Book of Abstracts Albany 2011: the 17Th Conversation June 14-18 2011

Book of Abstracts Albany 2011: The 17th Conversation June 14-18 2011

Journal of Biomolecular Structure & Dynamics Volume 28, Issue # 6 June 2011

Schedule of 17th Conversation ...... ii-iv Book of Abstracts: The 17th Conversation ...... 975- 1164 Index to Authors ...... 1165 - 1170 Registration form: The 17th Conversation ...... 1171 - 1172

Sponsored by:

University at Albany Department of Chemistry Department of Biology Office of the Dean, Arts and Sciences Vice President for Research National Institutes of Health (pending) JBSD Adenine Press Tuesday, June 14: You are arriving Today Thursday, June 16 1:00-11:00 pm Mount your posters around LC-18 concourse 6:15-9:15 am Breakfast, Hospitality Suites, Empire Commons 4:00-7:00 Dinner-Campus Center Cafe; you pay cash & buy 6:00-8:00 JBSD + Organizing Cmte Dinner, Sitar 8:00-9:55 Session 7: Prebiotic Evolution 8:30-12:00 am Wine & Cheese Reception, Empire Commons, Chair: Nobel Laureate Jack Szostak, Harvard 8:00-8:05 Remarks by the Chair Wednesday, June 15 8:05-8:25 Edward Trifonov, Haifa, Israel 7:00-8:30 am Breakfast, LC-18 Concourse 8:25-8:45 Uwe Meierhenrich, Univ. of Nice, France 8:45-9:05 Ernesto Di Mauro, Univ. of Rome, Italy 8:00-8:10 am Welcome by VPR Dias, and Chairs Paul Toscano & Chair: Jiri Sponer, IBP, Czech Republic Richard Zitomer 9:05-9:25 James Ferris, RPI 8:10-10:25 Session 1: Proteins-I: New in Dynamics & Folding 9:25-9:35 Christopher D. McFarland, Harvard Chair: Harold Scheraga, Cornell Univ. 9:35-9:55 Nick Hud, Georgia Tech 8:10-8:15 Remarks by the Chair. 9:55-10:05 Irena Mamajanov, Georgia Tech 8:15-8:35 Dave Thirumalai, UMD 10:05-11:00 Coffee and Poster Session III 8:35-8:45 Menahem Pirchi, Weizmann, Israel 8:45-8:55 Amrita Dasgupta, NCBS, India 11:00-12:30 Session 8: Protein Evolution 8:55-9:05 Ghosh Dastdar, Bose Insti, India Chair: Anna Panchenko, NCBI 9:05-9:25 Angel Garcia, RPI 11:00-11:20 Igor Berezovsky, Univ. of Bergen, Norway Chair: Aditya Mittal, IIT Delhi, India 11:20-11:30 Kosuke Hashimoto, NCBI 9:25-9:45 Ken Dill, Stony Brook 11:30-11:50 William Duax, Univ. at Buffalo 9:45-9:55 Sanjeev Singh, Alagappa Univ, India Chair: Dan Fabris, Univ. at Albany 9:55-10:05 Priya Banerjee, Univ. at Albany 11:50-12:00 Ivanka Besseva, IBP, Czech Republic 10:05-10:15 Adeleh Divsalar, Tarbiat Moallem Univ., Iran 12:00-12:10 Brandon Tolbert, Miami Univ. Oxford, OH 10:15-10:35 B. Jayaram, IIT, Delhi, India 12:10-12:30 Loren Williams, Georgia Tech 10:35-11:25 Coffee and Poster Session I 12:30-1:40 Lunch, Campus Center Cafeteria & Barbique Pit (open 11 am- 2 pm) you pay cash and buy 11:25-12:30 Session 2. Proteins - II: New in Design & Detection Chair: Neville Kallenbach, NYU 1:40-2:30 Session 9: Artficial DNA 11:25-11:30 Remarks by the Chair Chair: S.Wijmenga, Radboud Univ., The Netherlands 11:30-11:50 Irit Sagi, Weizmann, Israel 1:40-2:00 Peter Nielsen, Univ. of Copenhagen, Denmark 11:50-12:00 Hridoy Bairagya, NIT-Durgapur , India 2:00-2:20 Maxim Frank Kamenetskii, BU 12:00-12:10 Traaseth Nate, NYU, 2:20-2:30 Marcus Wilhelmsson, Chalmers Univ., Sweden 12:10-12:30 David Cowburn, Albert Einstein 2:30-3:15 Session 10: What is New in Z-DNA 12:30-1:40 Lunch, Campus Center Cafeteria & Barbique Pit Chair: Wolfram Saenger, Frie Univ. Berlin, Germany (open 11 am- 2 pm) you pay cash and buy 2:30-2:50 Alex Rich, MIT 2:50-3:10 Alpana Ray, Univ. of Missouri, Columbia MO 1:40-3:10 Session 3: Proteins-III: New Dynamics & Allostery Chair: Miroslav Fojta, IBP, Czech Republic 3:10-3:40 Session 11 (Mini): Telomeres and Quadruplexes 1:40-2:00 Richard Bryce, Univ. of Manchester, UK Chair: Nick Ulyanov, UCSF 2:00-2:20 Amnon Horovitz, Weizmann, Israel 3:10-3:20 Katie Castor, McGill Univ. 2:20-2:30 Stan George, Univ. of Cincinnati 3:20-3:30 Saptarpani Ghosh, Saha Insti, Calcutta, India 2:30-2:40 Moitrayee Bhattacharyya, IISc, Bangalore, India 3:30-3:40 Lim Kah Wai, Nanyang Univ., Singapore 2:40-3:00 Ivet Bahar, Univ. of Pittsburgh 3:40-4:25 Coffee & Poster Session IV 3:00-3:10 Sefica Banu Ozkan, ASU, Tempe, AZ 4:25-6:20 Session 12: DNA Repair 3:10-4:25 Session 4: Proteins-IV: Neurodegeneration Chair: Rick Cunningham, SUNY at Albany. Chair: Volodya Uversky, IUPUI 4:25-4:30 Remarks by the Chair 3:10-3:30 Gary Daughdrill, Univ South Florida 4:30-4:50 John A. Tainer, Scripts 3:30-3:50 David Eliezer, Cornell Univ 4:50-5:10 Dmitry Zharkov, ICB, Novosibirsk, Russia 3:50-4:00 Sai P. Srinivasan, RPI 5:10-5:20 Sarah Delaney, Brown Univ. 4:00-4:20 Akihiko Takashima, RIKEN, Japan Chair: Krystyna Zakrzewska, IBCP, France 4:20-5:15 Coffee & Poster Session II 5:20-5:40 Reuben S. Harris, Univ. of Minnesota, Minneapolis 5:40-6:00 V. Enrico Avvedimento, Univ. Federico II, Italy 5:15-6:35 Session 5: DNA Nanotechnology 6:00-6:20 Mike Fried, Univ. of Kentucky Chair:Ned Seeman, NYU 5:15-5:35 Ned Seeman, NYU 6:20-7:40 Dinner, Campus Center Cafeteria (open 4:00-7:10 5:35-5:55 Kurt Vesterager Gothelf, Aarhus Univ pm) you pay cash and buy 5:55-6:15 Friedrich Simmel, TU Munchen 7:40-9:05 Session 13: Chromosomes 6:15-6:25 Hari KK Subramaniam NYU Chair: Wilma Olson, Rutgers 6:25-6:35 Graham Hamblin, McGill Univ. 7:40-7:45 Remarks by the Chair, 6:35-8:00 Dinner, Empire Commons, (open 5:30-7:45 pm) 7:45-8:05 Thomas Cremer, LMU, Germany 8:05-8:15 Ekaterina Khrameeva, IITP, Moscow, Russia 8:00-9:10 Session 6: Nobel Laureate Evening Lecture 8:15-8:25 Khushhall Menaria, ANIT, Bhopal, India 8:00-8:05 Chair & Introduction: Alex Rich MIT 8:25-8:45 Kazuhiro Maeshima, NIG, Mishima, Japan 8:05-9:05 Jack Szostak, Harvard 8:45-9:05 Jonathan Widom, Northwestern Univ. 9:30- 12:00 am Reception for Jack Szostak, Empire Commons 9:05- 12:00 am Trifonov & Maxim host their Russian Party in their apartments

-ii- Friday, June 17 Saturday, June 18: You are going home today after lunch 6:15-9:15 am Breakfast, Hospitality Suites, Empire Commons 7:15-10:15 am Breakfast, Hospitality Suites, Empire Commons

8:00-9:50 Session 14: Replication Meets Transcription 9:00-10:35 Session 18: DNA-Protein-I Chair: Sergei Mirkin, Tufts Univ. Chair: Tom Tullius, BU 8:00-8:20 Sergei Mirkin, Tufts Univ. 9:00-9:05 Remarks by the Chair 8:20-8:40 Benedicte Michel, CNRS, Gif-sur-Yvette, France 9:05-9:25 Christoph W. Müller, EMBL, Heidelberg, Germany 8:40-9:00 Jorge B. Schvartzman, CIB, Madrid, Spain 9:25-9:45 Koby Levy, Weizmann, Israel 9:00-9:10 Boris Belotserkovskii, Stanford 9:45-9:55 Lydia-Ann Harris, Univ. at Buffalo 9:10-9:30 Sue Jinks-Robertson, Duke Univ. 9:55-10:05 Steve Parker, NIH 9:30-9:50 Michael O'Donnel, Rockefeller Univ. 10:05-10:15 Racca Joe, Case Wesrwern Reserve 9:50-10:00 Yayan Zhou, Wesleyan 10:15-10:35 Scot Wolfe, U Mass., Worcester 10:00-11:00 Coffee & Poster Session V 10:35-10:50 Coffee 11:00-12:30 Session 15: Cis Regulatory Modules; DNA Stuff Chair: Mikhail Gelfand, IITP, Moscow, Russia 10:50-11:55 Session 19: DNA-Protein-II 11:00-11:20 Martha Bulyk, Harvard Chair: Richard Mann, Columbia 11:20-11:40 Vsevolod Makeev, GosNIIGenetika, Moscow, Russia 10:50-10:55 Remarks by the Chair 11:40-12:00 Olga Ozoline, RAS, Pushchino, Russia 10:55-11:15 Remo Rohs, USC 12:00-12:10 Ivan Kulakovsky, EIMB, Moscow, Russia 11:15-11:25 Matt Slattery, Univ. of Chicago 12:10-12:20 Alexander Lomzov, ICB, Novosibirsk, Russia 11:25-11:35 Todd Riley, Columbia 12:20-12:30 Alexander Ivanov, ICBCRC, Moscow, Russia 11:35-11:55 Udo Heinemann, MDC, Berlin, Germany 12:30-1:40 Lunch, Campus Center Cafeteria & Barbique Pit (open 11 am- 2 pm) you pay cash and buy 11:55-12:45 Session 20: DNA-Protein-III Chair: Barry Honing, Columbia 1:40-3:40 Session 16: Molecular Simulation: Breakthroughs 11:55-12:00 Remarks by the Chair Chair: Valeri Barsegov, Univ. of Mass, Lowell 12:00-12:20 Victor Zhurkin, NIH 1:40-1:45 Remarks by Chair 12:20-12:40 Zippi Shakked, Weizmann, Israel 1:45-2:05 Klaus Schulten, UIUC 2:05-2:15 Ilya Kovalenko, Moscow Sate Univ. 12:40-12:45 Barry Honing Closes the Conversation 2:15-2:25 Lilian Chong, Univ. of Pittsburgh 2;30-2;50 Ruxandra Dima, Univ. of Cincinnati 12:45-2:00 Farewell Lunch Empire Commons 2:50-3:00 Peyel Das, IBM Go Home After Lunch 3:00-3:20 Gianni De Fabritiis, Univ Pompeu Fabra, Spain 3:20-3:30 Rajib Mukherjee, Tulane Univ. Chair: Tom Cheatham, Univ. of Utah 3:30-3:35 Remarks by Chair 3:35-3:55 David Beveridge, Wesleyan 3:55-5:00 Coffee & Poster Session VI 5:00-7:00 Session 17: Beveridge Celebrations Chair: B. Jayaram, IIT Delhi, India 5:00-5:05 Remarks by the Chair 5:05-5:25 Mihaly Mezei, Mount Sinai 5:25-5:45 Ishita Mukerji. Wesleyan Univ. 5:45-5:55 Elizabeth Wheatly, Wesleyan Chair: Manju Hingorani, Wesleyan 5:55-6:00 Remarks by the Chair 6:00-6:20 Matthew Young, Univ. of Michigan 6:20-6:30 Na Le Dang, Wesleyan 6:30-6:40 Tom Bishop, Tulane Univ. 6:40-7:00 David Beveridge

7:30-12 am Big Feast, Campus Center Ball Room

-iii- Albany 2011 The 17th Conversation State University of New York Albany NY USA June 14-18 2011

Director Hi Folks: Prof. Dr. Ramaswamy H. Sarma Chemistry Department In behalf of the University at Albany, State University of New York, I have the great pleasure of State University of New York welcoming all of you to our uptown Albany campus. Albany NY 12222 USA ph: 518-456-9362; fx: 518-452-4955 Have a great time, enjoy your stay, above all let us have a memorable Conversation in biological email: [email protected] structure, dynamics, interactions and expression.

Sincerely yours

Senior Organizing Committee Paul Agris. Univ. at Albay David. L. Beveridge, Wesleyan Tom Bishop, Tulane Univ. Maxim Frank-Kamenetskii, Boston U. Robert Jernigan. Iowa State Univ. Thomas Cheatham, Utah Dan Fabris, Univ. at Albany Udo Heinemann, Berlin, Germany Richard Lavery, IBCP, France Prof. Dr. Ramaswamy H. Sarma David Lilley Dundee, UK Chemistry, Sergei Mirkin, Tufts University at Albany -SUNY Dino Moras, Strasbourg, France Albany NY 12222 Bengt Norden, Nobel Cmte, Sweden Wilma Olson, Rutgers Ph: 518-456-9362; fx: 518-452-4955 Alex Rich, MIT Email: [email protected] Wolfram Saenger, Berlin, Germany Ned Seeman, New York Univ. April 2 2011 Zippi Shakked , Weizmann, Israel

Jiri Sponer, Czech Republic Ed Trifonov , Uni. of Haifa, Israel Volodya Uversky, IUPUI Sybrren Wijmenga, Univ. Nijmegen Krystyna Zakrzewska, IBCP, France Victor Zhurkin, NIH

Junior Organizing Committee Elena Bichenkova, Univ. of Manchester, UK; Calvin Yu-Chian Chen, China Medical Univ., Taiwan; Miroslav Fojta, Institute of Biophysics, Czech Republic; Yaakov (Koby) Levy, Weizmann Institute of Science, Israel; Vsevolod J. Makeev, Genetika, Moscow, Russia; Aditya Mittal, IIT-Delhi, India; Anna Panchenko, NCBI, NLM, NIH, USA; Remo Rohs, HHMI, Columbia Univ., USA; Dmitry O. Zharkov, SB RAS Institute of Chemical Biology, Russia. Journal of Biomolecular Structure & Dynamics, ISSN 0739-1102 Volume 28, Issue Number 6, (2011) ©Adenine Press (2011)

Book of Abstracts Albany 2011:17th Conversation

Resolving Fibrinogen Nanomechanics Using Dynamic Force Measurements In Vitro and In Silico 1 Mechanical functions of protein fibers are important in cytoskeletal support and Artem Zhmurov1,2 cell motility (1), cell adhesion and formation of extracellular matrix (2), and blood clotting (3). Due to their complexity (103−105 amino acids) and large size Andre Brown3 (~40−200 nm), experimental force measurements (4, 5) of their physical properties Rustem I. Litvinov3 yield results that are impossible to interpret without some input from the computer- Ruxandra I. Dima4 based modeling. Fibrinogen, the precursor of fibrin, provides building blocks for fibrin polymers, a scaffold of blood clots and thrombi. The mechanical properties John W. Weisel3 of fibrin(ogen), which control how clots and thrombi respond to external mechani- Valeri Barsegov1,2* cal factors, are essential for hemostasis. Yet, the complexity of fibrin(ogen) structure makes it difficult to uncover the unfolding mechanism using dynamic force measurements in vitro alone. We carried out combined experimental-theoretical 1Department of Chemistry, University of studies of the mechanical properties of fibrinogen, using AFM assays and Langevin Massachusetts, Lowell, MA 01854 simulations on Graphics Processing Units (GPUs). A combination of the Self- 2Moscow Institute of Physics and Organized Polymer (SOP) model (6) and simulations on GPUs (7) makes it possible to characterize the fibrinogen nanomechanics in the experimental 0.1-1s timescale. Technology, Moscow, Russia 141700 The mechanical unraveling of fibrinogen is determined by the microscopic transi- 3Department of Cell and tions that couple reversible extension-contraction of the coiled-coils and unfolding Developmental Biology, University of of the terminal γ C-domains. The coiled-coils play a role of the biomolecular storage of mechanical energy to amortize an external perturbation and to transmit and Pennsylvania School of Medicine distribute tension among the γ C-domains. Unfolding of the γ C-domains, stabilized Philadelphia, PA 19104 by domain interactions with the βC-domains, result in three force signals, which 4Department of Chemistry, University of are characterized by the average force of ~100 pN and peak-to-peak distance of ~30 nm. The results obtained provide important quantitative characteristics of the Cincinnati, Cincinnati, OH 45221 fibrinogen nanomechanics necessary to understand fibrin viscoelasticity at the fiber *[email protected] and whole clot levels.

