolymerase for applications such as scheduled biodegradation.

46

No. 12

Comparison of protein flexibility among human homologues of the RNase Asuperfamily

Donald Gagne & Nicolas Doucet

INRS-Institut Armand-Frappier, Universite du Quebec, 531 Boulevard des Prairies, Laval, QCH7V 1B7, CANADA

Atomic motions are an integral part of the biological function of several protein and enzymesystems. This molecular flexibility can be involved in the binding and discrimination of sub-strates, ligand positioning and/or product release. It is yet unknown whether structural andfunctional enzyme homologues rely on the same concerted residue motions to promote catalysisand/or biological function. It has been hypothesized that biologically relevant motions occurringon the millisecond timescale have evolved to promote and/or preserve optimal enzyme cataly-sis. We used NMR relaxation dispersion experiments (1H-15N CPMG) to successfully captureand compare the role of conformational flexibility among human members of the RNase A su-perfamily, which share a common fold and RNA degrading ability. We show that motions onthe millisecond timescale are maintained within similar functional regions displaying divergentsequences. Although motions are preserved, exchange rates vary considerably, consistent withdistinct catalytic activities. The study of functional dynamics opens the door to the design ofallosteric inhibitors that act by disrupting the dynamic equilibrium essential for enzyme cataly-sis. This promising path provides a complementary alternative to conventional inhibitors, whichoften fail due to the emergence of antibiotic resistance.

47

No. 13

Introducing New Functions into Old Proteins

Elizabeth Raymond, Jennifer Yoon, Korrie Mack, Yurii Moroz, Ivan Korendovych

Syracuse University

Generating new catalytic function in proteins has intrinsic practical utility and provides greaterunderstanding of enzymatic activity. However, the challenge of creating an efficient catalystfor a(un)natural chemical transformations remains largely unmet. We employ a minimalist ap-proach to protein design in order to determine the contribution of various parameters to catalyticfunction. Here we study the effects of placement of a single reactive lysine residue in a hydropho-bic pocket of calmodulin. Calmodulin is a non-enzymatic protein that is allosterically regulatedby calcium ions. We show that introducing a single lysine residue (F92K) into the C-terminaldomain of CaM’s hydrophobic pocket enables this protein to catalyze retro-aldol reaction. Theresulting catalyst retains calmodulin’s allosteric control: it is only active in the presence of Ca2+.

48

No. 14

Connectivity between catalytic landscapes of the metallo-β-lactamase superfamily

Florian Baier, Nobuhiko Tokuriki

Michael Smith Laboratories, University of British Columbia

The expansion of functions in an enzyme superfamily is thought to occur through recruitmentof latent, promiscuous functions within existing enzymes. Thus, the promiscuous activities ofenzymes represent connections between different catalytic landscapes and provide an additionallayer of evolutionary connectivity between functional families alongside their sequence and struc-tural relationships. Here, we describe a superfamily-wide analysis of evolutionary and functionalconnectivity in the metallo-β-lactamase (MBL) superfamily. We investigated evolutionary con-nections between functional families and related evolutionary to functional connectivity; 24enzymes from 15 distinct functional families were challenged against 10 catalytically distinctreactions. We revealed that enzymes of this superfamily are generally promiscuous, as eachenzyme catalyzes on average 1.5 reactions in addition to its native one. In addition, metal ionexchange substantial increases and alters the degree of catalytic promiscuity among several en-zymes. Interestingly, several evolutionary and functional connections (phosphotriesterase andlactonase as well as arylsulfatase and phosphodiesterase) were also observed in other superfami-lies, suggesting an universal relationship between these functions. In addition, many connectionsappear to be unrelated to recent evolutionary events and occur between chemically distinct re-actions. Consequently, catalytic landscapes in the MBL superfamily overlap substantially; eachreaction is connected on average to 3.7 other reactions, suggesting that the highly distinct reac-tions in the MBL superfamily are a consequence of divergent evolution. Our results show thatnew enzymatic function could evolve rapidly from the current diversity of enzymes and rangeof promiscuous activities, which has important implications for enzyme engineering and in ourunderstanding of enzyme evolution.

49

No. 15

Decoding signalling roles of diphosphoinositol polyphosphates

Gayane Machkalyan, Terry Hebert and Gregory J Miller

Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada

Inositol phosphates (IPs) are small molecules that regulate a variety of cellular signaling path-ways. The best characterized IP, inositol-1,4,5-triphosphate (IP3), triggers Ca2+ release from in-tracellular reservoirs; however, there are <30 differently phosphorylated IPs that perform diversesignaling roles. Diphosphoinositol polyphosphates (diIPs) are the most highly phosphorylatedIPs and are structurally distinct from IPs due to pyrophosphate moieties on 1 or more posi-tions of the inositol ring. They are implicated in regulating apoptosis, DNA repair, telomerasemaintenance, vesicle trafficking, phosphate sensing, and actin cytoskeleton dynamics (a). Twoclasses of IP kinases produce structurally distinct diIPs: inositol pyrophosphate synthetase (IPS)and inositol hexakisphosphate kinases (IP6Ks) (b). Although diIPs are implicated in regulatingvarious processes in the cells, very few mechanisms of their actions have been demonstrateddirectly. Proteomics approach has been applied on mammalian cells overexpressing IP6K andIPS enzymes and interacting proteins of each enzyme are identified by mass spectrometry. Iden-tification of protein complexes of IP6K and IPS is an important step towards our understandingof cellular signaling roles of diIPs.Reference:a. Miranda S. C. Wilson, Thomas M. Livermore, Adolfo Saiardi Biochem. J. (2013) 452 (369-379)b. Chakraborty A, Kim S, Snyder SH. (2011) Sci Signal. 4(188) 4(188): re1. doi:10.1126/scisignal.2001958.

50

No. 16

Intracellular protein expression as an efficient vessel for biosynthesis of flavoringesters

Guillaume Brault & Nicolas Doucet

INRS-Universite du Quebec

Flavors and fragrances are broadly exploited molecules in the food, cosmetic, detergent, chemicaland pharmaceutical industries. Finding better ways to answer the needs of the industry partlyrelies on metagenomic approaches to uncover new and versatile lipases. While these methodsare powerful biotechnological tools, the need for a simple and optimized expression system oftenremains a serious bottleneck for the production of industrial yields. Here, we present a simple E.coli recombinant expression model for two lipases (LipIAF5-2 and LipIAF1-6) that we previouslyextracted from a metagenomic study. Initial screening of these two enzymes for affinity toward18 flavoring esters showed promising versatility of the heterologously expressed proteins. Usingthe recombinant E. coli strain as an overexpression vessel for a whole-cell biocatalyst (WCB),we efficiently synthesized short-chain esters by transesterification and/or esterification reactionsin organic media. Lipase LipIAF5-2 showed good activity for the synthesis of isoamyl acetate,an important banana flavor, while LipIAF1-6 was more effective toward substrates containing a4-carbon acyl donor chain. Permeabilization of the cell membrane proved essential for specificapplications, and different compounds were tested to evaluate the impact of permeabilization onthe synthesis activity of the WCB. Out of eight compounds, 1% (p/v) Tween-20 was the mosteffective permeabilization agent for LipIAF5-2, while 1% (p/v) Triton-X100 gave the highest ac-tivity for LipIAF1-6. Impact of cell lyophilisation and cell freezing on activity was also assessedin the scope of a simple production protocol of WCB. The wet but frozen cells appeared to bethe most effective form of WCB, thus providing base for a simple biocatalyst production. Alto-gether, these results show promising potential for the industrial biosynthesis of short-chain esters.

51

No. 17

Enzymatic activity of Lecithin:retinol acyltransferase: A thermostable and highlyactive enzyme with a likely mode of interfacial activation

Habib Horchani, Sylvain Bussieres, Line Cantin, Mustapha Lhor, Jean-Sebastien Laliberte-Gemme, Rock Breton, Christian Salesse

Laval University

Lecithin:retinol acyltransferase (LRAT) plays a major role in the vertebrate visual cycle. Indeed,it is responsible for the esterification of all-trans retinol into all-trans retinyl esters, which canthen be stored in microsomes or further metabolized to produce the chromophore of rhodopsin.In the present study, a detailed characterization of the enzymatic properties of truncated LRAT(tLRAT) has been achieved using in vitro assay conditions. A much larger tLRAT activity hasbeen obtained compared to previous reports and to an enzyme with a similar activity. In addi-tion, tLRAT is able to hydrolyze phospholipids bearing different chain lengths with a preferencefor micellar aggregated substrates. It therefore presents an interfacial activation property, whichis typical of classical phospholipases. Furthermore, given that stability is a very important qual-ity of an enzyme, the influence of different parameters on the activity and stability of tLRAThas thus been studied in detail. For example, storage buffer has a strong effect on tLRAT ac-tivity and high enzyme stability has been observed at room temperature. The thermostabilityof tLRAT has also been investigated using circular dichroism and infrared spectroscopy. A de-crease in the activity of tLRAT was observed beyond 70 °C, accompanied by a modification ofits secondary structure, i.e. a decrease of its α-helical content and the appearance of unorderedstructures and aggregated β-sheets. Nevertheless, residual activity could still be observed afterheating tLRAT up to 100 °C. The results of this study highly improved our understanding ofthis enzyme.

52

No. 18

Expression, purification and characterization of an archaeal enzyme involved intyrosine biosynthesis

Irina Shlaifer, Joanne Turnbull

Concordia University

Prephenate dehydrogenase (PD) is a member of the TyrA protein family involved in the biosyn-thesis of L-tyrosine. This enzyme catalyzes the oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate in the presence of NAD+. This conversion, along with the rearrangementof chorismate to prephenate catalyzed by chorismate mutase (CM), constitutes two consecutivereactions that are essential for tyrosine biosynthesis in many bacteria and other microorganisms,yeast, and fungi. The enzymes CM and PD and are generally present as distinct proteins or asbifunctional fusions of the two activities, CM-PD. Accordingly, there is considerable interest inthe structural relationship between the sites at which the two reactions are catalyzed. Whileattempts to crystallize CM-PD from several mesophilic bacteria have failed, it has been reportedthat proteins derived from thermophilic organisms may be more amenable to crystallization. Assteps towards obtaining structural data on the bifunctional enzyme we present the first biochem-ical study on CM-PD from Ignicoccus hospitalis, a hyperthermophilic archaeon.In this report, the gene encoding a putative CM-PD was cloned from the genomic DNA derivedfrom an archaeal co-culture of Nanoarchaeum equitans and I. hospitalis. The bifunctional en-zyme was recombinantly expressed in Escherichia coli and purified to near homogeneity usingnickel affinity chromatography. The enzyme possesses CM and co-factor-dependent PD activitiesand is present as a dimer in solution. Interestingly, the enzyme is not inhibited by L-tyrosine asreported for other bacterial enzymes, and does not undergo a quaternary conformational changein the presence of NAD+ or L-tyrosine as reported for E. coli CM-PD. I. hospitalis CM-PDshares only modest amino acid sequence identity with the E. coli enzyme and possesses an el-evated pI. Our future studies will determine if I. hospitalis CM-PD, with its distinct features,will yield diffraction quality crystals.

53

No. 19

The Role of Intramembrane Chemistry in the Gating of Pentameric Ligand-GatedIon Channels

J.P. Daniel Therien, Peter F. Juranka, and John E. Baenziger

University of Ottawa; Faculty of Medicine; Biochemistry, Microbiology and Immunology

Pentameric ligand-gated ion channels (pLGICs) are responsible for fast synaptic communica-tion between neurons. The transmembrane domain (TMD) in each of the five pLGIC subunitsis comprised of 4 membrane-spanning α-helices, M1 to M4, with M2 lining the ion channel poreand both M1 and M3 forming a ring of α-helices surrounding M2. The fourth transmembraneα-helix M4 binds to both M1+M3, but is highly exposed to the lipid bilayer. Increasing evidencesuggests that altered M4 binding to M1+M3 modulates channel gating, and that some TMD al-losteric modulators (lipids, neurosteroids, and some pharmaceuticals) affect gating by influencingM4 interactions with M1+M3. Surprisingly, there is tremendous variation in the chemistry at theinterface between M1, M3, and M4, suggesting that different pLGICs exhibit variable affinitiesfor M4 binding to M1+M3 and thus variable efficiencies of channel gating. Using site-directedmutagenesis, we explore here the effects of variable chemistry at the M1/M3/M4 interface onthe gating of a prokaryotic pLGIC, called GLIC (a proton activated channel). Ala-scanningmutagenesis of residues at the interface between M1, M3, and M4 shows that most aromatic toalanine mutations result in a 5 to 10-fold reduction in the gating efficiency. The mutation ofpolar or charged residues to alanine also reduces gating efficiency, but the detrimental effectsof these mutations are less dramatic (3-fold or less reduction in gating). In contrast, variableeffects were observed with the mutation of aliphatic residues (both gain and loss of function).Our data highlight the role of different types of interactions in M4 binding to M1/M3 in GLIC,and provide a basis for predicting the efficiencies of M4 binding and thus channel gating inhuman neuronal pLGICs.

54

No. 20

Blue light induced domain swapping

Jakeb M. Reis, Darcy C. Burns, G. Andrew Woolley

Department of Chemistry, University of Toronto

The design of new optogenetic tools would be facilitated by the development of protein scaffoldsthat undergo large, well defined structural changes upon exposure to light. We describe here avariant of the blue light photoreceptor photoactive yellow protein (PYP), in which a surface loopis replaced by a heterodimeric coiled-coil forming sequence (E-helix). The protein forms domainswapped dimers with a dimerization affinity of Kd = 10 µM in the dark. These interconvertwith monomers on the timeframe of weeks. Blue light irradiation decreases the dimerizationaffinity (Kd = 300 µM) and dramatically enhances the rate of domain swapping, leading to theproduction of monomers on a time frame of >1 min. Whereas the dimer form of the proteinspecifically binds a partner K-helix sequence in a coiled-coil motif, the monomeric form is unableto do so. Blue light induced domain swapping thus provides a mechanism for control of proteinactivity with very low thermal background activation.

55

No. 21

Engineering D-amino acid aminotransferase mutants displaying increased activitytowards aromatic D-amino acids

Janet E.B. Barber, Adam M. Damry, Guido F. Calderini, Curtis J.W. Walton & Roberto A.Chica

Department of Chemistry, University of Ottawa, Ottawa, Ontario & Centre for Catalysis Re-search and Innovation, University of Ottawa, Ottawa, Ontario

D-Amino acid aminotransferase (DAAT) is a pyridoxal phosphate-dependent enzyme that cat-alyzes the synthesis of a broad range of D-amino acids, making it an attractive biocatalyst forthe production of enantiopure D-amino acids. To bolster its biocatalytic applicability, improvedvariants displaying increased activity towards non-native substrates are desired. Recently, wedeveloped a high-throughput, colorimetric, continuous coupled enzyme assay for the screening ofDAAT mutant libraries that is based on the use of D-amino acid oxidase (DAAO). In this assay,the D-amino acid product of DAAT is oxidized by DAAO with concomitant release of hydro-gen peroxide, which is detected colorimetrically by the addition of horseradish peroxidase ando-dianisidine. Using this assay, we identified two DAAT mutants (V33G and V33Y) displayingaltered substrate specificity via the screening of cell lysates in 96-well plates. The V33G mutantdisplays a ≈8-fold decrease in kcat/KM for pyruvate and a ≈3-fold increase in kcat/KM forphenylpyruvate, resulting in a ≈25-fold specificity switch that makes this mutant more specificfor phenylpyruvate than native substrate pyruvate, unlike wild-type DAAT which displays theopposite preference. On the other hand, the V33Y mutant displays a catalytic efficiency forthe transamination of pyruvate that is similar to that of the wild type, yet is also ≈3-fold moreefficient at transaminating phenylpyruvate, demonstrating a broadened substrate specificity. Weare currently characterizing the range of aromatic D-amino acid substrates that these mutantscan react with in order to evaluate their potential for the synthesis of these valuable chemicals.

56

No. 22

Structural dynamics of CuZn Superoxide dismutase monitored byhydrogen-deuterium exchange

Jessica A. Rumfeldt, Elizabeth M. Meiering

University of Waterloo

Methods used to determine protein structure tend to give a static picture of the native state;hydrogen-deuterium amide exchange (HX) monitored by NMR can reveal the more biologi-cally relevant dynamic nature of a protein under native conditions. CuZn superoxide dismutase(SOD1), an enzyme linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS),was studied using HX in order to see if structural fluctuations/local unfolding are plausible ini-tiators of potentially toxic SOD1 aggregation observed in the motor neurons of ALS patients.The wild-type protein in its fully mature state is a homodimer that binds one copper and onezinc per beta barrel monomer (holo). In the holo state, SOD1 is extremely stable with HXrevealing most of the core amides resist exchange with solvent water for greater than 80 days.Only the edges of the barrel and connecting loops show any dynamic nature as indicated bysolvent exchange. ALS-linked single site mutations of SOD1 were also studied by HX. All threeshow a greater degree of mobility in the regions surrounding their respective sites of mutationand, interestingly, compensating regions showing decreased mobility. In two of the mutants, theeffect of the amino acid change propagates quite far throughout the beta barrel. There is evenevidence that the G93A mutation, located in a tight turn near the beta barrel plug, is enoughto weaken the core of the protein so that one half of the barrel unfolds independently of theother, making aggregation of this protein a much more likely event. These studies suggest thatthe holo wild-type SOD1 is very compact and immobile and not likely to open up aggregationprone regions. ALS-associated mutations increase mobility to varying degrees suggesting thatALS-mutations in the holo form of the protein may make the protein more likely to aggregate.