975 976 This work has been supported by the American Heart Association grant (09SDG2460023) and by the Russian Ministry of Education grant (02−740− 11−5126).

References

1. T. P. Stossel, J. Condeelis, L. Cooley, J. H. Hartwig, A. Noegel, M. Schleicher, and S. S. Shapiro. Nat Rev Mol Cell Biol 2, 138-145 (2001). 2. V. Barsegov and D. Thirumalai. Proc Natl Acad Sci USA 102, 1835-1839 (2005). 3. J. W. Weisel. Biophys Chem 112, 267-276 (2004). 4. I. Schwaiger, C. Sattler, D. R. Hostetter, and M. Rief. Nature Mat 1, 232-235 (2002). 5. W. Liu, L. M. Jawerth, E. Sparks, M. R. Falvo, R. R. Hantgan, R. Superfine, S. T. Lord, and M. Guthold. Science 313, 634 (2006). 6. R. I. Dima and H. Joshi. Proc Natl Acad Sci USA 105, 15743-15748 (2008). 7. A. Zhmurov, R. I. Dima, Y. Kholodov, and V. Barsegov. Proteins 78, 2984-2999 (2010).

Phase Transition from a-Helices to b-Sheets in 2 Fibrinogen Coiled Coils 1,2 Mechanical functions of fibrin fibers are essential for hemostasis and wound heal- Artem Zhmurov ing (1). Fibrinogen, the precursor of fibrin, is a branched polymer that provides the Andre Brown3 scaffold for a thrombus in vertebrates. The physical properties of fibrin(ogen) are Rustem I. Litvinov3 essential for the ability of fibrin clots to accomplish hemostasis and are an impor- 4 tant determinant of the pathological properties of thrombi. Despite such critical Ruxandra I. Dima importance, the structural basis of fibrin clot mechanics is not well understood (2). John W. Weisel3 Graphics Processing Units (GPUs) are being used in a variety of scientific applica- Valeri Barsegov1,2* tions, including the biological N-body problem (3). We carried out theoretical studies of the mechanical properties of fibrinogen molecule, using all-atom Molecular Dynamics (MD) simulations in implicit water fully implemented on a GPU. When 1Department of Chemistry, University the α-helical regions in the coiled-coils are subject to an external mechanical per- of Massachusetts, Lowell, MA 01854 turbation, they undergo reversible phase transition to form the extended β-sheets (4). The D-regions of the molecule make several turns around the direction of force 2Moscow Institute of Physics and application to accommodate the mechanical unraveling of the coiled coils. As a Technology, Moscow, Russia 141700 result, the hydrophobic side-chains buried inside the α-helices in the fibrin(ogen) 3Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 4Department of Chemistry, University of Cincinnati Cincinnati, OH 45221 *valeri_barsegov@ uml.edu folded state become exposed to solvent. We argue that the observed increase in 977 the free energy of solvation might lead to protein aggregation, and hypothesize that these transitions provide the molecular mechanism for the negative compress- ibility observed in experiments on whole blood clots (5). These results provide important quantitative characteristics of the fibrinogen nanomechanics necessary to understand the viscoelastic properties of fibrin polymers at the fiber and whole clot levels.

This work has been supported by the American Heart Association grant (09SDG2460023) and by the Russian Ministry of Education grant (02− 740−11−5126).

References

1. J. W. Weisel. Science 320, 456-457 (2008). 2. A. E. X. Brown, R. I. Litvinov, D. E. Discher, and J. W. Weisel. Biophys J 92, L39-L41 (2007). 3. A. Zhmurov, R. I. Dima, Y. Kholodov, and V. Barsegov. Proteins 78, 2984-2999 (2010). 4. A. Zhmurov, A. E. X. Brown, R. I. Litvinov, R. I. Dima, J. W. Weisel, and V. Barsegov (manuscript in preparation). 5. A. E. X. Brown, R. I. Litvinov, D. E. Discher, P. K. Purohit, and J. W. Weisel. Science 325, 741-744 (2009).

Computer Simulations of Protein–Protein Complex Formation on Graphics Processing Units 3 Protein-protein interactions create new properties to the interacting components 1 and to the whole system, and it is an important aspect of the biological machinery Ilya Kovalenko * (1, 2). We present a new method for computer simulations of formation of protein- Alexandra Diakonova1 protein complexes in a cell environment. The method is based on Brownian dynam- Sergey Khrushchev1 ics, which makes it possible to simulate association reactions of several hundreds 1 of protein pairs in sub-cellular compartments, and to obtain the real-time dynamics Anna Abaturova of protein-protein interactions. The method allows us to explore the effect of elec- Galina Riznichenko1 trostatic forces on the protein-protein complex formation, evaluate the kinetic rate Andrei Rubin1 constants, and to unmask the molecular interactions (diffusion, electrostatic inter- 2 actions) underlying the dynamics of processes in a cell. Biomolecular simulations Sergey Trifonov were used to study the kinetics of protein-protein interactions between the electron Ivan Morozov2 transport proteins involved in photosynthesis, plastocyanin–cytochrome f complex, Yaroslav Kholodov2 both for wild-type and mutant plastocyanins. The method describes accurately bind- 3 ing interactions for different values of ionic strength in the solution (3) and in the Valeri Barsegov chloroplast thylakoid lumen (4), while taking into account (b) electrostatic interactions between proteins and in the thylakoid membrane (5), (c) kinetic characteristics 1Biology Department, Moscow State of ferredoxin and ferredoxin:NADP–reductase complex formation in solution (6), University, Moscow, Russia, 119992 and (d) complex formation of the transmembrane pigment-protein complex Photo- system I and proteins plastocyanin and ferredoxin (7). The developed method can 2Moscow Institute of Physics and also be used as a predictive tool to resolve the binding sites and to describe complex Technology, Moscow Region structures for a range of protein (8). Biomolecular simulation was accelerated on a Russia, 141700 Graphics Processing Unit (GPU) to describe interactions among a large number of proteins. The 10-100-fold computational acceleration, attained on the GPU device, 3Department of Chemistry, University enabled us calculate the kinetic rates of protein-protein complex formation under of Massachusetts, Lowell, MA 01854 the physiologically relevant conditions of sub-cellular environment. *[email protected] References

1. L. K. Chang, J. H. Zhao, H. L. Liu, J. W. Wu, C. K. Chuang, K. T. Liu, J. T. Chen, W. B. Tsai, and Y. Ho. J Biomol Struct Dyn 28, 39-50 (2010). 2. M. J. Aman, H. Karauzum, M. G. Bowden, and T. L. Nguyen. J Biomol Struct Dyn 28, 1-12 (2010). 978 3. I. B. Kovalenko, A. M. Abaturova, P. A. Gromov, D. M. Ustinin, E. A. Grachev, G. Y. Riznichenko, and A. B. Rubin. Phys Biol 3, 121-129 (2006). 4. I. B. Kovalenko, A. M. Abaturova, P. A. Gromov, D. M. Ustinin, G. Y. Riznichenko, E. A. Grachevo, and A. B. Rubin. Biophysics 53, 140-146 (2008). 5. O. S. Knyazeva, I. B. Kovalenko, A. M. Abaturova, G. Y. Riznichenko, E. A. Grachev, and A. B. Rubin. Biophysics 55, 221-227 (2010). 6. I. B. Kovalenko, A. N. Diakonova, A. M. Abaturova, G. Y. Riznichenko, and A. B. Rubin. Phys Biol 7, 026001 (2010). 7. I. B. Kovalenko, A. M. Abaturova, G. Y. Riznichenko, and A. B. Rubin. BioSystems, doi: 10.1016/j.biosystems.2010.09.013 (2010). 8. I. B. Kovalenko, A. M. Abaturova, G. Y. Riznichenko, and A. B. Rubin. Dokl Biochem Biophys 427, 215-217 (2009).

Computer Simulation of Plastocyanin Diffusion and 4 Interaction with Its Reaction Partners We present a Langevin dynamics computer model of limited diffusion of protein Ilya Kovalenko* plastocyanin and its interaction with transmembrane protein complexes photosys- Alexandra Diakonova tem 1 and cytochrome bf in the narrow chloroplast thylakoid lumen. The model Olga Knyazeva is multiparticle, it considers many plastocyanin molecules that compete to form complexes with numerous photosystem 1 and cytochrome bf complexes embedded Anna Abaturova in the photosynthetic membranes. The model takes into account the geometry of Galina Riznichenko the luminal space packed with many protein molecules and considers electrostatic Andrei Rubin interactions of plastocyanin with its reaction partners in the thylakoid membrane. The model uses a continuum electrostatic approach that describes molecules at the atomic level using a macroscopic description. The Poisson-Boltzmann formalism Biophysics Department, Biological was used to determine the electrostatic potentials of the electron carrier proteins Faculty, Moscow State University, and the thylakoid membrane at different ionic strengths. This work uses the model parameters of protein-protein association estimated in our papers (1-4). Leninskie Gory, Moscow 119992, Russia Calculations correctly reproduce the experimentally registered kinetic curves of *[email protected] redox changes of the reaction center P700 of photosystem 1 and cytochrome f. The model demonstrates non-monotonic dependences of complex formation rates on the ionic strength as the result of long-range electrostatic interactions. The simulation method presented in this work can be applied for the description of diffusion and functioning of many macromolecules that interact in the heterogeneous interior of subcellular systems.

References

1. I. B. Kovalenko, A. M. Abaturova, P. A. Gromov, D. M. Ustinin, E. A. Grachev, G. Y. Riznichenko, and A. B. Rubin. Phys Biol 3, 121-129 (2006). 2. I. B. Kovalenko, A. M. Abaturova, P. A. Gromov, D. M. Ustinin, G. Y. Riznichenko, E. A. Grachev, and A. B. Rubin. Biophysics 53, 140-146 (2008). 3. I. B. Kovalenko, A. M. Abaturova, G. Y. Riznichenko, and A. B. Rubin. BioSystems, doi: 10.1016/j.biosystems.2010.09.013 (2010). 4. O. S. Knyazeva, I. B. Kovalenko, A. M. Abaturova, G. Y. Riznichenko, E. A. Grachev, and A. B. Rubin. Biophysics 55, 221-227 (2010).

Computational Microscopy

Biomolecular modeling, taking advantage of ever increasing computer power, has 5 dramatically improved in accuracy as well as in time and size scale covered. For Klaus Schulten example, low resolution single molecule force measurements are imaged at atomic resolution through steered molecular dynamics simulations; crystallography and Department of Physics, electron microscopy data, combined through molecular dynamics flexible fitting calculations, are interpreted through atomic level structures of functional intermedi- University of Illinois at Urbana-Champaign, ates of large cellular machines; simulations at multiple length scales show movies Urbana, IL 61801, USA of peripheral membrane proteins sculpting cellular membranes. The lecture will [email protected] illustrate for the three examples atomic resolution images and movies provided by means of biomolecular modeling as a result of new simulation concepts, algo- 979 rithms, and technology.

A computational-experimental collaboration (with H. Gaub, Munich) discovered a possibly fundamental epigenetic mechanism of methylated DNA. Highly sampled chip-based stretching of double stranded DNA combined with simulation revealed that strand separation mechanics is strongly affected by epigenetic modification of DNA. The study, involving long time scale and large size simulations, resolved and explained the observed effect of DNA methylation.

Atomic resolution crystallographic structures and electron microscopy density maps at better than 10 Angstrom resolution (from R. Beckmann, Munich) were combined in a computational analysis employing a new simulation method, molecular dynamics flexible fitting, to construct an atomic resolution structure of a ribosome seen in the process of threading a nascent protein through a translocon into a biological membrane. The simulations revealed also in great detail the interaction between nascent protein and ribosomal exit channel as well as interaction with the translocon, for example the binding of the signaling element to the translocon.

The shape of cellular membranes can be induced by peripheral proteins for example N-BAR and F-BAR domains. The latter proteins have been observed in vitro to form tubular membranes from vesicles and a similar behavior has been seen in simulations. Employing a combination of coarse-grained and all atom molecular dynamics simulations, calculations offer movies revealing how the peripheral proteins, in forming regular lattices as observed by electron microscopy (Unger, Yale U.), bend flat membranes into tubes, the latter remaining stable after removal of protein. The simulations shed light on the protein-lipid interactions responsible for membrane bending.

Exploring the Role of Protein-Protein Interactions in the Mechanical Unfolding of Protein Assemblies 6 Dynamic force spectroscopy methods provide unique opportunities for directly probing the free energy landscape of complex biomolecules. However, even if they Ruxandra I. Dima provide information on the chain extension of the molecule, they cannot supply full details of the populated structures. This is especially true in the case of multi- Department of Chemistry, domain or multi-chain proteins for which the complexity of the fold translates into University of Cincinnati, many unfolding scenarios. An assumption employed by the majority of experiments for the interpretation of the force-unfolding of such proteins is that the behavior of Cincinnati, OH 45221 the ensemble is the sum of its parts. While this assumption is likely to apply to [email protected] tandems of identical units, for hetero-protein tandems or whenever units are connected by interfaces as in multi-domain or multi-chain complexes this assumption needs to be revisited.