57

No. 23

Biophysical characterization of recombinant carboxylesterase EstGtA2 fromGeobacillus thermodenitrificans

Jessica Kelly Moisan1, 2, Fatma Meddeb-Mouelhi1, 3 and Marc Beauregard1, 2

1 Centre de recherche sur les materiaux lignocellulosiques, Universite du Quebec a Trois-Rivieres,3351 Boul. Des Forges, C.P. 500 Trois-Rivieres (Quebec) G9A 5H7, Canada 2 PROTEO, Univer-site Laval, 2705 Boul. Laurier, Quebec (Quebec) G1V 4G2, Canada 3 Buckman North America,351 Joseph-Carrier, Vaudreuil-Dorion (Quebec) J7V 5V5, Canada

Carboxylesterases from thermophiles have become objects of special interest for structural in-vestigation and for a broad range of biotechnological applications. Their range of physico-chemical properties makes them enzymes of great interest for textile, food and pulp and paperindustries. Carboxylesterases (EC 3.1.1.1) belong to a class of hydrolases adopting the α/βhydrolase fold. These enzymes are active at alkaline pH and high temperature. Understandingthe relationship between structure and enzymatic properties is essential in order to producenovel biocatalysts mutants with desirable properties for a given application. Five mutants ofthe recombinant Geobacillus thermodenitrificans carboxylesterase (EstGtA2) were generated bysite-directed mutagenesis in order to study the contribution of specific salt bridges in the stabi-lization and refolding of EstGtA2. One particular mutant (M1a), in which a salt bridge has beendisrupted by a point mutation (R37A), was found to have an increased melting temperature.This is an unexpected change in enzymatic properties considering the fact that salt bridges oftencontribute to thermal stability. In order to better understand the impact of this point muta-tion (R37A), yield production has been optimized for both the wild type protein EstGtA2 andthe mutant M1a (R37A). Biophysical and biochemical studies were performed to enable betterunderstanding the structure-function relationships. The enzymes were characterized by circulardichroism for conformational analysis, by enzymatic assays for activity and specificity and bydynamic light scattering to determine solution conditions which are optimal for the sample tobe monodisperse. In the near future our goal is to determine the three dimensional structuresby X-ray crystallography, allowing for a detailed investigation of structural key elements whichcontrol the properties of the carboxylesterase EstGtA2.

58

No. 24

Engineering Lipid Sensitivity in Prokaryotic Ligand-Gated Ion Channels

Jiayin Sun, Casey L. Carswell, John E. Baenziger

University of Ottawa

Pentameric ligand-gated ion channels (pLGICs) play a crucial role in synaptic transmission,have been implicated in neurological disease, and are important drug targets. The prototypicpLGIC, the nicotinic acetylcholine receptor (nAChR), is particularly sensitive to allosteric mod-ulators, such as lipids, that act on the transmembrane domain. To understand the mechanismsof nAChR lipid sensing, we have focused on two prokaryotic homologues of the nAChR, calledELIC and GLIC. Membrane-reconstituted nAChR, ELIC and GLIC exhibit similar structures and biophysical properties, butdifferent thermal stabilities ( 53, 60, and 69 degrees Celsius, respectively). In simple membraneslacking activating lipids, the nAChR adopts a native-like uncoupled conformation that does notundergo agonist-induced conformational transitions. Although ELIC does not adopt a similaruncoupled conformation, channel gating is also sensitive to lipids. In contrast, the gating ofGLIC is relatively insensitive to bilayer lipid composition. Lipid sensing in all three pLGICsis likely mediated by M4, the most lipid-exposed transmembrane alpha-helix of each subunit.Structural comparisons of the three homologues suggest that GLIC’s increased thermal stabilityand relative lipid insensitivity may be due to increased aromatic interactions between M4 andthe rest of the transmembrane helical bundle, and thus tighter helical packing. Here, we useprotein engineering to probe the role of aromatic residues in transmembrane domain stabilityand lipid sensing in the nAChR, ELIC, and GLIC.

59

No. 25

Mitotic kinesin structure displays elusive near rigor state of the motor domain

Kritica Arora, Parker Anderson, Ana Asenjo, Lama Talje, Monika Joshi, Hernando Sosa, Ben-jamin Kwok, and John Allingham

Department of Biomedical & Molecular Sciences, Queen’s University, Kingston ON

Kinesins are a diverse class of ATP-powered motor proteins that perform a variety of vitaltasks in eukaryotic cells. Some use microtubules as a ‘rail system’ to drag intracellular cargos,while others create structural networks of microtubules that enable alignment and separation ofchromosomes when the cell divides. Kif14 is from the latter category that functions in mitosisand cytokinesis, and has a unique molecular structure compared to other known kinesins of itsclass . Kif14 binds tightly to microtubules and does not display typical nucleotide-dependentchanges in this affinity. We present a structural characterization of the mouse Kif14 motor do-main as a Maltose Binding Protein fusion. Superposition of the highly conserved P-loop motifof Kif14 onto available kinesin structures shows that the ATP binding pocket is open, as if readyto exchange its bound ADP for MgATP. In this state, the central β-sheet is twisted 10 degreesbeyond the maximal amount observed in other kinesins. This phenomenon has only been seenduring the transition between ATP-bound and nucleotide-free states of myosins – known as thenear rigor state . Kif14’s inclination to crystallize in this state could explain its microtubulebinding affinity and propensity for futile ATP hydrolysis, as well as the profound degree ofmicrotubule lattice decoration we observe on both intact microtubules and curled microtubulesheets using electron microscopy.

60

No. 26

Development of Computational Tools towards the Virtual Engineering ofBiocatalysts

Joshua Pottel, Anna Tomberg, Calem Bendell, Nicolas Moitessier

McGill University

Biocatalysis is a growing field, demonstrated by the increased use in industrial processes. En-zymes, the major class of biocatalysts, are attractive because of their intrinsic chemo-, regio-and stereo-selectivity when working with complex, functionalized molecules that may be unableto withstand harsh reaction conditions. However the main drawback of most biocatalysts istheir restricted substrate specificity, which significantly limits their applicability in catalyzingnew, industrially-relevant reactions. To address this issue, one can engineer an enzyme for agiven reaction and/or a given substrate. Enzyme engineering has been driven by experimen-tal techniques however advances have revealed that the combination of computational tools andexperimental validation may be more cost-efficient and effective. In this context, we are develop-ing computational tools to guide the engineering of cytochrome P450 enzymes (CYPs) towardsnew reactions. We will present our latest developments starting with the implementation of anefficient, accurate method to model transition states (TSs) of oxidation reactions catalyzed byCYPs in our software IMPACTS (In-silico Metabolism Prediction by Activated Cytochromesand Transition States). This has been followed by the development of additional software rou-tines to mutate residues and a statistical analysis of side-chain conformations to properly orientthese newly mutated residues.

61

No. 27

Filament assembly by CTP synthase: A novel mechanism for allosteric regulation ofenzyme activity

Justin Kollman

McGill University

A number of metabolic enzymes have recently been shown to have unanticipated patterns ofsubcellular localization, including filamentous localization patterns. CTP Synthetase (CtpS) isa metabolic enzyme that is essential for cell proliferation across all kingdoms. While many en-zymes form small oligomers, CtpS was recently shown to form large-scale filamentous structuresof unknown function in prokaryotes and eukaryotes. To determine why CtpS polymerizes, wesimultaneously monitored CtpS polymerization and enzymatic activity. Polymerization inhibitsactivity and CtpS’s product, CTP, induces assembly. To understand how assembly inhibitsactivity, we used electron microscopy to define the structure of CtpS polymers. This struc-ture, at 6.8 A resolution suggests that polymerization sterically hinders a conformational changenecessary for CtpS activity. Structure-guided mutagenesis and mathematical modeling fur-ther indicate that coupling activity to polymerization promotes cooperative catalytic regulation.This previously-uncharacterized regulatory mechanism is important for cellular function sincea polymerization-defective CtpS mutant disrupts E. coli growth and metabolism. We proposethat regulation by large-scale polymerization enables ultrasensitive control of enzymatic activitywhile storing inactive enzymes in a readily accessible form.