I will present our investigations into the role of protein-protein interactions through simulations of the biomechanical unfolding reactions of multi-domain and multi- chain protein complexes covering a range of fundamental cellular functions from fusion to cytoskeletal support in real (experimental) time (1, 2). To obtain the force extension curves using experimental pulling speeds, we employed a coarse- grained minimalist model (SOP model) (3) of proteins to carry out overdamped Langevin simulations implemented on Graphics Processing Units (GPUs). GPUs have unleashed tremendous computational power that has been utilized in a wide range of scientific applications. The system size dependent 10-90-fold computational speedup on a GPU, compared to an optimized CPU program, enabled us to follow the dynamics in the centisecond timescale (4). While our model reproduces 980 the experiments, we find that the independence assumption needs to be critically assessed. I will discuss the signature of the protein-protein interactions as a function of the applied vector force, and the connection between the shape of the force peaks and the degree of cooperativity in the protein complex. Remarkably, we find that the degree of stabilization conferred by the interactions between units is determined by a combination between the stability of the interface and the internal fluctuations of a module.

This research has been supported by NSF CAREER Award MCB-0845002.

References

1. R. I. Dima and H. Joshi. Proc Natl Acad Sci USA105, 15743-15748 (2008). 2. J. Y. Lee, T. Iverson, and R. I. Dima. J Phys Chem B 115, 186-195 (2011). 3. C. Hyeon, R. I. Dima, and D. Thirumalai. Structure 14, 1633-1645 (2006). 4. A. Zhmurov, R. I. Dima, Y. Kholodov, and V. Barsegov. Proteins 78, 2984-2999 (2010).

Efficient Explicit Solvent Simulations 7 of Molecular Association Kinetics Atomically detailed views of molecular association events are of great interest to a Matthew C. Zwier variety of research areas in biology and chemistry. A natural approach to providing Joseph W. Kaus such views is to use molecular dynamics (MD) simulations in explicit solvent which Lillian T. Chong* are quite routine for tens of nanoseconds in certain systems (1-4). However, it has been computationally prohibitive to perform MD simulations for a sufficiently long time (by “brute force”) to capture more complicated association events, e.g. Department of Chemistry, protein-protein associations, that require microseconds or beyond (5). Fortunately, University of Pittsburgh, the long timescales required are not necessarily because the actual events take a long time; instead the events may be fast but infrequent, separated by long waiting times. Pittsburgh, PA 15260 Path sampling approaches aim to capture rare events by minimizing the simulation *[email protected] of long waiting times between events [as reviewed by Zwier and Chong (6)]. In this work, we have combined the “weighted ensemble” path sampling approach (7) with explicit solvent MD simulations. This approach allows us to obtain accurate kinetics as well as ensembles of molecular association pathways. We have determined the efficiency of this approach relative to brute force simulations in sampling the molecular association events for a range of well-studied systems: methane/methane, Na+/Cl-, methane/benzene, and K+/18-crown-6 ether (pictured below from left to right). Relative to brute force simulation, we obtain efficiency gains of at least 1,100-fold for the most challenging system, K+/18-crown-6 ether, in terms of sampling the distribution of molecular association pathways. Our results indicate that weighted ensemble sampling is likely to allow for even greater efficiencies for more complex systems with higher barriers to molecular association. Applications of weighted ensemble sampling to explicit solvent MD simulations of protein binding events will also be discussed.

This research has been supported by NSF CAREER Award MCB-0845216. References 981 1. P. Sklenovský and M. Otyepka. J Biomol Struct Dyn 27, 521-539 (2010). 2. M. J. Aman, H. Karauzum, M. G. Bowden, and T. L. Nguyen. J Biomol Struct Dyn 28, 1-12 (2010). 3. C. Koshy, M. Parthiban, and R. Sowdhamini. J Biomol Struct Dyn 28, 71-83 (2010). 4. Y. Tao, Z. H. Rao, and S. Q. Liu. J Biomol Struct Dyn 28, 143-157 (2010). 5. K. A. Henzler-Wildman and D. Kern. Nature 450, 964-972 (2007). 6. M. C. Zwier and L. T. Chong. Curr Opin Pharmacol 10, 745-752 (2010). 7. G. A. Huber and S. Kim. Biophys J 70, 97-110 (1996).

Molecular Simulations Unravel Key Amino Acid Interactions Regulating Stability and Aggregation of Human Lens gD-crystallin 8 Payel Das1* The prevalent eye disease age-onset cataract is associated with aggregation of Jonathan A. King2 human γD-crystallins, one of the longest-lived proteins of the body. The molec- 1,3 ular determinants of the unusually high stability and (un)folding/aggregation of Ruhong Zhou γD-crystallin remain to be unidentified. Determination of complex dynamics during protein (un)folding and aggregation events require molecular simulations at 1IBM Thomas J. Watson Research long time scales (micro to milliseconds). Even though simulations that are tens of nanoseconds long for certain systems (1-4) are routine, it remains challenging to Center, Yorktown Heights, NY 10598 perform them at longer time scales for atomistic models of biologically relevant 2Department of Biology, proteins. In this study, we perform extensive atomistic molecular dynamics simula- Massachusetts Institute of Technology, tions using massively parallel IBM Blue Gene/L supercomputer (5) to characterize unfolding (6) and oligomerization (7) of human gamma D crystallin to advance our Cambridge, MA 02139 current understanding of cataract. 3Department of Chemistry, Columbia University, New York, Using large-scale atomistic simulations (6), we have shown that the isolated N-terminal domain (N-td) of γD-crystallin is less stable than its isolated C-terminal NY 10027 domain (C-td), in addition to being the less stable domain in the full-length protein (Figure 1A and B), in agreement with biochemical experiments. Sequential unfolding of individual Greek key motifs was revealed within each isolated domain. Our simulations strongly indicate that the stability and the folding mechanism of the

Figure 1: Schematic summary of human γ D-crystallin polymerization. 982 N-td are regulated by the interdomain interactions, consistent with experimental observations. We also found that the a and b strands from the Greek Key motif 4 comprising the interdomain interface are the most stable structures within the full protein. Detailed analysis uncovers a surprising Glu-Arg salt-bridge at the topologically equivalent positions of residues E135 and R142 that plays a significant role in determining the stability of a Greek Key motif (see Figure 1A). Disrupt- ing the E135-R142 salt-bridge in silico resulted in destabilizing the inter-domain interface and facilitated the N-td unfolding. These findings (6) indicate that certain highly conserved charged residues, that is, Glu135 and Arg142, of γD-crystallin are crucial for stabilizing its hydrophobic domain interface in native conformation, and disruption of charges on the γD-crystallin surface might lead to unfolding.

Identification of the aggregation precursors of γ-crystallins is extremely crucial for developing strategies to prevent and reverse cataract. We have used large-scale simulations to determine the structural basis of the pathogenic monomeric state and the intermolecular association of γD-crystallin. Our microseconds of atomistic molecular dynamics simulations (7) uncover the molecular structure of the experimentally detected aggregation-prone folding intermediate species of monomeric native γ D-crystallin with a largely folded C-terminal domain and a mostly unfolded N- terminal domain (see Figure 1B). About 30 residues including a, b, and c strands from the Greek Key motif 4 of the C-terminal domain experience strong solvent exposure of hydrophobic residues as well as partial unstructuring upon N-terminal domain unfolding. Those strands comprise the domain-domain interface that is crucial for the unusually high stability of γ D-crystallin. We further simulate the intermolecular linkage of these monomeric aggregation precursors (7), which reveals domain- swapped dimeric structures (Figures 1C and D). In the simulated dimeric structure, the N-terminal domain of one monomer is frequently found in contact with residues 135-164 encompassing the a, b, and c strands of the Greek Key motif 4 of the second molecule. The present results suggest that γD-crystallin polymerize through successive domain swapping of those three C-terminal β-strands leading to age-onset cataract, as an evolutionary cost of its very high stability (Figure 1). These findings (7) thus provide critical molecular insights onto the initial stages of age-onset cataract formation, which is important toward understanding protein aggregation diseases.

References

1. P. Sklenovský and M. Otyepka. J Biomol Struct Dyn 27, 521-539 (2010). 2. M. J. Aman, H. Karauzum, M. G. Bowden, and T. L. Nguyen. J Biomol Struct Dyn 28, 1-12 (2010). 3. C. Koshy, M. Parthiban, and R. Sowdhamini. J Biomol Struct Dyn 28, 71-83 (2010). 4. Y. Tao, Z. H. Rao, and S. Q. Liu. J Biomol Struct Dyn 28, 143-157 (2010). 5. S. Kumar, et al. IBM J Res Dev 52, 177-188 (2008). 6. P. Das, J. A. King, and R. Zhou. Prot Sci 19, 131-140 (2010). 7. P. Das, J. A. King, and R. Zhou. Proc Natl Acad Sci, USA, under review (2011).

Reconstructing an Enzyme-Inhibitor Binding Process 9 by Molecular Dynamics Simulations Gianni De Fabritiis The understanding of protein-ligand binding is of critical importance for biomedical research, yet the process itself has been very difficult to study due to its Research Group of biomedical intrinsically dynamic character. In this talk, we will go through the quantitative Informatics (GRIB-IMIM) reconstruction of the complete binding process of the enzyme-inhibitor complex Trypsin-Benzamidine performed with molecular dynamics simulations of free Universitat Pompeu Fabra inhibitor binding. The binding events obtained are able to capture the kinetic path- Barcelona Biomedical Research way of the inhibitor diffusing from solvent to bound passing for few metastable Park (PRBB), C/ Dr. Aiguader 88 intermediate states. Unexpectedly, rather than directly entering the binding pocket, the inhibitor appears to roll on the surface of the protein to the final binding pocket. 08003, Barcelona, Spain The trajectories are analysed via a Markov state model-based analysis which addi- [email protected] tionally yields the kinetic parameters and binding affinity of the interaction. These results show an impressive predictive power for unconventional high-throughput 983 molecular simulations. At the same time, the general methodology is easily applicable to other molecular systems becoming of interest to biomedical and pharmaceutical research.

The Nucleosome Simulator: 100 Nucleosomes; 2 Microseconds and Counting 10 Molecular simulation is an effective tool to study structure function relationships in 1 biomolecules. A modest molecular dynamics simulation today includes ~100,000 Rajib Mukherjee * atoms and represents over 100 ns of time. The computational effort requires nearly Hideki Fujioka1 one hundred processors in order to complete in less than one week. Today’s super- Abhinav Thota2 computers contain up to 200,000 processors, too many for a single simulation of 2 modest size. However, it is reasonable to simulate 10’s to 100’s of structures simul- Shantenu Jha 1 taneously on a single supercomputer or to distribute them to any suitable comput- Thomas C. Bishop ing resources as they become available. Such high throughput high performance simulations require careful coordination and strategy. The ManyJobs and BigJobs 1Center for Computational Science, tools provide this functionality. Tulane University, New Orleans, Our present efforts are directed toward the investigation of nucleosome position- LA 70118, USA ing and stability as a function of DNA sequence using all atom molecular dynam- 2Center for Computation & Technology, ics simulation. Nucleosome positioning is one of the current ‘hot’ issues and the various approaches and hypotheses on what positions nucleosomes are discussed Johnston Hall, Louisiana State extensively in recent publications, particularly in an issue of this Journal devoted University, Baton Rouge, exclusively to this subject (1-14). One of the major factors affecting the nucleosome LA 70803, USA positioning is DNA sequence. Nucleosomes consist of 147 base pair (bp) of DNA wrapped ~1.7 left handed superhelical turns around a histone core. This study *[email protected] requires simulations of hundreds of nucleosomes with different sequences. Our chosen sequences are divided into four broad categories: naturally occurring positioning sequences, artificial positioning sequences, sequences from theSaccharomyces cerevisiae genome and sequences used for control purposes, e.g. homopolymers. For the S. cerevisiae derived sequences, we model 336 nucleosomes. The collection represents 16 of the most well positioned nucleosomes and their immediate neighbors in sequence space. Each neighborhood spans two turns of the DNA, one upstream turn and one downstream turn. The 21 individual neighbors contain only 147 bp, each created by threading the appropriate sequence onto the histone core. Thus each neighborhood has a common segment of 126 bp located at 21 successive positions on the histone core. To date, we have simulated over 100 nucleosomes, including 4 separate neighborhoods, and accumulated over 2 microseconds of nucleosome dynamics. Our high throughput approach requires significant computational power, constant scheduling, monitoring, and efficient utilization of resources in order to achieve the shortest time to completion.

To manage the workflow we have utilized two scheduling tools: ManyJobs and BigJobs. ManyJobs is a portable tool written in Python. ManyJobs maintains a database of all compute tasks and the dependencies between tasks. At the beginning of a run, ManyJobs submits requests for resources to all computers listed by the user. Once a resource is allocated and a job starts. ManyJobs assigns a task to the resource and requests additional resources in anticipation of the next task. Upon job completion, the task is marked complete in the ManyJobs database. The process repeats until all tasks in the database are completed. The current version of ManyJobs uses secure shell for communications between the machine maintaining the task database and the various compute resources. BigJobs is a Simple API for Grid Applications (SAGA) based implementation of the pilot job concept. SAGA provides alternate methods for authentication and communication than secure shell. Another distinction of BigJobs is the ability to dynamically bundle individual tasks into a container with multiple tasks, the pilot job. 984 We will discuss implementation and proper utilization of these tools and their pros and cons. A meta-analysis of simulation results is conducted to identify features of nucleosome positioning and stability. We focus attention on DNA structural deformations, in the form of kinks, their location, sequence dependencies, and the timescale associated with kink formation and healing.

References

Exploring Fast Protein Folding Funnel Using Replica Exchange Molecular Dynamics at 11 Different Temperature Ranges Vinod Jani Shruti Koulgi Protein folding is a biological process through which one dimensional sequence acquires its three dimensional structure. During past four decades various aspects Uddhavesh Sonavane of protein folding have been explored experimentally and theoretically. There have Rajendra Joshi* been some success in predicting 3D structure of proteins using statistical potentials (for example see references 1, 2). Reaching the experimental time scale of millisecond is a grand challenge for protein folding simulations. With the development of Bioinformatics Team, SECG advanced Molecular Dynamics (MD) techniques like Replica Exchange Molecular Centre for Development of Advanced Dynamics (REMD), simulations can possibly reach to the experimental timescales. Computing, Pune University Campus, Pune – 411 007, India *[email protected]

Figure 1: NMR structure showing the three helices along with the amino acid sequence of same (pdb code 1VII). The major difficulty in experimental studies of protein folding lies in capturing 985 of transient intermediates and events, which is possible via performing very long simulations. Here an attempt has been made to reach the multi-microsecond simulation time scale by carrying out folding simulation on a fast folding three helix bundle protein using REMD. REMD based folding simulation has been carried out on Villin headpiece (PDB code 1VII, Figure 1), a 36-residue small protein (3) and Engrailed Homeodomain (PDB ID: 1ENH), a 54 residue protein (4) starting from its extended structure. The multiple REMD simulations were carried out at moderate and broader temperature range. The population landscape has been built using segment wise Root Mean Square Deviation (RMSD), Principal Com- ponent Analysis (PCA) as reaction coordinates. The REMD has helped to carry out very long time scales simulations where results are close to the experimental findings. Also the effect of temperature range on the population landscape has been discussed in detail.

References

1. P. Sklenovský and M. Otyepka. J Biomol Struct Dyn 27, 521-539 (2010). 2. M. J. Aman, H. Karauzum, M. G. Bowden, and T. L. Nguyen. J Biomol Struct Dyn 28, 1-12 (2010). 3. V. Jani, U. B. Sonavane, and R. Joshi. J. Biomol Struct Dyn 28, 845-860 (2011). 4. S. Koulgi, U. B. Sonavane, and R. Joshi. J Mol Graph Model, 29, 481-491 (2010).

Exploring Folding Funnel of Villin Headpiece Using Replica Exchange Molecular Dynamics and Amber United Atom Model 12 Vinod Jani Understanding protein folding has been a scientifically and computationally chal- Uddhavesh Sonavane lenging task till date. The question, “How does an amino acid sequence dictate the structure of a protein?” has been of major interest and different theories have Rajendra Joshi* been put forward to answer it. Despite the success of deriving the 3 D structure of a protein (for example see 1, 2) from our conventional understanding of the prefer- Bioinformatics Group ential interactions between certain amino acids, recently questions have been raised regarding the use of statistical potentials and preferential interactions (3). Reaching Centre for Development of Advanced the experimental time scale of millisecond is a grand challenge for protein folding Computing, Pune University Campus, simulations. The major challenges involved in the use of molecular dynamics (MD) Pune – 411 007, India simulations are to explore folding landscape for fast folding proteins and give an atomic level understanding of the folding process (4). The advanced methodologies *[email protected] like REMD (5) and Coarse-grained MD and computational power enable one to carry out long simulations. The advanced methods like REMD have significantly contributed to the understanding of the folding landscape of various ultrafast folding proteins. The advancement of force field to handle Coarse Grained models over all atom model system may be needed in achieving the goal of very long time scale folding simulation. Here an attempt has been made to reach the multi-microsecond simulation time scale by carrying out folding simulation on a fast folding three Helix bundle protein. A combination of REMD and Amber United atom model (6) has been employed. Two folding simulations based on REMD have been carried out on Villin headpiece (PDB code 1VII), a 36-residue small three Helix bundle protein (7) starting with an extended conformation. The protein folding funnel has been explored using segment wise Root Mean Square Deviation (RMSD) and Principal Component Analysis (PCA) as reaction coordinates. The combination of REMD and CGMD has helped to carry out very long time scales simulations where the results are close to the experimental findings. The present study is targeted to explore the folding funnel as well as to study the effect of temperature range in REMD simulations. 986 References 1. P. Sklenovský and M. Otyepka. J Biomol Struct Dyn 27, 521-539 (2010). 2. M. J. Aman, H. Karauzum, M. G. Bowden, and T. L. Nguyen. J Biomol Struct Dyn 28, 1-12 (2010). 3. A. Mittal, B. Jayaram, S. Shenoy, and T. S. Bawa. J Biomol Struct Dyn 28, 133-142 (2010). 4. K. A. Dill, S. B. Ozkan, T. R. Weikl, J. D. Chodera, and V. A. Voelz. Current Opinion in Structural Biology 342-346 (2007). 5. Y. Sugita, and Y. Okamoto. Chem Phys Lett 329, 261-270 (1999). 6. L. Yang, C. H. Tan, M. J. Hsieh, J. Wang , Y. Duan, P. Cieplak, J. Caldwell, P. A. Kollman, and R. Luo. J Phys Chem B, 110, 13166-76 (2006). 7. V. Jani, U. Sonavane, and R, Joshi. J Biomol Struct Dyn 28, 845-860 (2011).

Computational Investigation of the Free Energy Landscape of the) of the Four Stereomers of 13 Ac-L-Pro-c3Phe-NHMe (c3Phe=2, Juan J. Perez* 3-Methanophenylalanine) in Explicit Alex Rodriguez and Implicit Solvent Francesc Corcho The prediction capabilities of atomistic simulations of peptides are hampered by different difficulties including, the reliability of force fields, the treatment of the Department of Chemical Engineering, solvent or the adequate sampling of the conformational space. The present report Technical University of Catalonia, regards a computational study aimed at assessing the conformational profile of Barcelona, Spain the four stereoisomers of the peptide Ace-Pro-c3Phe-NMe, previously reported to exhibit β-turn structures in dichloromethane with different type I/type II β-turn *[email protected] profiles [1]. For this purpose, we carried out a thorough sampling of the conformational space of the four peptides in explicit solvent using the replica exchange molecular dynamics method as a sampling technique and compared the results with simulations of the system modeled using the analytical linearized Poisson- Boltzmann (ALPB) method with two different AMBER force fields: parm96, and parm99SB.

The free energy landscapes of the different peptides computed in explicit solvent show two minima separated by high barriers and agree well with the published experimental results. The calculations carried out in implicit solvent do not describe the system in the same manner. Moreover, it is shown that implicit solvent calculations carried out with the parm96 force field agree better with those obtained with the parm99SB force field in explicit solvent [2,3]. The results of the simulations suggest that the balance between intra- and intermolecular interactions is the cause of the differences between implicit and explicit solvent simulations in this system, stressing the role of the environment to define properly the conformational profile of a peptide in solution.

References

1. A. I. Jimenez, C. Cativiela, A. Aubry, and M. Marraud. J Am Chem Soc 120, 9452-9459 (1998). 2. A. Rodriguez, J. Canto, F. Corcho, and J. J. Perez. Biopolymers 92, 518-524 (2009). 3. A. Rodriguez, P. Mokoema, F. Corcho, K. Bisetty, and J. J. Perez. J Phys Chem B, 115, 1440-1449 (2011). Folding Globular Proteins: Collapse Kinetics 987 and Chevron Plots

Quantitative description of how proteins fold under experimental conditions remains a challenging problem. Experiments often use urea and Guanidinium Chloride (GdmCl) to study folding whereas the natural variable in simulations is temperature. 14 To bridge the gap, we use the Molecular Transfer Model that combines measured D. Thirumalai denaturant-dependent transfer free energies for the peptide group and amino acid residues, and a coarse-grained model for polypeptide chains to simulate the folding Institute for Physical Science and mechanism of src SH3. Stability of the native state decreases linearly as [C] (the concentration of GdmCl) increases with the slope that is in excellent agreement with Technology University of Maryland, experiments. We show that lnk (k is the sum of folding and unfolding rates) as a obs obs College Park, function of [C] has the characteristic V (Chevron) shape. In the dominant transition state, which does not vary significantly at low [C], the core of the protein and certain MD 20742, USA loops are structured. Besides solving the long-standing problem of computing the [email protected] Chevron plot, our work lays the foundation for incorporating denaturant effects in a physically transparent manner either in all atom or coarse-grained simulations.

Single-molecule Fluorescence Maps the Folding Landscape of a Large Protein 15 A substantial body of information, both theoretical and experimental, has accumulated over the last years on protein folding, including a provocative recent idea that Menahem Pirchi it is driven by amino acid stoichiometry (1), as opposed to preferred interactions (e.g., hydrophobic interactions) between specific amino acids (2, 3). Particularly Israel Perlman Chemical Sciences advanced is our understanding of the principles that govern the folding of small, Building 603, Weizmann Institute single-domain proteins (4). But can we apply the same principles in order to decipher the folding mechanisms of larger, multi-domain proteins? More than 70% of of Science, the eukaryotic proteins belong to this group, yet rather little is known about their P.O.Box 26, Rehovot 76100, Israel folding reactions (5). [email protected] We propose here that high-throughput single-molecule fluorescence spectroscopy, combined with statistical analysis, can be used to study folding dynamics of large proteins (6). As a proof-of-concept, we studied the folding landscape of adenylate kinase (AK), a 214-residue protein from Escherichia coli. AK molecules were double-labeled for FRET at two sites in their CORE domain. The molecules were individually trapped within surface-tethered lipid vesicles (7, 8) in the presence of different concentrations of guanidinium chloride (GdmCl), a chemical denaturant. The trapped molecules were examined using an automated single-molecule fluorescence microscope, which allowed us to obtain data sets consisting of many thousands of short FRET trajectories of individual molecules. The availability of this massive amount of data enabled us to construct a detailed map of the folding landscape of AK, using hidden Markov modeling (HMM) (9). We found that the folding dynamics of AK could be described in terms of transitions between six quasi-stable states, one of which is probably misfolded. The folding reaction involves many parallel pathways connecting these states in jumps of various sizes (See Figure). This folding pattern differs markedly from the well-known two-state model of small proteins. Future work, involving both experiments and computation, will attempt Figure: 1D projection of the energy landscape of adenylate to structurally characterize the intermediate states discovered here. kinase molecules, based on analysis of single-molecule fluorescence trajectories measured at 0.5 M GdmCl. Shown are the References relative stabilities of the observed states, as well as free energy barriers corresponding to transitions between them. The amount 1. A. Mittal, B. Jayaram, S. Shenoy, and T. S. Bawa. J Biomol Struct Dyn 28, 133-142 of flux carried by each transition is represented by its line width. (2010). The figure represents transitions which carry at least 10% of the 2. B. W. Matthews. J Biomol Struct Dyn 28, 589-591 (2011). folding flux. 988 3. S. Rackovsky and H. Scheraga. J Biomol Struct Dyn 28, 593-594 (2011). 4. E. Shakhnovich. Chem Rev 106, 1559-1588 (2006). 5. J. H. Han, S. Batey, A. A. Nickson, S. A. Teichmann, and J. Clarke. Nat Rev Mol Cell Biol 8, 319-330 (2007). 6. V. Ratner, D. Amir, E. Kahana, and E. Haas. J Mol Biol 352, 683-699 (2005). 7. E. Boukobza, A. Sonnenfeld, and G. Haran. J Phys Chem B 105, 12165-12170 (2001). 8. E. Rhoades, E. Gussakovsky, and G. Haran. Proc Natl Acad Sci USA 100, 3197-3202 (2003). 9. L. R. A. Rabiner. Proc IEEE 77, 257-286 (1989).

Evidence for Initial Non-specific Polypeptide Chain Collapse During the Refolding of the SH3 16 Domain of PI3 Kinase Amrita Dasgupta Jayant B. Udgaonkar Proteins are evolutionarily selected heteropolymers, but their response to solvent change appears to be very similar to that of simple homopolymers (1, 2). The unfolded protein chains undergo global contraction when transferred from a good National Centre for Biological Sciences, to a bad solvent (3, 4). This ultrafast compaction of the unfolded state of a protein (collapse) channels it to the unique native structure by reducing its conformational Tata Institute of Fundamental Research, space. The collapse reaction of any polypeptide chain has been assumed to be GKVK Campus, Bellary Road, driven by non-specific hydrophobic interactions (3, 5). Alternatively, the driving Bangalore 560065, India force of the collapse reaction could also be the formation of backbone hydrogen bonds at low concentration of denaturant (6, 7). For some recent provocative dis- [email protected] cussion on protein folding, see Mittal et al. (8), Matthews (9) Scheraga (10) and [email protected] others published in the February 2011 issue of this Journal.

In the present study, the refolding of the PI3K SH3 from the guanidine hydro- chloride (GdnHCl)-unfolded state was probed with millisecond (stopped flow) and sub-millisecond (continuous flow) measurements of the change in tyrosine fluorescence, circular dichroism, ANS fluorescence and three-site fluorescence resonance energy transfer (FRET) efficiency (11). Previously, the refolding of this protein appeared two-state (12). Presently, our studies show that the folding of the protein commences via the rapid (complete within 150 μs) formation of a collapsed ensemble in a transition that is gradual and without any significant accumulation of secondary structure. All three intra-molecular distances collapsed to the same extent indicating that the compaction was synchronous as that expected for a coil to globule transition of a simple homopolymer as a consequence of solvent change (13). These results highlight the homopolymer nature of the unfolded polypeptide chain of the PI3K SH3. Furthermore to investigate the role of intra-chain hydrogen bonding in the collapse reaction, the folding was initiated by dilution of the urea- unfolded state (11). The extent of compaction seen for one of the intra-molecular distance was similar to that observed for the GdnHCl induced unfolded state.

To elucidate the importance of a non-specific collapsed ensemble in the subsequent structure formation of the PI3K SH3, an attempt was made to tune folding conditions such that a specific structured component of the collapsed ensemble could be preferentially populated. With the above objective, the effect of 500 mM sodium sulphate on the refolding of the PI3K SH3 was studied using multiple spectroscopic probes. Results indicate the formation of a specifically collapsed intermediate that has greater ANS binding than the previously reported non-specific ensemble (8). Two intra-molecular distances in this collapsed ensemble show greater contraction than just a solvent induced compaction, indicating the formation of a specific structured ensemble, before the rate limiting step of folding. Interestingly, one of the FRET pairs indicates the formation of native-like distance in the first millisecond of the refolding reaction. Abbreviations: FRET: Fluorescence resonance energy transfer; PI3K SH3: SH3 989 domain of PI3 kinase; ANS: 1-anilino-naphthalene-8-sulfonate.

References

1. P. G. de Gennes. J Phys Lett 46, L639-L642 (1985). 2. I. C. Sanchez. Macromolecule 12, 980-988 (1979). 3. V. R. Agashe, M. C. Shastry, and J. B. Udgaonkar. Nature 377, 754-757 (1995). 4. K. K. Sinha and J. B. Udgaonkar. J Mol Biol 353, 704-718 (2005). 5. K. A. Dill. Biochemistry 29, 7133-7155 (1990). 6. D. W. Bolen, G. D. Rose. Annu Rev Biochem 77, 339-362 (2008). 7. L. M. Holthauzen, J. Rosgen, and D. W. Bolen. Biochemistry 49, 1310-1318 (2010). 8. A. Mittal, B. Jayaram, S. Shenoy, and T. S. Bawa. J Biomol Struct Dyn 28, 133-142 (2010). 9. B. W. Matthews. J Biomol Struct Dyn 28, 589-591 (2011). 10. S. Rackovsky and H. Scheraga. J Biomol Struct Dyn 28, 593-594 (2011). 11. A. Dasgupta and J. B. Udgaonkar. J Mol Biol 403, 430-445 (2010). 12. J. I. Guijarro, C. J. Morton, K. W. Plaxco, I. D. Campbell, C. M. Dobson. J Mol Biol 276, 657-667 (1981). 13. C. Williams, F. Brochard, H. L. Frisch, Annu. Rev. Phys. Chem. 32, 433-451 (1981).

Simulations of the Folding Unfolding of Proteins Under Different Solvent Condition 17 Proteins exhibit marginal stability, determined by the balance of many competing 1,2 effects. This stability can be perturbed by changes in temperature, pH, pressure, and Deepak R. Canchi other solvent conditions. Osmolytes are small organic compounds that modulate the Camilo Jimenez-Cruz1,3 conformational equilibrium, folded (F) and unfolded (U), of proteins as cosolvents. Angel E. Garcia1,2,3* Protecting osmolytes such as trimethylamine N-oxide (TMAO), glycerol, and sugars that push the equilibrium toward F play a crucial role in maintaining the function of intracellular proteins in extreme environmental conditions. Urea is a denaturing 1 Center for Biotechnology and osmolyte that shifts the equilibrium toward U. Here we report the reversible fold- Interdisciplinary Studies ing/unfolding equilibrium, under various solution conditions that include urea, high 2 Department of Chemical and pressure, and different charge states of the Trp-cage miniprotein (1-4). The folding/ unfolding equilibrium is studied using all-atom Replica exchange MD simulations. Biological Engineering For urea, the simulations capture the experimentally observed linear dependence of 3 Department of Physics, unfolding free energy on urea concentration. We find that the denaturation is driven Applied Physics and Astronomy, by favorable direct interaction of urea with the protein through both electrostatic and van der Waals forces and quantify their contribution. Though the magnitude of Rensselaer Polytechnic Institute, direct electrostatic interaction of urea is larger than van der Waals, the difference Troy, NY 12180, USA between unfolded and folded ensembles is dominated by the van der Waals interac- *[email protected] tion. We also find that hydrogen bonding of urea to the peptide backbone does not play a dominant role in denaturation. The unfolded ensemble sampled depends on

Figure 1: Preferential binding of urea to the protein side chains over the backbone. The left figure shows the binding of urea around the Trp-cage protein. Red spheres represent urea molecules closer to the backbone atoms, while blue spheres represent molecules closer to the sidechains. The figure on the left shows the preferential interaction of urea to the side chains and backbone. 990 urea concentration, with greater urea concentration favoring conformations with greater solvent exposure. The m-value is predicted to increase with temperature and more strongly so with pressure (3, 4).