62

No. 28

Fluorescence-based monitoring of PYP photo-switching in vivo

Katherine Brechun, Vitali Borisenko, Lori Yin, Asim Rashid, Huixin Lu, Andrew Woolley

University of Toronto, Department of Chemistry

Photoactive yellow protein (PYP) is a bacterial protein that changes its structure in responseto irradiation with blue light. From a biotechnological standpoint, PYP is useful because itcan be used to develop synthetic proteins with activity that can be controlled by light; theselight-controlled proteins would enable in vivo studies with a high degree of spatial and tempo-ral control. In this project, a system was developed to monitor the photo-cycle of PYP usingfluorescence, allowing the activity of PYP to be confirmed in eukaryotic cell culture. This is animportant step to confirm the feasibility of in vivo studies with PYP-controlled proteins.To monitor the photo-cycle, PYP was fused with blue fluorescent protein (BFP). When PYP isin its dark-adapted state, it absorbs blue light; therefore, in the constructed fusion protein theblue fluorescence from BFP is absorbed by PYP resulting in an observed quench of fluorescence.When PYP absorbs blue light it changes conformation, assuming its light-adapted state. In thislight-adapted state, the absorbance characteristics of PYP change and the protein no longerabsorbs blue light. Therefore after the initial quench in blue fluorescence, PYP light-adapts andceases to quench the blue fluorescence, resulting in a corresponding increase in blue fluorescencefrom BFP. The time-dependent changes in fluorescence produced by this BFP-PYP fusion pro-tein were characterized in vitro, as well as in E. coli and in eukaryotic cell culture. The changesin blue fluorescence were able to confirm the PYP photocycle in vivo.

63

No. 29

Towards Integrating Calcium Sensors for In vivo Neuronal Activity Imaging

Landon Zarowny and Robert E. Campbell

University of Alberta

Currently, there are several genetic and physical methods for investigating neuronal networks inthe context of a live small model animal. The most promising of these are genetically encodablecalcium or voltage sensors, based on fluorescent proteins, which report activity as a change inrelative fluorescence. The development of these sensors has been extensive and their activity andperformance thoroughly characterized. However, due to their mechanism, these biosensors mustbe continuously imaged which presents a challenge for recording activity over extended periods oftime or deep in the brain where light does not penetrate. Our goal is to correlate calcium influx,which is associated with membrane depolarization during action potentials, with an increase inthe concentration of a long-lived fluorescent molecule. Here we propose a novel biosensor designcapable of integrating calcium fluctuations in living models. The substrate for this biosensoris a ubiquitous metabolite of heme synthesis, precorrin-2, which can be enzymatically methy-lated at one additional site to produce a novel fluorescent product: trimethylpyrrocorphin. Theaccumulation of this heme derivative can be observed through whole cell fluorescence whichindicates increased levels of synaptic activity. Several calcium sensing motifs and topologicalvariations have been tested to assess the characteristics of each design. We are currently work-ing towards the rational development of a first generation biosensor which will subsequently beevolved through directed evolution to enhance its performance.

64

No. 30

Use of enzymes to improve the refining and physical properties of kraft pulp

Li Cui, Fatma Meddeb, Marc Beauregard

Universite du Quebec a Trois-Rivieres

Over the last few years, the importance of enzymes as biotechnological catalysts for the pulp andpaper industry has been demonstrated. Among the enzymes mostly studied, hydrolases such ascellulases and hemicellulases have been investigated for their potential impact on refining energycosts. Unfortunately, energy reduction by prior enzyme treatments often had a negative impacton certain paper properties. In this study, five different commercial cellulase formulations wereused for the modification of fiber properties. PFI refining was employed at 3000 and 4500 revolu-tions to mimic the impact of various levels of refining, particularly on fiber size and morphology.With the five enzyme preparations, it was possible to decrease the number of PFI revolutionsby 50% and achieve the same target freeness value (decrease in pulp CSF by approximately200 mL) afforded by more intense refining without enzyme. The fiber morphology changed todifferent extent according to various enzymatic treatments. Subsequently, the carbohydrates inthe filtrate released during enzymatic treatment was studied by ion chromatography (IC). ICresults showed good agreement with the enzyme activity measured independently. The impactof enzymatic treatment on physical properties of handsheet was also investigated. The enzymeimpact on tear index was exceptional compared to most properties measured in this study. Aslight decrease in tear strength was observed with enzyme C1 and C4 at pH 7 after mechanicalrefining (less than 10%) while the most important decrease in tear was observed after C2, C3,C5 treatments. The reason for this phenomenon appears to be that C1 and C4 had xylanaseactivity. We conclude that xylanase activity could preserve and/or improve the properties ofhandsheet made from enzyme treated pulp, and that the balance between cellulolytic and hemi-cellulolytic activities is the key to optimization of biorefining, leading to energy reduction andimproving handsheet properties.

65

No. 31

Decoupling epistatic relationships that maintain structural stability in a β-propellerprotein

Loretta Au1,2 and David F. Green1,2,3

1. Department of Applied Mathematics and Statistics, 2. Laufer Center for Physical andQuantitative Biology, 3. Graduate Program in Biochemistry & Structural Biology, Stony BrookUniversity, Stony Brook, NY 11794-3600, USA

β-propeller proteins are a highly evolved family of repeat proteins which interact with verydiverse binding partners, despite having very similar folds, accounting for their involvement inmany cellular pathways. As for all families of repeat proteins, motifs remain consistent betweenrepeating units, in addition to the entire family, but how these motifs provide a template for evo-lutionary selection remains unclear. Comparative sequence analysis techniques can be successfulin identifying conservation between closely related proteins, but the reasons underlying amino-acid conservation cannot be determined without further experimentation. Additional challengesarise with repeat proteins: the appropriate definition for aligning reoccurring regions becomesless obvious if the number of repeats vary, and contextual information may be lost if entiresequences are decomposed into their repeating regions for alignment. To overcome these obsta-cles, we have devised a computational approach to perform large-scale mutagenesis by adaptingthe dead-end elimination and A* search algorithms, and demonstrate: (1) how the involvementof individual amino acids to overall protein fitness (defined by structural stability and bindinginteractions) can be deconvolved, and (2) how epistatic interactions between them contribute tostructural stability. As a model system, we focus on the β-subunit of a G-protein heterotrimer.Our computational approach can identify important patterns of interaction for this protein, andprovide insight on how associations between and within repeats contribute to overall fitness.

66

No. 32

Characterization of hydrolytic enzyme-producing bacteria isolated from paper mill

Manel Ghribi1, 2, Fatma Meddeb- Mouelhi1, 3 and Marc Beauregard1 2

1 CRML, Centre de recherche sur les materiaux lignocellulosiques, Universite du Quebec aTrois- Rivieres, (Quebec) Canada 2 PROTEO, Universite Laval, Quebec, (Quebec) 3 BuckmanNorth America

Enzymes act as biocatalyst in many industries, such as textiles, detergent, food, animal feed,bio-fuel, paper and pulp, pharmaceutical, to name a few. Cellulases and hemicellulases are effi-cient hydrolytic enzymes used in the pulp and paper industry to reduce the cost of production.In addition, industrial enzymes reduce the environmental impact by replacing harmful chemi-cals. The leading industrial enzyme suppliers (Novozymes, Genencor) offer a limited library ofenzymes. Thoses enzymes are poorly adapted to the need of the paper industry. Therefore, ourpartner, Buckman North America, wants to expand his enzymes portfolio by characterizing newenzymes-producing bacteria.Pulp and paper mills offer untapped biodiversity for microorganisms that use cellulose-basedsubstrates as nutriments.In this project, we will isolate and characterize cellulose and hemicellulose degrading microor-ganisms from paper mill sludges.To conserve microorganism’s biodiversity, we used two temperatures (37 oC and 50 oC) for theisolation step. Detection of extracellular enzymatic activities was carried out on minimum agarplate medium supplemented with either cellulose (carboxylmethylcellulose or Avicel) or beech-wood xylans. Bacteria strains showing extracellular cellulase and/or xylanase activities wereisolated from various sludges (primary, secondary, presses and machines) found in a paper mill.These bacteria were identified based on their morphology, biochemical characterization and DNA16s sequencing. The biorefining potential of these enzymes will be evaluated.