We also explored the effect of protonation of charged groups in protein and found that the unfolded state ensemble changes little as a function of protonation. However, charge-charge interactions in the folded state ensemble are responsible for the change in stability of the protein. Our results show how atomic level simulations with explicit solvent models can be used to characterize the stability of proteins.

This research has been supported by grants from NSF MCB-0543769 and MCB-1050966.

References

1. D. Paschek, S. Hempel, and A. E. Garcia. Procs Natl Acad Sci (USA) 105, 17754-17759 (2008). 2. R. Day, D. Paschek, and A. E. Garcia. Proteins 78, 1889-1899 (2010). 3. D. Canchi, D. Paschek, and A. E. Garcia. J Amer Chem Soc 132, 2238-2244 (2010). 4. D. Canchi and A. E. Garcia. Biophys J 100, (2011) doi:10.1016/j.bpj.2011.01.028.

Flexibility and Modulations in Protein-Protein Interactions: Mechanistic Insights from Molecular 18 Dynamics Simulations of MDM2 and P53 Shubhra Ghosh Dastidar The p53 protein provides a protection mechanism to cells against cancer (1). In normal cells p53 is complexed and down-regulated by another protein MDM2. In dam- Bose Institute, Kolkata 700054, India aged cells the process is interrupted by stress signals that activate p53’s network, [email protected] whose dysfunction is associated with cancers. Inhibiting over-expressed MDM2 to up-regulate p53’s function is a promising therapeutic strategy which will involve design of ligands to inhibit MDM2. In order to accomplish this one can employ modeling and molecular dynamics (2-5) approaches.

In this presentation, it will be shown how modeling and MD simulations have extracted the dynamics of interactions between MDM2-p53 in atomistic detail and have revealed the mechanisms that yield tight binding peptides to inhibit MDM2 (6). The simulations have attributed the flexibility and the conformational modulations to be key players which lead to the multiple mechanisms of binding associated with varying thermodynamic origins (6). Switching between electrostatics and van dar Waals components of interactions bring the uncomplexed MDM2 and p53 to each other to encounter, leading to transition to the complexed state (7, 8). Taking this system as an example, MD studies suggest that consideration of the detailed dynamics is essential to gain mechanistic insights about the plasticity of such systems and to yield improved strategies for ligand design. Atomistic insight into the effect of post-translational modifications of MDM2 on p53 regulations will also be discussed (9).

References

1. B. Vogelstein, D. Lane, and A. Levine. Nature 408, 307-310 (2000). 2. P. Sklenovský and M. Otyepka. J Biomol Struct Dyn 27, 521-539 (2010). 3. M. J. Aman, H. Karauzum, M. G. Bowden, and T. L. Nguyen. J Biomol Struct Dyn 28, 1-12 (2010). 4. C. Koshy, M. Parthiban, and R. Sowdhamini. J Biomol Struct Dyn 28, 71-83 (2010). 991 5. Y. Tao, Z. H. Rao, and S. Q. Liu. J Biomol Struct Dyn 28, 143-157 (2010). 6. S. G. Dastidar, D. P. Lane, and C. S. Verma. J Am Chem Soc 130, 13514-13515 (2008). 7. S. G. Dastidar, D. P. Lane, and C. S. Verma. BMC Bioinformatics 10 (Suppl 15):S6 (2009). 8. S. G. Dastidar et al. Theor Chem Acc 125, 621 (2010). 9. S. G. Dastidar et al. Cell Cycle (In Press).

Homology Modeling and Molecular Dynamics Simulations of RNA Polymerases 19 Although transcription is of central importance to cell survival, only few antimi- crobial agents have been directed towards the RNA polymerase (RNAP) enzyme. Ivan Barvik* Rifampicin, one of the most potent and broad spectrum antibiotics and a key com- Vlastimil Zima ponent of anti-tuberculosis therapy, binds in a pocket of the RNAP deep within the Kamil Malac DNA/RNA channel, but more than 12 Å away from the active site. Unfortunately, binding of Rifampicin can be easily disturbed by enzyme mutations. Therefore, we are interested in blocking of active sites of bacterial RNAPs directly using analogs Charles University, of NTPs. Such approach was found as very potent in the case of viral infections. Faculty of Mathematics and Physics, Here, we present results of homology modeling (using the MODELLER software Institute of Physics, package), ab initio and QMMM calculations (GAUSSIAN03, ONIOM) and classical molecular dynamics simulations (AMBER and NAMD software packages). Ke Karlovu 5, RNAPs in complex with nucleic acids (template DNA strand, RNA transcript, Prague 2, 121 16, Czech Republic, NTPs – either natural or chemically modified) were investigate in detail. *[email protected] Support from the Ministry of Education, Youth and Sports of the Czech Republic (Project No. MSM 0021620835 and Project No. NPVII 2B06065) is gratefully acknowledged.

Method of Molecular Dynamics for Proteins in the Ionization-Conformation Phase Space at Equilibrium Conditions at Constant pH 20 Y. N. Vorobjev In view of the extensive interest in molecular dynamics simulations (1-4), a new realization of the constant-pH molecular dynamics simulation method (5) is proposed. Molecular dynamics simulation is performed in the potential of mean force Institute of Chemical Biology and of protein molecule in the water-proton bath at equilibrium titration conditions Fundamental Medicine, (MD-pH-ET). It is shown that: i) the algorithm of MD-pH-ET which delivers the Siberian Division, Russian Academy highest numerical accuracy of the MD-pH-ET method, is the simulation of protein in its instant most probable physical ionization microstate with additional potential of Sciences, Novosibirsk, 630090, Russia of mean force of equilibrium titration, which depends on a deviation of the most [email protected] probable physical ionization microstate from an equilibrium ensemble of ionization microstates for a given protein conformation; ii) the new method MD-pH-ET allows one to carry out the optimization of protein structure and the total free energy of a protein in the aqueous solution at constant pH, and the calculation of the pH-dependent properties. Method MD-pH-ET possesses unique features such as: i) it uses precise and computationally effective realization of calculation of ionization equilibrium and electrostatic energy and atomic forces for protein in water solution by the model of continuous dielectric media with Poisson equation solved by the Fast Adaptive Multigrid Boundary Element (FAMBE) method (6). Coupling of FAMBE method with Generalized Born method by definition of the Poisson “ideal” Born atomic radii allows one to accelerate an accurate calculation of forces and energies in the MD-pH-ET method; ii) it uses the same model of the potential energy surface in the ionization-conformational phase space, both for the calculation 992 of the potential energy of the protein and atomic forces and for determining the ionization states; iii) it calculates the total free energy of the protein in the aqueous solution in proton reservoir under the conditions of equilibrium titration. The work- ability of the new method MD-pH-ET is demonstrated on a set of proteins.

This research has been supported by grant of Russian Fond of Basic Research #09-04-00136 and by projects #09-26 and # 09-119 of the Siberian Brunch of the RAS.

References

1. P. Sklenovský and M. Otyepka. J Biomol Struct Dyn 27, 521-539 (2010). 2. M. J. Aman, H. Karauzum, M. G. Bowden, and T. L. Nguyen. J Biomol Struct Dyn 28, 1-12 (2010). 3. C. Koshy, M. Parthiban, and R. Sowdhamini. J Biomol Struct Dyn 28, 71-83 (2010). 4. Y. Tao, Z. H. Rao, and S. Q. Liu. J Biomol Struct Dyn 28, 143-157 (2010). 5. S. L.Williams, C. A. F. Oliveira, and J. A. McCammon. J Chem Theory Comput 6, 560-568 (2010). 6. Y. N. Vorobjev, J. A. Vila, and H. A. Scheraga. J Phys Chem 112, 11122-11136 (2008). 7. A. Onufriev, D. A. Case, and D. Bashford. J Comput Chem 23, 1297-1304 (2002).

Molecular Dynamics Simulations on Structural 21 Heterogeneity States of Protein Well-defined structure proteins can turn into all kinds of structural heterogeneity Jihua Wang states such as intermediate state, transition state or unfolded state under differ- Liling Zhao ent circumstance. Another type of protein, namely intrinsically disordered protein Zanxia Cao (IDPs), is lacking independently folded states at physiological condition. These states have highly structural heterogeneity (1) and they play central roles in a range of important biological process. It is a formidable challenge to reveal structural Key Lab of Biophysics in Shandong character of the flexible and conformatically highly heterogeneous states. Some (Dezhou University), effective sampling methods such as replica exchange molecular dynamics (REMD) and restraint molecular dynamics simulation (restraint MD) are used to study the Dezhou, China, 253023 disorder states. Here, we gave two cases to study IDPs and protein intermediate [email protected] state by REMD and restraint MD.

Case 1: Human α-synuclein protein, a presynaptic protein of 140 amino acid residues, is the major component of Lewy bodies (LBs) deposited in the brains of patients with Parkinson’s disease, and it usually has extensive intrinsically disordered regions (IDRs). The N-terminal 12 residues peptide of the α-synuclein (α-syn12) was choosen. The structural and thermodynamics character of α-syn12 peptide in aqueous solution have been investigated by temperature replica exchange molecular dynamics (T-REMD) simulations. The structural and thermodynamic characters of α-syn12 peptide at different pH and temperatures have also been studied by temperature replica exchange molecular dynamics (T-REMD) simulations (2).

Case 2: The other case is to study the thermal intermediate state of high mobility group (HMG) box 5 of human upstream binding factor using the restraint MD simulations. The thermal intermediate state of high mobility group (HMG) box 5 of human upstream binding factor was detected at 55ºC by experimental technique (3), but could not be defined for the sparse data. We performed ensemble-averaged MD simulations to study the intermediate state ensemble of HMG box-5 at 328K with forty-eight replicas for 2.88 ms. 421 inter-atomic distances derived from NOE and PRE were used as restraints. The results indicated that the intermediate state ensemble presented the structural characteristic of box-5 protein intermediate state and were consistent with experimental results. The two cases show that molecular dynamics simulation is a powerful route to 993 study structural and thermodynamics characters of the conformational heterogeneous states of protein.

Financial support from the Chinese Natural Science Foundation and Shandong Natural Science Foundation (30970561and 31000324) are acknowledged.

References

1. M. Vendruscolo. Current Opinion in Structural Biology 17, 15-20 (2007). 2. Z. Cao, L. Liu, and J. Wang. Journal of Biomolecular Structure and Dynamics 28, 343-353 (2010). 3. Z. Liu, J. Zhang, X. Wang, Y. Ding, J. Wu, and Y. Shi. Proteins: Structure, Function and Bioinformatics 77, 432-447 (2009).

Swarm Intelligence: Cooperative Replica Methods for Prediction of Protein Structure 22 The use of atomistic simulation techniques to directly resolve protein tertiary structure from primary amino acid sequence is hindered by the rough topology of Neil J. Bruce the protein free energy surface and the resulting simulation timescales required. Richard A. Bryce* An interesting approach to improve the exploration of this surface, termed the SWARM-MD method, was proposed by Huber and van Gunsteren (1). Their School of Pharmacy and approach is inspired by particle swarm methods. Introduced in 1995 and originally applied to electrical circuit design, the particle swarm algorithm mimics the Pharmaceutical Science, social behaviour of bird flocking or fish schooling. The efficiency of thealgo- University of Manchester, rithm’s search behaviour, which exhibits remarkable organization and planning, Manchester, M13 9PT, UK can be traced to cooperativity of individual members of the swarm, influenced by their memory and that of their peers. In SWARM-MD, the free energy surface is *[email protected] smoothed through the use of a swarm of multiple interacting simulation replicas that are driven towards the average conformation of the swarm members. We have successfully applied this approach to prediction of the native states of a series of model peptides (2), including Trp-cage miniprotein in aqueous solvent. In each case, the cooperation between replicas was found to improve the convergence of the simulations towards the native state. Limitations and future directions of this method will be discussed.

This research has been supported by the EPSRC.

References

1. T. Huber and W. F. van Gunsteren. J Phys Chem A 102, 5937-5943 (1998). 2. N. J. Bruce and R. A. Bryce. J Chem Theory Comput 6, 1925-1930 (2010).

Thermodynamics of Waters Sequestered Inside Macromolecules 23 The talk will discuss application of concepts developed while working in the Beveridge Laboratory. Grand-canonical ensemble simulations (1, 2) were shown to Mihaly Mezei be well suited for the exploration of solvent occupancy of isolated pockets inside Department of Structural and macromolecules (3). From the ensemble of configurations thus generated, the con- Chemical Biology, cept of generic sites (4) serves to identify distinct water sites inside proteins. The Mount Sinai School of Medicine, thermodynamics if different waters will be characterized both using the technique developed in the Lazaridis Laboratory (5, 6) and by comparison of occupancies NY 10029, New York, from simulations run at different chemical potentials. The work is done in collabo- [email protected] ration with Roman Osman. 994 References 1. D. J. Adams. Molec Phys 29, 307-311 (1975). 2. M. Mezei. Mol Phys 61, 565-582 (1987); Erratum, 67, 1207-1208 (1989). 3. H. Resat and M. Mezei. J Am Chem Soc 116, 7451-7452 (1994). 4. M. Mezei and D. L. Beveridge. J Comp Chem 6, 523-527 (1984). 5. Z. Li and T. Lazaridis. J Phys Chem B 109, 662-670 (2005). 6. Z. Li and T. Lazaridis. J Phys Chem B 110, 1464-1475 (2006).

Role of Disulfide Bonds in Structural Stability and Flexibility of Cuticle-degrading Proteases from 24 Nematophagous Fungi—A Molecular Dynamics Lian-Ming Liang1 Simulation Study Ke-Qin Zhang1 Shu-Qun Liu1,2* It has been shown that disulfide bonds play an important role in the stability of some proteins by an entropic effect (1), usually the globular proteins secreted to extracellular medium (2). Two cuticle-degrading proteases, Ver112 and PII, which were 1 Laboratory for Conservation and derived respectively from nematode-parasitic and nematode-trapping fungi, belong Utilization of Bio-Resources & Key to the subtilisin family sharing relatively high sequence identity (45.7%). Ver112 is an alkaline protease and has two disulfide bonds, C35-C124 and C179-C250 (3); Laboratory for Microbial Resources of PII is a neutral protease and has no disulfide bond. Despite the minor structural the Ministry of Education, Yunnan difference between them (root mean square deviation (RMSD) is ~0.6 Å), Ver112 University, Kunming 650091, displays higher thermal stability and stronger nematicidal/catalytic activity than PII does (4). P. R. China 2 Sino-Dutch Biomedial and Information In order to investigate how the disulfide bonds influence structural stability Engineering School, Northeastern and flexibility of these two proteases, molecular dynamics simulations on their structures of wild-type and disulfide bond-disrupted mutant (Ver112_124C/A, University, Shenyang 110003, Ver112_179C/A, and Ver112_124C/A_179C/A) were performed at temperatures P. R. China 300 K and 400 K, respectively. Analyses of the geometrical properties along the 300 K MD trajectories indicate that PII has higher average values of C RMSD and *[email protected] α solvent accessible surface area (SASA) while lower average values of number of native hydrogen bonds (NNH) and number of native contacts (NNC), suggesting a higher flexibility and less compact equilibrium structure of PII in comparison with Ver112. This may be caused by the lack of equivalent disulfide bonds in PII. The geometrical properties of Ver112 are similar on average to those of its three mutants during simulations at 300 K, while at 400 K the wild-type Ver112 presents more NHB and NNC and less SASA than its mutants suggesting that disulfide bonds contribute to the global stability of Ver112 at high temperature. Additionally, the stability of local structures within 5 Å of the two disulfide bonds C35-C124 and C179-C250 was also enhanced, as indicated by their increased RMSF and decreased NNC values upon disulfide bond breaking. Analyses of the average RMSF values of the S1 and S4 substrate-binding pockets show that upon disruption of C35-C124, RMSF of S1 pocket decreased by 21.2% while that of S4 pocket showed almost no change; upon disruption of C179-C250, the relatively large reduction in flexibility of both S1 and S4 pockets was observed; and the most pronounced reduction (30.7% and 17.2%) occurred when both disulfide bonds were broken. According to these results, we can conclude that i) the presence of disulfide bonds enhances not only the local but also the global stability of the protease, thus explaining the higher thermal stability of the alkaline protease Ver112 compared to that of the neutral protease PII; ii) the presence of disulfide bonds increases the flexibility of substrate-binding pockets located relatively far from disulfide bonds, thus explaining why alkaline proteases have higher substrate affinity (5, 6) and catalytic activity than neutral proteases. This research was supported by grants from NSFC (No. 30860011) and Yunnan 995 province (2007PY-22), and foundation for Key Teacher of Yunnan University.

References

1. M. Matsumura, G. Signor, and B. W. Matthews. Nature 342, 291-293 (1989). 2. C. S. Sevier, and C. A. Kaiser. Nat Rev Mol Cell Biol 3, 836-847 (2002). 3. L. M. Liang, Z. Y. Lou, F. P. Ye, J. K. Yang, S. Q. Liu, Y. N. Sun, Y. Guo, Q. L. Mi, X. W. Huang, C. G. Zou, Z. H. Meng, Z. H. Rao, and K. Q. Zhang. FASEB J 24, 1391-1400 (2010). 4. L. M. Liang, S. Q. Liu, J. K. Yang, Z. H. Meng, and K. Q. Zhang. FASEB J (2011) (in press). 5. S. Q. Liu, Z. H. Meng, Y. X. Fu, and K. Q. Zhang. J Mol Model 17, 289-300 (2011). 6. Y. Tao, Z. H. Rao, and S. Q. Liu. J Biomol Struct Dyn 28, 143-157 (2010).

Thinking into Mechanism of Protein Folding and Molecular Binding 25 Protein folding and molecular binding provide the basis for life on earth. The native 1,2 3D structure of a protein is a prerequisite for its function; and the molecular binding Xing-Lai Ji is the fundamental principle of all biological processes (1). Therefore unraveling Shu-Qun Liu1,2* the mechanisms of protein folding and binding is fundamental to describing life at molecular level. Of particular interest is that protein folding and binding are similar 1Laboratory for Conservation and processes because the only difference between them is the presence and absence of the chain connectivity. Among many models (such as diffusion-collision (2), Utilization of Bio-Resources & Key hydrophobic collapse (3) and stoichiometry (4) models) proposed to describe the Laboratory for Microbial Resources mechanism of these two processes, the “folding funnel” (5) model (Figure 1) is of the Ministry of Education, most widely accepted. In this model, protein folding can be viewed as going down the free energy hill through multiple parallel pathways towards the bottom of the Yunnan University, Kunming 650091, funnel (6); and molecular binding can occur along rough free energy surface around P. R. China the funnel bottom, especially for binding between flexible proteins/molecules. 2 Sino-Dutch Biomedial and Information These are essentially thermodynamically controlled processes involving various types of driving forces, including the enthalpic contribution of noncovalent bond Engineering School, Northeastern formations, entropic effects such as solvent release and burial of apolar surface University, Shenyang 110003, area (hydrophobic effect), restrictions of degrees of freedom of protein/ligand, and P. R. China loss of rotational and translational freedom of interacting partners. Briefly, these two processes, which are driven by a decrease in total Gibbs free energy (ΔG), are *[email protected] dictated by the mechanism of a delicate balance of the opposing effects of enthalpic (ΔH) and entropic (ΔS) contributions (equation 1).

DG 5 DH2TDS 1

Here we emphasize that it is the thermodynamically driven subtle enthalpy- entropy compensation that leads to the global free energy minimum of the protein/ligand-solvent system (7), and that the specific inter-atomic interactions observed in the folded or complexed structure are to large extent the consequence of thermodynamic equilibrium but can not fully define the driving forces for folding and binding interactions.

Interestingly, we speculate that many other processes can be explained by thermodynamic enthalpy-entropy compensation, i.e., the Yin and Yang balance in traditional Chinese medicine theory could correspond to the enthalpy and entropy compensation of the second law of thermodynamics; global warming can be considered as the consequence of excessive production of positive entropy (carbon dioxide) from chemically ordered fossil fuel, urg- ing people to slow resource consumption to delay the inevitable death by Figure 1: Schematic 2D funnel of protein folding and entropy. binding (modified from (6)). 996 A deeper understanding of mechanism of biological processes from thermodynamic point of view can facilitate greatly the understanding of life and rational drug design in the post-genomic times.

This research was supported by grants from NSFC (No. 30860011) and Yunnan province (2007PY-22), and foundation for Key Teacher of Yunnan University and SRF for ROCS, SEM.

References

1. R. Perozzo, G. Folkers, and L. Scapozza. J Recept Signal Transduct Res 24, 1-52 (2004). 2. M. Karplus and D. L. Weaver. Protein Sci 3, 650-668 (1994). 3. V. R. Agashe, M. C. Shastry, and J. B. Udgaonkar. Nature 377, 754-757 (1995). 4. A. Mittal, B. Jayaram, S. Shenoy, and T. S. Bawa. J Biomol Struct Dyn 28, 133-142 (2010). 5. P. E. Leopold, M. Montal, and J. N. Onuchic. Proc Natl Acad Sci USA 89, 8721-8725 (1992). 6. J. M. Yon. J Cell Mol Med 6, 307-327 (2002). 7. X. L. Ji and S. Q. Liu. J Biomol Struct Dyn 28, 621-623 (2011).

Molecular Motions of Proteins Play Crucial Role in 26 their Function 1,2 Proteins, which are the materials central to cellular function, should not be regarded Shu-Qun Liu * simply as static pictures as determined by X-ray crystallography. They are dynamic Shi-Xi Liu3 entities in cellular solution with functions governed ultimately by their dynamic Zhao-Hui Meng1,4 character (1). Therefore a complete understanding of the structure-function rela- 1 tionship of a protein requires an analysis of its dynamic behavior and molecular Yan Tao motion. Ke-Qin Zhang1 Yun-Xin Fu1,5 Using molecular dynamics (MD) simulation or CONCOORD (2) approach, the dynamic behaviors of HIV-1 gp120 envelope glycoprotein and serine protease proteinase K were investigated. Apart from analyses of the conventional structural 1 Laboratory for Conservation and properties during simulations, the essential dynamics analysis method was used to Utilization of Bio-Resources & Key study the large concerted motions of these two proteins, including the influence of ligand bindings or residue mutations on molecular motions. The results revealed Laboratory for Microbial Resources of that i) the proteinase K shows relatively rigid internal core with some highly flex- the Ministry of Education, Yunnan ible surface loops forming the substrate-binding region, supporting the induce-fit University, Kunming 650091, P. R. China or conformational selection mechanism of substrate binding (3); ii) the removal of Ca2+ cations from proteinase K increases the global conformational flexibility, 2 Sino-Dutch Biomedial and Information decreases the local flexibility of substrate-binding region and does not influence the Engineering School, Northeastern thermal motion of catalytic triad, thus explaining the experimentally determined University, Shenyang 110003, decreased thermal stability, reduced substrate affinity and almost unchanged catalytic activity upon Ca2+ removal (4); iii) the substrate binding affects the large P. R. China concerted motions and flexibility behavior of proteinase K suggesting that the vari- 3 School of Chemical Science and ations in substrate-pocket motions can be connected to substrate binding, cataly- Technology, Yunnan University, sis and product release (5); amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on molecular motions of gp120 (6), facilitating 375 S/W Kunming 650091, P. R. China mutant to adopt the CD4-bound conformation while 423 I/P mutant to prefer for 4 Department of Cardiology, No. 1 CD4-unliganded state (7). Analyzing the dynamic character of proteins not only is Affiliated Hospital, Kunming Medical important for the characterization of the functional properties of proteins but also facilitates the reasonable interpretation of experimentally determined structural, College, Kunming 650032, P. R. China biochemical and biological data. 5 Human Genetics Center, The University of Texas Health Science Center, Houston, This research was supported by grants from NSFC (No. 30860011) and Yunnan province (2007PY-22), and foundation for Key Teacher of Yunnan University. TX 77030, USA *[email protected] References 997 1. K. Henzler-Wildman and D. Kern. Nature 450, 964-972 (2007). 2. B. L. de Groot, D. M. F. van Aalten, R. M. Scheek, A. Amadei, G. Vriend, and H. J. C. Berendsen. Proteins 29, 240-251 (1997). 3. S. Q. Liu, Z. H. Meng, Y. X. Fu, and K. Q. Zhang. J Mol Model 16, 17-28 (2010). 4. S. Q. Liu, Z. H. Meng, Y. X. Fu, and K. Q. Zhang. J Mol Model 17, 289-300 (2011). 5. Y. Tao, Z. H. Rao, and S. Q. Liu. J Biomol Struct Dyn 28, 143-157 (2010). 6. S. Q. Liu, S. X. Liu, and Y. X. Fu. J Mol Model 14, 857-870 (2008). 7. S. Q. Liu, C. Q. Liu, and Y. X. Fu. J Mol Graphics Modell 26, 306-318 (2007).

Origins of the Mechanical Stability of the C2 Domains in Human Synaptotagmin 1 27 Synaptotagmin 1 (Syt1) induces the buckling of plasma membrane during neu- 1 rotransmitter release at the synapse (1). Therefore, elucidating the mechanical Li Duan * properties of Syt1 is essential for understanding its biological function in synaptic Artem Zhmurov2 response. Syt1 contains two homologous cytoplasmic domains, C2A and C2B. We Valeri Barsegov2 employed a self-organized polymer (SOP) model of a protein chain (2) to carry out 1 molecular simulations, implemented on a CPU and on a GPU (Graphics Processing Ruxandra I. Dima Unit) (3), using experimental pulling speeds. The forced unfolding of isolated C2A and C2B domains occurs under comparable forces starting from their C-terminal 1 Department of Chemistry, ends, but according to different pathways. Our results for the behavior of the C2A University of Cincinnati, domain correlate very well with dynamic spectroscopy experimental studies (4, 5), but no direct measurements of the mechanical behavior of the isolated C2B domain OH 45221, Cincinnati, exist to date. Thus, to confirm the presence of the pathways generated with the 2Department of Chemistry, SOP model, we also carried out implicit solvent model simulations. Atomic force University of Massachusetts, microscopy (AFM) experiments found an increase in the critical unfolding force of C2B when joined with C2A in the Syt1 molecule (4), which was proposed to Lowell MA 01854, Lowell, result from the contribution of the C2A-C2B interface. However, our simulations *[email protected] reveal that the presence of an intact interface does not lead to the unfolding of Syt1 according to the AFM experiments. In contrast, we discovered that the presence of linkers used in the experimental set-up plays a crucial role in the behavior of this synaptic protein complex and, their inclusion in simulations as well leads to data that fully matches the experiments. Interestingly, we found that the stabilization effect of the linker on the C2B domain alters not only the critical force, but also the unfolding pathways of both C2 domains. Our findings provide insights into the relative conformation variability of the C2 domains and the origins of stability of the Syt1 protein. 998 References 1. S. Martens, M. M. Kozlov, and H. T. McMahon. Science 316, 1205-1208 (2007). 2. C. Hyeon, R. I. Dima, and D. Thirumalai. Structure 14, 1633-1645 (2006). 3. A. Zhmurov, R. I. Dima, Y. Kholodov, and V. Barsegov. Proteins 78, 2984-2999 (2010). 4. K. L. Fuson, L. Ma, R. B. Sutton, and A. F. Oberhauser. Biophys J 96, 1083-1090 (2009). 5. M. Carrion-Vazquez, P. E. Marszalek, A. F. Oberhauser, and J. M. Fernandez. PNAS 96, 11288-11292 (1999).

Universality of the Spatial Distribution of the Backbones and a Narrow Band of Amino Acid 28 Stoichiometries Amidst the Structural and Functional B. Jayaram Diversity of Folded Proteins A. Mittal Is there a universal principle guiding protein folding besides the thermodynamic hypothesis of Anfinsen? Rigorous analyses of several thousand crystal structures of Department of Chemistry & School of folded proteins reveal a surprisingly simple unifying principle of backbone organi- Biological Sciences, zation (1, 2). We find that protein folding is a direct consequence of a narrow band Indian Institute of Technology, of stoichiometric occurrences of amino-acids in primary sequences, regardless of the size and the fold of the protein. Furthermore, the amino acid residues in folded Hauz Khas, New Delhi-110016, India proteins display novel and invariant neighborhoods, independent of their locations [email protected] (e.g. center vs. periphery) in contrast to anticipations from preferential interaction [email protected] theories (3). These findings present a compelling case for a newer view of protein folding which takes into account solvent mediated and amino acid shape and size assisted optimization of the tertiary structure of the polypeptide chain to make a functional protein.

References

1. A. Mittal, B. Jayaram, S. R. Shenoy, and T. S. Bawa. J Biomol Struct Dyn 28, 133-142 (2010). 2. A. Mittal and B. Jayaram. J Biomol Struct Dyn 28, 669-674 (2011) and references therein. 3. A. Mittal and B. Jayaram. J Biomol Struct Dyn 28, 443-454 (2011). Largescale Protein Properties: Crystallization, 999 Amyloid Formation, and the Stability of the Proteome

We are interested in largescale protein behaviors. By largescale, we mean processes such as amyloid aggregation or protein crystallization that involve multiple proteins associating into complexes, or collective properties of the thousands of 29 proteins in whole proteomes. There are many experimentally measurable properties Ken A Dill1* for which some basic insights do not require large computer simulations of atomi- 2 cally detailed models. Kings Ghosh Jeremy Schmit3 Protein crystallization. Why do proteins crystallize? Under typical crystallization conditions, two protein molecules have charges of the same sign, so they should 1 repel. So, the usual explanation for protein crystallization is that proteins also have Stony Brook University a neutral sticking energy that overcomes the charge-charge repulsion. However, 2Denver University more is needed in order to account for the strong observed dependence of crystal 3University of California at San Francisco stability on salt, which is of practical importance for crystallizing proteins. We treat the crystallization of proteins, such as lysozyme, as an association of charged *[email protected] spheres in the presence of salts (1). The model is in good agreement with lysozyme crystal solubility data as functions of temperature, pH, and salt. A key conclusion is that because the crystal is macroscopic, it must be electroneutral, so: (a) increasing the protein’s charge has little effect on its crystal stability because counterions are sequestered in proportion to the charge, and (b) the reason that crystals melt upon heating is because it melts the counterion `glue’, not because it melts the protein- protein interactions.