67

No. 33

Examining the substrate and carrier protein specificity of the Bacillaene thioesterase

Mark Horsman, Christopher Boddy

University of Ottawa Department of Chemistry

Bacillaene is a linear free-acid polyketide produced by a trans-AT modular PKS pathway inBacillus sp.; bacillaene contains an unusual terminal beta-gamma unsaturation that may beisomerized either before or after thioesterase-catalyzed release. Recent mutagenesis work hasdemonstrated that mutation of the penultimate PKS module inhibits full-length bacillaene pro-duction.The thioesterase domain and cognate acyl carrier protein (ACP) were heterologously expressedin E. coli for the use in Ellman’s colorimetric kinetic assays as well as LC-MS monitored acyl-ACP hydrolysis end-point assays. Three truncated bacillaene homologues were synthesized inboth n-acetyl cysteamine and coenzyme-A form to determine the thioesterase substrate speci-ficity with regards to the position of the terminal alkene.The results from these studies support the theory that the thioesterase is capable of toleratingboth the conjugated and unconjugated equivalents of the tethered bacillaene product. The ex-perimental design will be expanded to examine whether the thioesterase’s compatability withother Bacillus ACPs is comparable to other transAT thioesterase family members in an effortto genetically engineer bacillaene truncants and hybrids.

68

No. 34

Tandem dimer fluorescent proteins

Matthew Wiens, Robert Cambell

University of Alberta

In engineering fluorescent proteins one focus is enhancing the brightness, to facilitate detec-tion in various environments. Our starting point is tdTomato, an already bright red fluorescentprotein tandem dimer, and using this as a template for novel tandem dimer fluorescent proteins.The tdTomato fluorescent protein has an interesting maturation pathway that goes through agreen fluorescent state. Though rational and directed evolution we have stopped this maturationpathway at the green state creating a green dTomato that still dimerizes with both itself and reddTomato. With green dTomato we are creating and evolving a green green tandem dimer andgreen red tandem dimer. These variants have potential to be used in single molecule detectionsystem as they can be very bright. The green red tandem dimer exhibits very strong FRETefficiency and can be very useful in 2-photon tissue imaging, as both the 2-photon excitationof the green fluorophore and the emission of the red fluorophore occur in the available opticalwindow.

69

No. 35

Structural analysis and molecular dynamics of the self-sufficient P450 CYP102A5and CYP102A1: A a combined computational/experimental approach to increase

the efficiency of biocatalyst engineering

Maximilian Ebert1,4, Brahm Yachnin2,4, Guillaume Lamoureux3,4, Albert Berghuis2,4, JoellePelletier1,4

1Universite de Montreal, 2McGill University, 3Concordia University, 4PROTEO

P450s catalyze the oxidation of non-activated carbon atoms, which is chemically demanding.Members of the CYP102 family are termed “self-sufficient P450s”, meaning that they containall the machinery necessary to ensure the electron transfer and active site regeneration in onesingle protein. However, the macromolecular assembly remains unknown. Here we report resultsof SAXS analysis that bring new insights into the formation of the active complex. The recentlyreported new member CYP102A5 is highly interesting due to its sequence similarity to the todate most active member BM3, however with a significant increase in electron transfer rateand improved regioselectivity. Homology models were generated, compared and used to identifystructural reasons for these differences in catalytic activity. Based on this result we were ableto identify important residues for the gating and substrate capturing mechanism in CYP102A5.The model can reasonably interpret previously published results and support proposed hypoth-esis. Traditional molecular dynamic simulations were performed to investigate the structuralstability of our models. Predictions of differences in substrate incorporation and product releasefrom the active site were computed using the adaptive biasing force (ABF) method. With thisfirst time application of ABF in enzyme engineering we were able to predict all known importantresidues for substrate binding for BM3. This newly developed computational biology approach,in addition to conformational studies, will help to guide directed evolution efforts towards theoxidation of non-native substrates.

70

No. 36

Optimization of a fluorogenic labelling technique for in cellulo protein visualization

Miroslava Strmiskova, Natalie K. Goto, Jeffrey W. Keillor

University of Ottawa

Fluorescent protein labelling is a powerful tool for the sensitive visualization of proteins inliving cells, allowing the elucidation of their localization, trafficking and ultimately their cellularfunction. We have developed a novel labelling technique based on the genetic fusion of a proteinof interest to a small helical peptide sequence containing two Cys residues (dC10). This tag canundergo an efficient reaction with small fluorogenic labelling agents composed of a fluorophoreand a dimaleimide core (dM10) that confers high reaction specificity, and quenches the latentfluorescence through photo-induced electron transfer, until both of its maleimide groups haveform robust covalent bonds with the tag Cys thiol groups.Our initial efforts at intracellular protein labelling demonstrated the importance of the selectivityof the labelling reaction, which is dependent on the reactivity of the dC10 tag. To that end, were-engineered the dC10 tag through semi-rational protein design. Mutant libraries were preparedthrough combinatorial mutation at specific positions of the helical tag sequence, and screenedfor their fluorogenic reactivity. In this way, we identified a novel sequence for a next-generationdC10 tag that confers 10-fold greater selectivity. Subsequent mechanistic studies revealed thebasis for this dramatic increase in reactivity.Current applications of this powerful labelling technique will also be briefly introduced. In addi-tion to the fluorescent labelling of specific proteins in living cells, these include the site-specificchelation of lanthanide ions for NMR spectroscopy and site-specific covalent heavy-atom labellingfor X-ray crystallography.

71

No. 37

Effect of removal of free and bound phenolic compounds on molecular, chemicaland biological properties of separated peptides from hydrolyzed protein fractions

from Black cumin.

Muhammad Hussein Alu’datt

Department of Nutrition and Food Technology, Faculty of Agriculture, Jordan University ofScience and Technology, P.O. Box 3030, Irbid 22110, Jordan.

The objective of this project was to investigate the molecular, chemical and biological propertiesof separated peptides from hydrolyzed protein fractions from Black cumin. Protein fractionsincluding albumin, glutein-1, glutein-2, prolamin and globulin fractions were extracted fromblack cumin flour. The peptides from hydrolyzed crude protein fractions from two varieties ofblack cumin flour were purified using reversed phase high performance liquid chromatography(RP-HPLC). Free phenolic compounds were removed from protein fractions of two varieties ofblack cumin using methanol extraction. While the bound phenolic compounds extracted usingacid and base hydrolysis followed by methanol extraction. Protein fractions after removal offree and bound phenolic compounds from black cumin were subjected to pancreatic hydrolysisto obtain hydrolyzed peptides. Peptides were subjected to evaluate their potential to inhibitthe angiotensin converting enzyme (ACE)-inhibitory activity. Indicated results showed that theseparated peptides using RP-HPLC in hydrolyzed and non-hydrolyzed protein fractions beforeremoval of free and bound phenolic compounds has a potential inhibitory activity in all fractions.The optimum ACE inhibitory activity of RP-HPLC separated peptides were obtained in albuminand glutein-2 for both of cultivars. While the maximum inhibitory activity of ACE for separatedhydrolyzed peptides using RP-HPLC were found in albumin for both of cultivars. The ACE in-hibitory activity of peptide obtained from hydrolyzed of all protein fractions after removal of freeand bound phenolic compounds in cultivar 1 and 2 were varied significantly at different times ofhydrolysis. The optimum values of ACE inhibitory activity for peptide were obtained in cultivar2 for all times. The RP-HPLC chromatograms of hydrolyzed protein fractions before removalof free and bound phenolic compounds exhibited differences in number of major and minor peaks.

72

No. 38

Retinol dehydrogenase 11 membrane anchoring is mediated by its N-terminalα-helix with a preferential binding to phosphoethanolamine lipids

Mustapha Lhor, Habib Horchani, Mario Methot and Christian Salesse

CUO-Recherche, Centre de recherche du CHU de Quebec, Hopital du St-Sacrement; Departementd’ophtalmologie, Faculte de medecine, PROTEO, Universite Laval

Retinol Dehydrogenase 11 (RDH11) is presumed to be bound to the microsomal membranesof the Retinal Pigment Epithelial (RPE) cells where it participates to the visual cycle. Theanalysis of the primary sequence of RDH11 revealed the presence of a N-terminal hydrophobicsegment that was postulated to be necessary for its membrane attachment. To investigate theregion required to confer membrane localization of RDH11, two N-terminal peptides with dif-ferent lengths have been studied given that no clear information is available on the size of thesegment allowing membrane anchoring of this protein. The predicted secondary structure ofboth peptides was first analyzed using online tools. The results of this computational approachwas confirmed by spectroscopic methods including Fourier transform infrared (FT-IR) and Cir-cular dichroism (CD) spectroscopy. We found that regardless to the type of solvent used, theLong-RDH11-Nter (35 amino acids) peptide bears a more extensively structured a-helix thanthe short one (Short-RDH11-Nter, 25 amino acids). Langmuir monolayers have been used asmembrane models to study lipid-peptide interactions. The values of maximum insertion pressure(MIP) and synergy obtained by surface pressure measurements suggest that RDH11 membraneanchoring is likely mediated by the Long-RDH11-Nter peptide with a preferential binding tophospholipids with a phosphoethanolamine head group, which are known to be abundant inthe RPE. Changes in the conformation and orientation of the long- and short-peptides in thepresence or in the absence of phospholipid monolayers was also studied by infrared spectroscopy.

73

No. 39

Structural investigations of bacterial magnetotactic organelle formation andpositioning

Nancy Hom, Ertan Ozyamak, Arash Komeili, Justin Kollman

McGill University; UC, Berkeley

A unique group of magnetotactic bacteria are able to create multiple internal compartments,each of which serves as an enclosed space for controlled formation of a single magnetic nanocrys-tal. These organelles, called magnetosomes, are positioned in a line running the length of thebacterium, where they act essentially as a compass needle to align the cell along the earth’smagnetic field. The magnetic response appears to allow these bacteria to swim in a directedsearch toward regions with optimal growth conditions. Though the protein components, ge-netics and biochemistry of magnetosomes have been extensively studied, the three-dimensionalstructure of the magnetosome is not well-characterized. Using cryo-electron tomography, we areinterested in obtaining a high-resolution map of the intact organelle in order to gain insightinto the global magnetosome architecture. Additionally, we are investigating the structures ofindividual, purified, protein components necessary for magnetosome assembly and organization.The actin homolog MamK is required for the linear positioning of magnetosomes. We havedetermined the structure of MamK filaments by cryo-EM and homology modeling, revealing aunique filament structure with two twisted strands that are in register, rather than staggeredas in other actin homologs. We are now interested in elucidating the specific molecular contactsfor MamK assembly. Using MamK mutants, we performed negative-stain EM and biochemicalcharacterizations to determine sites of important filament interactions. We found that mutationsat the cross-strand interface lead to increased filament dynamics, though the filament structureis the same as wildtype. In contrast, mutations at the intersubunit interface along each strandcompletely abolish filament assembly. We are also studying the structures of other magnetosomeprotein components with the goal of gaining a complete view of the mechanisms of magnetosomedevelopment.

74

No. 40

Probing the clamping movement of xylanase B by NMR spectroscopy

Nhung Nguyen Thi & Nicolas Doucet*

INRS-Institut Armand-Frappier, Universite du Quebec, 531 Boul. des Prairies, Laval, Quebec,Canada.

The present work describes NMR assignments and relaxation dispersion experiments probingthe dynamics of apo and ligand-bound xylanase B (XlnB) from Streptomyces lividans. Evidencefrom mutagenesis, crystal structures and molecular dynamics simulations previously suggestedthat the “thumb-loop” motion of XlnB might play a major role in substrate binding/catalysis[1,2,3,4,5]. Our 15N-CPMG data show similar millisecond time-scale motions for active-site fin-gers in the free and xylobiose-bound enzyme. However, in the presence of the longer xylopentaoseligand, conformational exchange emerges on the thumb loop, along the active-site cleft, and onthe opposite side of the fingers. 1H-15N HSQC titration data also indicates the involvement ofthumb loop and binding cleft residues in substrate recognition. For the first time, our resultsilluminate the atomic-scale dynamics of XlnB on the millisecond time-scale, suggesting a globalclamping movement during catalysis.

References: [1]. Paes G., Berrin J.G., and Beaugrand J. Biotechnology Advances. (2012) 30:564–592. [2] Pollet A., Lagaert S., Eneyskaya E., Kulminskaya A., Delcour J.A., and CourtinC.M. Biochimica et Biophysica Acta (2010) 1804:977–985. [3]. Murakami M.T., Arni R.K.,Vieira D.S., Degreve L., Ruller R., and Ward R. FEBS Letter (2005), 579:6505–10. [4]. VieiraD.S, Degreve L., and Ward R.J. Biochimica et Biophysica Acta-Gen Subject (2009); 1790:1301–6.[5]. Hakulinen N., Turunen O., Janne Janis J., Leisola M. and Rouvinen J. European Journalof Biochemistry (2003), 270, 1399–1412.

75

No. 41

A Unified Framework for Computer Aided Biologics Design

Christopher Corbeil, Alain Ajamian, Paul Labute

Chemical Computing Group

Protein engineering plays a pivotal role in modulating the function, activity and physical prop-erties of biological entities. Representative strategies employed in protein engineering includerational protein design and directed evolution. In general, disparate work has been done inapplying computer-aided biologics design (CABD) to protein engineering for the developmentof novel biological therapeutics. Here, we establish a uniform framework for protein engineer-ing tools and investigate their applicability to modulate protein properties: affinity and stability.

76

No. 42

Combinatorial Active-Site Variants Confer Sustained Clavulanate Resistance inBlaC β-Lactamase from Mycobacterium tuberculosis

Philippe Egesborg1,�, Helene Carlettini1,�, Jordan P. Volpato1, Nicolas Doucet1,2,3*

1 INRS-Institut Armand-Frappier, Universite du Quebec, 531 Boul. des Prairies, Laval, QuebecH7V 1B7, Canada. 2 PROTEO, the Quebec Network for Research on Protein Function, Struc-ture, and Engineering,1045 Avenue de la Medecine, Universite Laval, Queb

Bacterial resistance to β-lactam antibiotics is a global issue threatening the success of infec-tious disease treatments worldwide. Mycobacterium tuberculosis has been particularly resilientto β-lactam treatment, primarily due to the chromosomally encoded BlaC β-lactamase, a broad-spectrum hydrolase that renders ineffective the vast majority of relevant β-lactam compoundscurrently in use. Recent laboratory and clinical studies have nevertheless shown that specificβ-lactam–BlaC inhibitor combinations can be used to inhibit the growth of extensively drugre-sistant strains of M. tuberculosis, effectively offering new tools for combined treatment regimensagainst resistant strains. In the present work, we performed combinatorial active-site replace-ments in BlaC to demonstrate that specific inhibitor-resistant substitutions at positions 69, 130,220 and/or 234 can act synergistically to yield active-site variants with several thousand foldgreater in vitro resistance to clavulanate, the most common clinical β-lactamase inhibitor. Whilemost single and double variants remain sensitive to clavulanate, double mutants R220S-K234Rand S130G-K234R are substantially less affected by time-dependent clavulanate inactivation,showing residual β-lactam hydrolytic activities of 46% and 83% after 24h incubation with theinhibitor. These results demonstrate that active site alterations in BlaC yield resistant variantsthat remain active and stable over prolonged bacterial generation times compatible with my-cobacterial proliferation. These results emphasize the formidable adaptive potential of inhibitor-resistant substitutions in β-lactamases, potentially casting a shadow on specific β-lactam–BlaCinhibitor combination treatments against M. tuberculosis.

77

No. 43

Structural insights into the interaction between the Fur family of metalloregulatorsand DNA

Sabina Sarvan, William Lam and Jean-Francois Couture

University of Ottawa

Metalloregulators are an important family of transcriptional factors controlling a myriad ofbiological processes in regulating gene expression. Despite several structural studies, the mecha-nistic basis underlying the binding of metalloregulators to DNA has remained elusive. To addressthis important question, we solved the crystal structure of the Ferric Uptake Regulator (FUR)and the PERoxyde stress Regulator (PerR); two representative members of metalloregulators.Comparative analysis revealed that both proteins adopt a V-shaped conformation harboring anevolutionary conserved cluster of positively charged residues on the surface of both proteins.Using an extensive library of mutants and electrophoretic mobility shift analysis, we found thatsubstituting residues forming the positively charged surface is detrimental for PerR and FURinteraction with DNA. Furthermore, our in vivo studies suggest that these positively chargedresidues are important for the repression of CjFur target genes. Overall, our structural studiessuggest that metalloregulators employ a common surface to bind DNA, regulate gene expressionand contribute to bacterial pathogenicity

78

No. 44

Unusual crystal packing highlights dynamic flexibility of an enzymatic kinasedomain

Shane J. Caldwell and Albert M. Berghuis

Department of Biochemistry, McGill University

The aminoglycoside phosphotransferase enzyme APH(2”)-Ia forms the C-terminal domain of thebifunctional antibiotic resistance enzyme AAC(6’)-Ie/APH(2”)-Ia. In crystal structures of thisenzyme domain, we observe four copies of the protein packed as two head-to-head pairs, buryingsurface consistent with packing in the full-length bifunctional protein. In these structures, threeprotein molecules form most crystal contacts, leaving the fourth molecule largely unaffected bycrystal packing. This fortuitous condition allows us to compare the effects of ligand binding inmolecules that are restricted by crystal packing, to a molecule that has much greater freedom ofmovement. Comparison of these structures give us an improved understanding of the dynamicsand flexibility of the enzyme, which is central to its versatility as an antibiotic resistance enzyme.