Amyloid aggregation. What are the forces of amyloid aggregation? We consider 3 states in equilibrium: monomeric amyloid peptide molecules, oligomers (micelle- like loose clusters of a few peptide chains), and fibrils (ordered fibers of many chains). We suppose the chains are driven by hydrophobic interactions into the oligomeric state and by additional interactions due to steric zipping (packing plus hydrogen bonding) into the fibrillar state (2). The model predicts two transitions, monomer to oligomer, and oligomer to fibril, the latter of which, interestingly, is predicted to be essentially independent of peptide concentration. The model predicts that if the oligomers are the toxic species, then the fibrils are ‘good guys’, because they soak up oligomeric chains and buffer their concentration. The model resolves an experimental puzzle from two research groups regarding denaturant disruption. Slightly different peptide concentrations lead to either stable oligomeric intermediates, or none. Model predictions are in good agreement with dependences on salt and pH.

Proteome stability. We compute the stabilities of all the proteins in various proteomes, including Ecoli, worm and yeast based on a simple thermodynamic param- eterization of the database of known protein stabilities (3). We find that proteomes are marginally stable. That is, even though thousands of proteins have a stability averaging around 6 kcal/mol, about 650 of Ecoli’s proteins are less stable than 4 kcal/mol. Stability distributions such as these are useful for computing the temperature at which the whole proteome denatures, which coincides closely with the experimental temperatures of cell death.

References

1. 1. J. D. Schmit and K. A. Dill. J Phys Chem B 114, 4020-4027 (2010). 2. 2. J. Schmit, K. Ghosh, and K. A. Dill. Bio phys J 100, 450-458 (2011). 3. 3. K. Ghosh and K. A. Dill. Bio phys J 99, 3996-4002 (2010). 1000 Virtual Screening and Theoretical Activity Prediction of Idenopyrazole Derivatives of CDK2 Inhibitors: A QPLD and MM-GBSA approach

Cyclin-dependent kinases (CDKs) are core components of the cell cycle machinery 30 that govern the transition between phases during cell cycle progression. Genes Sanjeev Kumar Singh* involved in cell cycle are frequently mutated in human cancer and deregulated CDK activity represents a characteristic of malignancy. Among them CDK2 is essential Sunil Kumar Tripathi in the mammalian cell cycle and is required to complete G1 and to activate the S phase (1). Lately there have been successful implementation of computer-aided drug design to develop new therapeutics (2-5), and we have employed these designs Department of Bioinformatics, to develop anticancer drugs targetting CDK2. In this study we have taken 119 com- Alagappa University, pounds of idenopyrazole derivatives, with in vitro biological activity data for CDK2 Karaikudi, Tamil Nadu, 630003, India inhibition (6). Here, we used Virtual screening protocol to obtain effective idenopyrazole derivatives of CDK2 via VS workflow of Schrodinger package, ADME and *[email protected] Lipinski- filter options. 17 top-ranked molecules obtained from this screening were subjected for Molecular docking with GLIDE module and Quantam Polarized Ligand Docking (QPLD) using QPLD module of Schrodinger package (7, 8).

The top-ranked 17 compounds were post-scored using molecular mechanics and continuum solvation (MM-GBSA) (9, 10). The validity of the virtual screening protocol was supported by (i) Testing of the MM-GBSA procedure (ii) Agreement between predicted and crystallographic binding poses (iii) Recovery and identification of top-scoring potent idenopyrazole derivatives. With these combined approach of ADME, Docking, QPLD and MM-GBSA, we found that compound 13, 24, 25 and 46 having properties essential for effective drugs so biological activity testing can be carried out on selected leads, for getting effective and potent anticancer drug.

Figure: Compound 48 showing interaction with Glu12, Asn132 and Asp145 of CDK2. References 1001 1. M. Malumbres and M. Barbacid. Nat Rev Cancer 9(3), 153-166 (2009). 2. C. Y. Chen. J Biomol Struct Dyn 27, 627-640 (2010). 3. C. Y. Chen, Y. H. Chang, D. T. Bau, H. J. Huang, F. J. Tsai, C. H. Tsai, and C. Y. C. Chen. J Biomol Struct Dyn 27, 171-178 (2009). 4. H. J. Huang, K. J. Lee, H. W. Yu, C. Y. Chen, C. H. Hsu, H. Y. Chen, F. J. Tsai, and C. Y. C. Chen. J Biomol Struct Dyn 28, 23-37 (2010). 5. H. J. Huang, K. J. Lee, H. W. Yu, H. Y. Chen, F. J. Tsai, and C. Y. C. Chen. J Biomol Struct Dyn 28, 187-200 (2010). 6. S. K. Singh, N. Dessalew, P. V. Bharatam. Eur J Med Chem 41(11), 1310-1319 (2006). 7. R. A. Friesner, J. L. Banks, R. B. Murphy, T. A. Halgren, J. J. Klicic, D. T. Mainz, M. P. Repasky, E. H. Knoll, M. Shelley, J. K. Perry, D. E. Shaw, P. Francis, P. S. Shenkin. J Med Chem 47, 1739-1749 (2004). 8. A. E. Cho, V. Guallar, B. J. Berne, R. Friesner. J Comput Chem 26, 915-931 (2005). 9. P. D. Lyne, M. L. Lamb, J. C. Saeh. J Med Chem 49, 4805-4808 (2006). 10. G. Barreiro, C. R. Guimarães, I. Tubert-Brohman, T. M. Lyons, J. Tirado-Rives, W. L. Jorgensen. J Chem Inf Model 47, 2416-2428 (2007).

Homologous and Heterologous Crystallin Interactions in Cataract 31 Age-related cataract is the most common cause of blindness worldwide. Nearly fifty percent of Americans above the age of 75 are diagnosed with this disease, Priya R. Banerjee and surgical intervention is the sole method of treatment at present. In the devel- Jayanti Pande* oping world, even this treatment is not readily available. These are compelling reasons to search for better treatments to delay, prevent or arrest cataract for- Department of Chemistry, mation. Recent evidence suggests that age-related cataracts also have a genetic component (1). Therefore, determining the mechanisms underlying genetic cata- University at Albany, racts with a known association to a protein-mutation is one important strategy Albany, NY 12222 towards understanding the molecular basis for cataract formation. This approach [email protected] has the added advantage of addressing mechanisms of congenital and childhood cataracts which are difficult to treat because surgical intervention frequently *[email protected] leads to serious consequences (2). For these reasons, we have been determining the molecular mechanisms underlying a number of genetic cataracts by studying the mutant proteins associated with them.

The most common human cataract-associated mutations occur in γD-crystallin (HGD). In the singly-substituted HGD mutants we have studied so far, we find that the protein structure and stability do not change, despite a significant lowering in protein solubility in most cases, which leads to the formation of a condensed phase and consequent light scattering and opacity. One example is the Pro23 to Thr (P23T) mutation in HGD in which the mutant protein shows a dramatically lowered solubility, which is about 1/100th of that of the normal protein. Moreover, the solubility profile of P23T is “retrograde”  i.e. it increases as the temperature decreases. For this mutant we have shown that hydrophobic surface patches emerge as a result of the mutation, which are largely responsible for its retrograde solubility and aggregation (3). Recently, we have identified the residues, Tyr16, His22, Asp21, and Tyr50, in the P23T mutant that give rise to novel hydrophobic surface, as well as several residues where backbone fluctuations in different time-scales are restricted, providing a comprehensive understanding of how lens opacity could result from this mutation. For P23T and several other mutants, we have shown that changes in the homologous protein-protein interactions (or self-association) are responsible for the formation of the distinct condensed phase, which in turn is likely to lead to light scattering in the cataractous lens. 1002 In an interesting recent development, we found that changes in homologous interactions leading to protein condensation and light scattering may not be the only mechanism leading to cataract (4). In our study of the Glu107 to Ala (E107A) mutant, we found that not only is the mutant protein very similar in structure and stability to the normal protein  as in all the previous cases we studied  but it is also as soluble as the normal protein (5). In fact, the E107A mutant does not form a condensed protein phase that could account for light scattering and cataract. However, mixtures of the mutant with another lens crystallin, namely α−crystallin, show increased light scattering compared to normal α−γ−crystallin mixtures. There is also a striking difference in the liquid-liquid phase separation behaviors: The two coexisting phases in the E107A−α mixtures differ much more in protein density than those that occur in HGD−α mixtures. In HGD−α mixtures, the de-mixing of phases occurs primarily by protein type while in E107A−α mixtures it is increasingly governed by protein density. Therefore, here it is clearly the heterologous attractive interactions between two different crystallins in the lens that lead to increased light scattering. It has been known for some time that the attractive interactions between various crystallins are optimized for stability, and any change  attractive or repulsive  is likely to lead to instability (5). Our data (5) support these theoretical predictions and emphasize the importance of examining detailed molecular mechanisms to build a comprehensive understanding of a complex disease. A brief commentary on our work is presented in (6).

References

1. C. J. Hammond, H. Snieder, T. D. Spector, and C. E. Gilbert. N Engl J Med 342, 1786-1790 (2000). 2. C. Zetterstrom, A. Lundvall, and M. Kugelberg. J Cataract Refract Surg 31, 824-840 (2005). 3. A. Pande, K. S. Ghosh, P. R. Banerjee, and J. Pande. Biochemistry 49, 6122-6129 (2010). 4. P. R. Banerjee, A. Pande, J. Patrosz, G. M. Thurston, and J. Pande. Proc Natl Acad Sci USA 108, 574-579 (2010). 5. A. Stradner, G. Foffi, N. Dorsaz, G. Thurston, and P. Schurtenberger. Phys Rev Lett 99, 198103 (2007). 6. N. Asherie. Proc Natl Acad Sci USA 108, 437-438 (2010).

Improving in Digestion and Antioxidant Activity of b-lactoglobulin Using Newly Designed Copper 32 Complexes as Artificial Proteases Adeleh Divsalar1* Sajedeh Ebrahim-Damavandi2 Beta-lactoglobulin (BLG) is a major protein of whey; it is a major carrier protein and is known to interact with Pd(II) anti-tumor compounds (1). It has wide appli- 2 Ali Akbar Saboury cation as a food ingredient (2-3). Stable structure of bovine whey proteins, espe- Hassan Mansouri- Torshizi3 cially BLG, restricts their hydrolysis by proteases and it leads to allergy reactions (4). Now-a-days, the design of synthetic metallo-proteases that cleave proteins at a specific site has elicited much interest (5). Hence, in the present investigation, 1Department of Biological Sciences, we have decided to design and synthesize a new class of cooper(II) complexes

Tarbiat Moallem University, Tehran, Iran ({Cu(bpy)Cl2}, {Cu(bpy)2}Cl2, {Cu(dien)OH2}(NO3)2 and {Cu(trien)}(NO3)2) as artificial protease in order to hydrolyze resistant BLG. We also examine the anti- 2Institute of Biochemistry and oxidant activity of fragmented BLG using SDS-PAGE and different spectropho- Biophysics, University of Tehran, tometric methods (Fluorescence and UV-Visible). Incidentally copper complexes Tehran, Iran are very important in biological systems; they have been used to locate nucleosome positioning (6), they occur widely in oxidative proteins such as laccases (7), and 3Department of Chemistry, University of participate in many many cellular processes and are implicated in pathogenesis of Sistan & Baluchestan, Zahedan, Iran many diseases (8). *[email protected] SDS -PAGE of BLG incubated with different Cu(II) complexes for 30 h repre- 1003 sented fragmentation of the protein. Also, increasing fluorescamine intensity measurements prove hydrolysis or fragmentation of protein in the presence of different Cu(II) complexes. SDS-PAGE and fluorescamin studies show that complex 3 has higher protease activity against BLG. The antioxidant activities of native and hydrolyzed BLG were determined by an ABTS°+ radical cation assay. The degree of decolorization of ABTS radical induced by native and hydrolyzed BLG resulting from different Cu(II) complexes were compared to that induced by Trolox. Results have represented that hydrolyzed BLG using complexes 2 and 3 exhibit significantly greater antioxidant activity than the other hydrolyzed or native BLG, probably signifying the greater number of solvent-exposed amino acids available for scavenging of the free radicals.

From above results, it can be concluded that our new designed Cu(II) complexes have artificial protease activities against model protein of BLG. Also, hydrolysing of BLG can enhance its fragmentation by proteases and increase its antioxidant activity.

References

1. A. Divsalar, A. A. Saboury, H. Mansoori-Torshizi, M. I. Moghaddam, F. Ahmad, G. H. Hakimelahi. J Biomol Struct Dyn 26, 587-597 (2009). 2. S. C. Cheison, M. Schmitt, L. Elena, T. Letzel, and U. Kulozik. Food Chemistry 121, 457- 467 (2010). 3. L. Tavel, C. Moreau, S. Bouhalla, and E. Li-Chan. Food Chemistry 119, 1550-1556 (2010). 4. M. Besler, P. Eigenmann, and R. H. Schwartz. Food Allergens 4, 199-106 (2002). 5. M. S. Kim and J. Suh. Bull Korean Chem Soc 26, 1911-1919 (2005). 6. A. Travers, E. Hiriart, M. Churcher, M. Caserta, and E. Di Mauro. J Biomol Struct Dyn 27, 713-724 (2010). 7. M. T. Cambria, D. Di Marino, M. Falconi, S. Garavaglia, and A. Cambria. J Biomol Struct Dyn 27, 501-509 (2010). 8. H. F. Ji and H. Y. Zhang. J Biomol Struct Dyn 26, 197-201 (2008).