79

No. 45

Phytoremediation of nitrous oxide: Expression of nitrous oxide reductase frompseudomonas stutzeri in transgenic plants

Shen Wan1, Illimar Altosaar2, and Joann K. Whalen1

1McGill University; 2University of Ottawa,

As the third most important greenhouse gas, nitrous oxide (N2O) is a stable greenhouse gasand also plays a significant role in stratospheric ozone destruction. The primary anthropogenicsource of N2O stems from the use of nitrogen in agriculture, with soils being the major con-tributors. Currently, the annual N2O emissions from this “soil–microbe-plant” system is morethan 2.6 Tg (one Tg equals a million metric tons) of N2O-N globally. My studies aimed toexplore innovative strategies for N2O mitigation, in the context of environmental microbiology’spotential contribution to alleviating global warming. The bacterial enzyme nitrous oxide reduc-tase (N2OR), naturally found in some soils, is the only known enzyme capable of catalyzingthe final step of the denitrification pathway, conversion of N2O to N2. Therefore, to “scrub” orreduce N2O emissions, bacterial N2OR was heterologously expressed inside the leaves and rootsof transgenic plants. Others had previously shown that the functional assembly of the catalyticcentres (CuZ) of N2OR is lacking when only nosZ is expressed in other bacterial hosts. There,coexpression of nosZ with nosD, nosF and nosY was found to be necessary for production of thecatalytically active holoenzyme. The activity of N2OR expressed in transgenic plants, analyzedwith the methyl viologen-linked enzyme assay, showed detectable N2O reducing activity. TheN2O-reducing patterns observed were similar to that of the positive control purified bacterialN2OR. The data indicated that expressing bacterial N2OR heterologously in plants, withoutthe expression of the accessory Nos proteins, could convert N2O into inert N2. This suggeststhat atmospheric phytoremediation of N2O by plants harbouring N2OR could be invaluable inefforts to reduce emissions from crop production fields.

80

No. 46

Making an ice-binding protein de novo

Shuaiqi Guo, Christopher Garnham, and Peter Davies

Queen’s University

The ice-binding activity of a 1.5-MDa RTX-adhesin solely resides in a 322-aa domain (MpAFP RIV)located towards its C terminus. The crystal structure of MpAFP RIV was previously determinedto 1.7 A, and revealed a Ca2+-dependent β-helix with a flat ice-binding site comprised of twoparallel rows of Thr and Asx residues. These ice-binding residues help anchor an extensive arrayof ice-like surface waters (termed anchored clathrate waters) that match to, and merge with, theplanes of ice, suggesting how ice-binding proteins (IBPs) might interact with their ligand. Hereour goal is to engineer an IBP out of a non-IBP, which has never been accomplished before,and if successful, would demonstrate a comprehensive knowledge of protein structure-functionrelationships necessary to bind to ice. A BLASTp search with MpAFP RIV identified a suitabletarget, which is a putative adhesin protein produced by the Gram-negative bacterium Magne-tospirillum magneticum. A homology model showed that one domain (AFLD) has a similarfold to MpAFP RIV but lacks its N-terminal capping structure and the appropriate Thr andAsx residues on its potential ice-binding site (IBS) for antifreeze activity. To determine if theN-terminal cap of MpAFP RIV could stabilize the fold of AFLD, a chimera was made. Also,residues deemed necessary for ice binding were introduced. The mutant chimeric AFLD genewas synthesized and then expressed in E. coli. The protein demonstrated moderate antifreezeactivity, which is about 10-fold less when compare to MpAFP RIV. Future structural studieswill help identify residues that spoil the IBS of AFLD, and we will then improve its activity byconducting mutagenesis.

Supported by CIHR

81

No. 47

Structure-based recombination of β-lactamases: functional, structural and dynamicinvestigation of artificially-evolved enzymes

Sophie Gobeil1,2, Jaeok Park2,3, Christopher Clouthier2,4, Donald Gagne2,5, Nicolas Doucet2,5,Albert Berghuis2,3 and Joelle Pelletier1,2,4

1Departement de biochimie, Universite de Montreal, QC, Canada; 2PROTEO, the QuebecNetwork for Research on Protein Structure, Function and Engineering; 3Department of Bio-chemistry, McGill University, QC, Canada; 4Departement de chimie, Universite de Montreal,QC, Canada; 5INRS-Institut Armand-Frappier, Universite du Quebec, QC, Canada

Is the structure/function/dynamics correlation conserved in highly blended chimeras? As ourmodel system, two structurally similar class A β-lactamases, TEM-1 and PSE-4, sharing 40% se-quence identity, were recombined using structure-based method to create a series of TEM-1/PSE-4 chimeras. Our analysis aims to understand the effect of this recombination on the functional,structural and dynamics realms of the resulting artificially evolved enzymes. Chimera cTEM-17m is harboring 17 TEM-1-to-PSE-4 substituted residues in the active site region. cTEM-19mharbour the same 17 substitutions plus two other neighbouring the catalytic nucleophile. Theturnover rate of cTEM-17m is native-like indicating that its 17 mutations are functionally tol-erated. However, the additional 2 mutations of cTEM-19m decreased the turnover rate by morethan 20-fold. To probe for synergistic or additive effects of these mutations, we kinetically char-acterized deconvolution mutants in both the chimeric and wild-type contexts. High-resolutioncrystal structures of chimeras cTEM-17m, cTEM-19m and a deconvoluted mutant were obtainedto verify the structural impact of the 2 substitutions. The overall structure of the chimeras andthe deconvoluted mutant were similar to the parents. However, two structures of cTEM-19m ob-tained in similar crystallisation conditions showed different active site conformations, suggestingincreased dynamics. NMR relaxation experiments on a fast (ps-ns) and slow (µs-ms) time scalesconfirmed new broadly distributed molecular dynamics in chimeras cTEM-17m and cTEM-19mnot observed in either the naturally-evolved TEM-1 or PSE-4 β-lactamase. The results pre-sented here shows that the structure can be conserved while introducing new large-scale motionsin functional artificially evolved enzymes.

82

No. 48

HIGH-THROUGHPUT ASSAYS AND IN SILICO DESIGN ACCURATELYPREDICT NOVEL SMYD2 SUBSTRATES

Lanouette S., Davey J., Chica R., Figeys D., Couture J.-F.

Ottawa Institute of Systems Biology, University of Ottawa

Methylation of lysine residues is a ubiquitous post-translational modification (PTM) known tomodulate many biological processes, including gene transcription, protein stability and cytoskele-tal organization. In contrast with other key PTMs however, pan-specific antibodies and massspectrometry have thus far been unsuccessful in characterizing methyl-lysine residues across thecell’s landscape. To chart and validate the substrates for a specific protein lysine methyltrans-ferase (PKMT), we developed a method based on high-throughput systematic biochemical assayscoupled with protein modelling and systems biology. Our methodology was validated using thePKMT SMYD2, a methyltransferase already known to methylate HSP90, p53, pRb and his-tone H3. Multi-State Design (MSD) of SMYD2 bound to a peptide substrate identified residuesimportant for substrate recognition and predicted the amino acids necessary for methylation.Peptide- and protein- based substrate libraries further confirmed that SMYD2 methylation activ-ity is primarily dictated by 3 positions which define the motif [KLM]-K*-[FYHKRSMAL]-[KLY]around the target lysine (K*). Comprehensive motif based searches and mutational analysisfurther established SIN3B, DHX15, SIX1 and SIX2 as novel SMYD2 substrates. Our findingsreveal intricate substrate selectivity by SMYD2 which provides a molecular rationale for its in-volvement and regulation in apoptosis, transcriptional regulation and muscle development. Inperspective, our methodology paves the way to systematically predict and validate novel lysinemethylation sites while simultaneously pairing those with their associated methyltransferase.

83

No. 49

LeSTATIUM: Incorporating a treatment of protein conformational space in theSTATIUM statistical potential

Vincent Frappier1,2, Laurent Bruneau-Cossette1, Joe DeBartolo2, Rafael Najmanovich1 andAmy E. Keating2

1 - Universite de Sherbrooke, Faculte de Medecine, Departement de Biochimie 2- MIT De-partment of Biology

Bcl-2 family proteins are involved in regulating cell death and are frequently responsible forthe failure of cells to die appropriately in many cancers. Bcl-2 family protein-protein interac-tions are critical for the cell-fate decision. At a structural level, pro-death Bcl-2 family proteinsdock as short alpha helices into a hydrophobic groove in prosurvival Bcl-2 family receptor pro-teins. A better understanding of the structural determinants of these interactions is critical tothe development of new therapeutic agents that can restore cell death signaling. The design ofnovel peptides that mimic pro-death proteins can be used to explore the specificity and func-tional mechanisms of different Bcl-2 family interactions.

To guide the design of new sequences, previous work in the Keating group has used STATIUM, asequence-scoring statistical model derived from structure data. STATIUM works by consideringeach interacting pair of amino acids between a receptor and a peptide within a template struc-ture. A database of structures is searched for structurally identical residue pair configurations,and the frequency of observed amino acid identities at the peptide position is used to calculatea score for an amino acid substitution.

Although STATIUM has shown good predictive capability on various benchmarking tests, thereis room for improvement. Proteins are dynamic entities, and a more realistic model of proteinstructure should account for that property. To explore whether a treatment of protein motioncan be used to improve STATIUM, we used ENCoM, a normal mode analysis model developin the Najmanovich group, to explore the whole conformational space of the structure databasethat is used to derived STATIUM potentials. Using the framework of ENCoM, we computed theprobability of an interacting pair in the database having the exact same conformation as a pairin the template. Our new model, called LeSTATIUM, is being tested on various benchmarksand is compared to static STATIUM.

84

No. 50

The Structural Basis for BVMO Substrate Profile and Stereospecificity

Yachnin, Brahm J., McEvoy, Michelle B., MacCuish, Roderick J. D., Morley, Krista L., Mitter-maier, Anthony K., Lau, Peter C. K., Berghuis, Albert M.

McGill University

The Baeyer-Villiger monooxygenases (BVMOs) are a group of bacterial enzymes that are ableto catalyze the synthetically useful Baeyer-Villiger oxidation reaction. As such, these enzymeshave attracted considerable attention as potential industrial biocatalysts. The interest in theseenzymes has led to a desire to be able to rationally design them for tailored biocatalytic ap-plications. While recent years have seen the publication of a number of crystal structures,1-3we have been lacking a structure of a BVMO that has its native substrate or product bound ina conformation that will allow the determination of substrate specificity and stereospecificity.Without such a structure, progress towards tailored BVMOs has been hampered.

We have been able to solve two crystal structures of cyclohexanone monooxygenase (CHMO)with its lactone product, ε-caprolactone, bound. These structures place the lactone in an idealposition for the determination of its substrate specificity and stereospecificity. These structureshave provided us with a structural framework for understanding the enzyme’s substrate profileand stereospecificity, paving the way for the rational design of tailored BVMOs.

At the same time, we have pursued small-angle X-ray scattering (SAXS) and nuclear mag-netic resonance (NMR) studies to better understand the dynamic nature of the enzyme. Thesestudies have allowed us to explain the relationship between the various crystallized states ofBVMOs and their complex, fourteen step enzyme mechanism.

[1] B. J. Yachnin, T. Sprules, M. B. McEvoy, et al., J. Am. Chem. Soc., 2012, 134, 7788-7795.[2] I. A. Mirza, B. J. Yachnin, S. Wang, et al., J. Am. Chem. Soc., 2009, 131, 8848-8854.[3] E. Malito, A. Alfieri, Fraaije, M. W., A. Mattevi, Proc. Natl. Acad. Sci. U. S. A., 2003,101, 13157-13162.

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No. 51

Tracking wood fibers decrystallization with carbohydrate binding module

Yannick Hebert-Ouellet 1,2, Vinay Khatri 1,2, Fatma Meddeb-Mouelhi 1,3 and Marc Beauregard1,2

1. Centre de recherche sur les materiaux lignocellulosiques, Universite du Quebec a Trois-Rivieres, Trois-Rivieres (Quebec) G9A 5H7, Canada 2. PROTEO, Universite Laval, Quebec(Quebec) G1V 4G2, Canada. 3. Buckman North America, Vaudreuil-Dorion (Quebec) J7V5V5, Canada.

There are many steps in the complex process of converting wood into paper. One of thesesteps is refining. Refining is essential for modifying the characteristics of wood fibers so thatit may form paper sheet with a specific set of properties. One important modification of woodfibers is the fibrillation of the exposed S2 layers which promote the formation of hydrogen bondsand increase the available bonding surface. This refining modification also partially convertscrystalline cellulose into amorphous cellulose. On the other hand, the majority of the energydevoted to paper manufacturing is consumed by the refining of wood fibers. Therefore, improv-ing energy efficiency through the understanding of wood fibers refining at a molecular level isof paramount importance for the pulp and paper industry. In this study, we specifically trackthe decrystallization of cellulose on the surface of refined paper sheets through the utilization ofClostridium thermocellum CipA carbohydrate binding module 3a fused to eGFP. Correlationsbetween crystalline cellulose quantification, energy of refining and paper physical properties areshown. Advantages, limitations and applications of this assay to enzymatic prerefining are alsodiscussed.

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No. 52

Structural insight into IFIT3 domain swapping

Yazan M. Abbas, Irene Xie, Bhushan Nagar

McGill University and Recherche Axe sur la Structure des Proteines

IFIT proteins are interferon-inducible, antiviral effectors that, together with other host fac-tors, form a multiprotein complex with the ability to recognize markers of viral infection andsubsequently restrict viruses. There are 4 well characterized IFITs in humans: IFIT1, IFIT2,IFIT3 and IFIT5; with crystal structures available for N-terminal IFIT1, full-length IFIT2 andfull-length IFIT5.IFIT1, 2 and 3 are at the heart of the complex forming what is known as the IFIT interactome.Central to their ability to form this interactome is the tetratricopeptide repeat (TPR) motif,a general protein- protein interaction module comprising of a pair of antiparallel alpha helices.Due to their degeneracy, TPR motifs are attractive tools for generating designer proteins withengineered specificities to modulate signalling pathways. Mammalian IFIT proteins can be di-vided into IFIT1-like, which are generally monomeric and bind virus-derived single strandedRNA; or IFIT2-like, which form domain- swapped dimers. IFIT2 dimerization may underlie itsability to bind double-stranded RNA.We have recently determined the crystal structure of N-terminal IFIT3, which reveals an IFIT2-like domain-swapped dimer. Sequence conservation and structural analysis suggests a mechanismfor domain swapping in IFIT proteins, and will form the basis of future mutational analysis toengineer IFITs with altered oligomerization states and RNA binding specificities. Ultimately,we hope this work will provide a framework for engineered multimerization in TPR proteins tocreate higher-avidity targetted interactions.

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No. 53

Comparison of predicted structures and dynamics of various mammalian EDN andECP orthologues

David Bernard, Donald Gagne & Nicolas Doucet

INRS-Institut Armand-Frappier, Universite du Quebec, 531 Boul. des Prairies, Laval, Quebec,Canada.

Enzymes are becoming more popular with time in pharmaceutical and industrial environments,particularly as greener and more efficient alternatives to organic solvent catalysts. However,engineering new enzyme reactions is an arduous and inefficient process, mainly because the pre-dictable outcome of protein engineering on 3D structure, function and dynamics remains elusive.Recent experimental evidences suggest that concerted molecular motions promote catalysis inmany enzymatic systems. However, the mechanisms underlying this catalysis-coupled atomicflexibility are still unclear. It is also unknown whether molecular motions are conserved inenzymes displaying similar structures and/or function. Moreover, the effect of the sequenceon dynamic transmission remains unclear. In line with these interrogations, this project willcompare mammalian orthologues of the human enzymes EDN and ECP, two major eosinophildegranulation toxins and members of the ribonuclease A superfamily. We will monitor the effectsof sequence and function on specific time scale motions to test whether motional behaviour islinked with biological function. Here we present homology models of all selected orthologues,allowing us to compute their relative accessible surface area and help predict their motionalbehaviour.

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