SANJEEVINI-A Lead Molecule Design Software

The ability of macromolecules to bind to their substrates in a highly specific manner is an important feature in many biological processes. The challenge for computer 33 aided drug discovery is to achieve this specificity - with small molecule inhibitors - Goutam Mukherjee in binding to their biomolecular targets, at reduced cost and time while ensuring Tanya Singh synthesizability, novelty of the scaffolds and proper ADMET profiles. Sanjeevini is a drug design software suite (1-7) developed to provide a computational pathway B. Jayaram* for automating lead molecule design. A sample of the various current approaches to drug design can be found in many of the recent articles published in this Jour- Department of Chemistry and nal, for example references 8-11. Our methodology treats macromolecular target and the lead compound at the atomic level and solvent as a dielectric continuum. Supercomputing Facility for It comprises several modules for diverse functionalities such as automated identi- Bioinformatics and Computational fication of potential binding sites (active sites) for the ligands (5), a rapid screen- Biology, Indian Institute of Technology, ing for identifying good candidates for any target protein from a million molecule database (6), optimization of their geometries and determination of partial atomic Hauz Khas, New Delhi-110016, India charges using quantum chemical / in-house methods (7), docking the candidates *[email protected] in the active site of target via Monte Carlo methods (4-5), estimating binding free energies through empirical scoring functions (1-2), followed by rigorous analyses of the structure and energetics of binding for further lead optimization. Each module is individually validated on a large data set of protein-ligand and DNA-ligand complexes with known structures and binding affinities. TheSanjeevini software is freely accessible at http://www.scfbio-iitd.res.in/sanjeevini/sanjeevini.jsp. 1004 References 1. T. Jain and B. Jayaram. FEBS Letters 579, 6659-6666 (2005). 2. S. A. Shaikh and B. Jayaram. J Med Chem 50, 2240-2244 (2007). 3. S. A. Shaikh, T. Jain, G. Sandhu, N. Latha, and B. Jayaram. Current Pharmaceutical Design 13, 3454-3470 (2007). 4. A. Gupta, A. Gandhimathi, P. Sharma, and B. Jayaram. Protein and Peptide Letters 14, 632-646 (2007). 5. T. Singh, D. Biswas, and B. Jayaram. “An Automated Active Site Identifier”, Manuscript in preparation. 6. G. Mukherjee and B. Jayaram. “A Rapid Identification of Hit Molecules for Target Proteins via Physico-Chemical Descriptors”, Manuscript in preparation. 7. G. Mukherjee, N. Patra, P. Barua, and B. Jayaram. Journal of Computational Chemistry, 32, 893-907 (2011). 8. T. T. Chang, H. J. Huang, K. J. Lee, H. W. Yu, H. Y. Chen, F. J. Tsai, M. F. Sun, and C. Y. C. Chen. J Biomol Struct Dyn 28, 309-321 (2010). 9. A. K. Kahlon, S. Roy, and A. Sharma. J Biomol Struct Dyn 28, 201-210 (2010). 10. T. C. Ramalho, M. V. J. Rocha, E. F. F. da Cunha, L. C. A. Oliveira, and K. T. G. Carvalho. J Biomol Struct Dyn 28, 227-238 (2010). 11. L. I. D. S. Hage-Melim, C. H. T. D. P. Da Silva, E. P. Semighini, C. A. Taft, and S. V. Sampaio. J Biomol Struct Dyn 27, 27-35 (2009). Inferred Biomolecular Interaction Server–A Method 1005 and a Server to Analyze and Predict Protein Interacting Partners and Binding Sites Recently we have developed a new database and method called “IBIS” (Inferred 34 Biomolecular Interaction Server, http://www.ncbi.nlm.nih.gov/Structure/ibis/ibis. cgi) which enables to conveniently study biomolecular interactions that have been Benjamin A. Shoemaker observed in protein structures. Moreover, through inference by homology IBIS Dachuan Zhang allows to formulate predictions/hypotheses for biomolecular interactions, even if Ratna R. Thangudu the data for specific biomolecules is not available. Similar binding sites are clustered together based on their sequence and structure conservation. To emphasize Manoj Tyagi biologically relevant binding sites, several algorithms are used for verification in Jessica H. Fong terms of evolutionary conservation, biological importance of binding partners, size Aron Marchler-Bauer and stability of interfaces, as well as evidence from the published literature. IBIS organizes, analyzes and predicts interaction partners and locations of binding sites Stephen H. Bryant in proteins for five different types of binding partners (protein, chemical, nucleic Thomas Madej acid, peptides and ions), and facilitates the mapping of a comprehensive biomo- Anna R. Panchenko* lecular interaction network for a given protein query. The method was validated by comparison to other binding site prediction methods and to a collection of manu- ally curated annotations. It has been shown that it achieves a sensitivity of about National Center for Biotechnology 72-75% at predicting biologically relevant sites for protein-small molecule and Information, National Institutes of protein-protein interactions. Health, Bethesda, MD 20894, USA *[email protected] 1006 This research has been supported by the National Institutes of Health/DHHS (Intra- mural Research program of the National Library of Medicine).

References

1. Shoemaker, et al. Nucleic Acids Res Jan;38:D518-24 (2010). 2. Thangudu, et al. BMC Bioinformatics Jul 1;11:365 (2010).

Integrating CADD Methodologies for the Design of 35 Novel COMT Inhibitors Catechol-O-methyltransferase (COMT) catalyzes the methylation of cate- Nidhi Jatana cholamines, including neurotransmitters like dopamine, epinephrine and norepi- Aditya Sharma nephrine, leading to their degradation. COMT has been a subject of study as it N. Latha* has implications in numerous neurological disorders like Parkinson’s disease (PD), depression, schizophrenia and several mood disorders (1).

Bioinformatics Infrastructure Facility, Availability of crystal structure of human soluble-COMT (S-COMT) (2) has helped Sri Venkateswara College (University of us in modeling of membrane-bound COMT (MB-COMT) and thus paved the way for the discovery of a novel lead molecule using integrated computer aided drug Delhi), Benito Juarez Road, design (CADD) methodologies (3-8). Combination of both structure-based (molec- Dhaula Kuan, New Delhi 110021, India ular docking, de novo ligand design) and ligand-based (QSAR, pharmacophore *[email protected] modeling) approaches that models separate facets of the natural system, will allow us to use all available information to screen a chemical database in a more objective and meaningful way.

In this study, we examine the structure of MB-COMT refined by molecular dynamics simulations. We have also conducted detailed computational modeling studies to understand the molecular mechanisms for the catalytic behavior of human MB-COMT with respect to AdoMet (the methyl donor) and various substrates. We have also carried out docking studies with known COMT inhibitors to (5, 9) understand physico-chemical interactions in the protein-ligand complex in order to identify the optimal binding geometry. The virtual screening procedure has been implemented in a docking pipeline that performs a step-by-step, target specific, filtering approach for data reduction to discover inhibitors with novel scaffolds. We hope that the combination of energy terms from structure-based docking studies with the chemical knowledge of a ligand-based pharmacophore search will lever- age the strengths of both approaches to produce a good diversity of active molecules. Results of these studies will be discussed and presented.

References

1. P. T. Mannisto and S. Kaakkola. Pharmacol Rev 51, 593-628 (1999). 2. K. Rutherford, I. L. Trong, R. E. Stenkamp, and W. W. Parson. J Mol Biol 380, 120-130 (2008). 3. P. G. Mezey. J Mol Model 6, 150-157 (2000). 4. S. Y. Yang. Drug Discov Today 15, 444-50 (2010). 5. H. C. Guldberg and C. A. Marsden. Pharmacol Rev 27, 135-206 (1975). 6. A. Kaur Kahlon, S. Roy, and A. Sharma. J Biomol Struct Dyn 28, 201-210 (2010). 7. H. J. Huang, K. J. Lee, H. W. Yu, C. Y. Chen, C. H. Hsu, H. Y. Chen, F. J. Tsai, and C. Y. C. Chen. J Biomol Struct Dyn 28, 23-37 (2010). 8. T. T. Chang, H. J. Huang, K. J. Lee, H. W. Yu, H. Y. Chen, F. J. Tsai, M. F. Sun, and C. Y. C. Chen. J Biomol Struct Dyn 26, 309-321 (2009). 9. M. J. Bonifacio, P. N. Palma, L. Almeida, and P. S. da Silva. CNS Drug Rev 13, 352-379 (2007). Liquid State NMR Spectroscopy as a Tool to Obtain 1007 Structural and Binding Information of Drug Molecules Metabolized by P450BM3 Enzymes Cytochrome P450 is a heme protein family ubiquitous in all domains of life and 36 involved in many biological processes, including the degradation and activation of 1 drugs by catalyzing the monooxygenation of a ligand (1). Cytochrome P450BM3 A. J. Kolkman * from Bacillus megaterium is one of the most promising monoxygenases for bio- V. Rea2 technological applications because it is the most active P450 so far identified. J. Draaisma1 Mutated forms of P450BM3 are highly efficient biocatalysts to metabolize steroids 1 and other drugs in-vitro in order to produce potential new lead compounds (2). In M. Tessari this contribution, liquid state NMR spectroscopy is shown to be a valuable tool to E. V. Vottero2 provide a structural basis for drug metabolism by P450BM3 enzymes. First, the K. A. M. Ampt1 regioselective hydroxylation of steroids by P450BM3 enzymes will be discussed. 2 Secondly, a new methodology for acquiring homonuclear decoupled proton NMR J. N. M. Commandeur spectra of drug metabolites will be discussed. H. Irth3 N. P. E. Vermeulen2 We show that by a single mutation (A82W) in the substrate binding pocket of 4 P450BM3 mutants, the regioselective hydroxylation of steroids at position 16-ß is M. Honing largely increased. Moreover, this enhanced regioselective hydroxylation is further S. S. Wijmenga1 investigated by means of binding affinity, as studied by UV-VIS spectroscopy, and the orientation of the steroid testosterone in the heme-active sites of these mutants, as studied by T1 paramagnetic relaxation NMR. It is shown that testosterone has 1Department of Biophysical Chemistry, an increased binding affinity and preferred orientation in the A82W mutant when Radboud University Nijmegen compared to its wild-type counterpart. 2LACDR division of Molecular NMR spectroscopy is not only a very powerful tool to provide information on Toxicology, VU University Amsterdam ligand binding, but its main power is to provide structural information on large mol- 3LACDR division of Biomolecular ecules, like RNA’s, DNA’s and enzymes, and small molecules. The determination Analysis, VU University Amsterdam of structures of small molecules, i.e. metabolites which are formed by P450BM3 enzymes, by liquid state NMR is a standardized but complicated procedure (2). 4MSD Research Institute, Small molecules often have a dense network of J-coupled protons, which hampers Medicinal Chemistry Oss the assignment of NMR spectra, due to broad multiplets and subsequent spectral *[email protected] overlap. We will show a novel methodology to acquire homonuclear decoupled proton NMR spectra with a high resolution (~1 Hz) and higher sensitivity than currently reported for these types of NMR experiments.

References

1. A. W. Munro and D. G. Leys, et al. Trends Biochem Sci 27, 250-257 (2002). 2. J. S. B. de Vlieger and A. J. Kolkman, et al. J Chromatogr B 878, 667-674 (2010).

Structure-function Investigation of Metalloproteinases Provides Novel Insights into Drug Design 37 Matrix metalloproteinases (MMPs) and disintegrin metalloproteinases (ADAMs) are endopeptidases central to the degradation and remodeling of the extracellular Netta Sela-Passwell matrix (ECM). These proteases also exhibit regulatory activity in cell signaling path- Alla Trahtenhercts ways and thus tissue homeostasis under normal conditions and in many diseases (1). Irit Sagi* Consequently, individual members of the MMP and ADAM protein families were identified as important therapeutic targets. However, designing effective inhibitors in vivo for this class of enzymes appears to be extremely challenging. This is attributed Department of Biological Regulation, to the broad structural similarity of their active sites and apparently to the dynamic The Weizmann Institute of Science, functional interconnectivity of MMPs with other proteases, their inhibitors, and sub- Rehovot, 76100, Israel strates (the so-called degradome) in healthy and disease tissues, demonstrated to be detrimental to the therapeutic rationale (2-3). We present recent advancements in *[email protected] 1008 our understanding of MMPs structures, dynamics and their function as master regulators (4-7). We highlight the use of structural-kinetic experimental approach for protein-based drug design strategies, e.g. antibodies and protein scaffolds, targeting extracatalytic domains, which are central to proteolytic and non-proteolytic enzyme functions (3). Such rationally designed function blocking inhibitors may create new opportunities in disease management and in emerging therapies that require control of dysregulated MMP activity without causing severe side-effects.

References

1. N. Sela-Passwell, G. Rosenblum, T. Shoham, and I. Sagi. Biochim Biophys Acta 1803, 29-38 (2010). 2. I. Sagi and M. Milla. Anal Biochem 372, 1-10 (2008). 3. N. Sela-Passwell, A. Trahtenhercts, A. Krüger, and I. Sagi. Expert opinion on Drug Discovery (2011) In Press. 4. A. Solomon, B. Akabayov, M. Milla, and I. Sagi. PNAS U S A 104, 4931-4936 (2007). 5. G. Rosenblum, P. Van den Steen, S. R. Cohen, G. J. Grossmann, A. Frenkel, R. Sertchook, N. Slack, R. W. Strange, G. Opdenakker, and I. Sagi. Structure 10, 1227-36 (2007). 6. G. Rosenblum, P. Van den Steen, S. Cohen, A. Bitler, A. D. D. Brand, G. Opdenakker, and I. Sagi. PLos One 5, 9-15 (2010). 7. M. Grossman, D. Tworowski, M. Lee, Y. Levy, G. Murphy, and I. Sagi. Biochemistry 49, 6184-6192 (2010).

Conserved Water Mediated H-bonding Dynamics of Carboxamide group in NAD to Catalytic Asp 274 and 38 His 93 in Human IMPDH Hridoy R Bairagya* The Inosine Monophosphate Dehydrogenase (hIMPDH)-II of human is led to Bishnu P Mukhopadhyay** new interest as an excellent target for the design of isoform specific anti leuke- Payel Mallik mic agent (1, 2). The stereodynamics and recognition of mono and di nucleotide 1 binding pockets (3) through one conserved water molecular center (4) and the par- Archana K. Srivastava ticipation of catalytic Arg 322 through a conserved water molecular triad (5) seem to be important concerning to water mimic drug design. Conserved water mol- Department of Chemistry, National ecules are also played a significant role in conformational transition or flexibility of “loop” and “flap” regions in unliganded form of enzyme, and was also used for anti Institute of Technology–Durgapur, leukemic drug design (6). Involvement of Asp–Gly–Gly–Ile–Gln (S1: 364–368) W.B., 713 209, India and Gly–Ser–Leu (S2: 387–389) sequences in the recognition of inhibitor CPR *[email protected] (6-Chloropurine Riboside 50-Monophosphate) through conserved water molecules have also indicated the rationality for modified CPR ligand (7). Attempt has been **[email protected] employed to investigate the role of water molecules in the recognition of NAD or its structural analogs to conserved catalytic D 274 of hIMPDH, which may be useful for IMPDH inhibitor design.

Extensive MD-Simulation studies of solvated x-ray structures of hIMPDH (1B3O, 1NFB, 1NF7 and 1JCN) and their complexes with NAD or its analogs have revealed the presence of conserved water mediated interaction of those ligand to carboxylate center of catalytic Asp 274 in the different isoforms of enzyme (Figure 1). In the unliganded IMPDH, one conserved water molecule (W1) forms H- bond to D 274 (OD2) and H 93. However, during the complexation of NAD or its analogs with IMPDH, that W1 retains its position and two other water molecules (W2 and WC) are also observed to form H-bond with the carboxyl group of Asp 274. Position of these three water molecules (WC, W1 and W2) are also be conserved during MD- simulation. All the conserved water molecules have stabilized through H-bonds and occupied the three corners of a distorted trigonal pyramid with carboxyl oxy-

gen (OD2) at apex (Figure 2). The water molecule WC interacts to carboxamide 1009

Figure 1: Presence of conserved water molecules ( W1, W2,WC ) and water mediated recognition of NAD to D 274 and H93 in the MD-simulated structure of 1B3O, 1NFB, 1NF7 and 1JCN. *MYD( NAD analog inhibitor ): C2 mycophenolic adenine dinucleotide

group of nucleotide or its analogs. The recognition geometry of carboxamide NN7 of NAD or analogs to carboxyl group of Asp 274 through conserved water (WC) mediated interaction and their stereochemical features may be used for IMPDH inhibitor design. Possibly, the chemical signals of cofactor binding region (NAD or its structural analogs) could be transmitted toremote part of protein (Figure 3) through Base (NAD NN7) ----- Water (WC) ----- Acid (D 274 OD2) ----- Base (H93 NE2) H-bonding interaction. 1010

Figure 2: The transition of unliganded IMPDH to NAD –bound IMPDH structure in human.*DN= Di-nucleotide Ligand (NAD/structural analogs)

Figure 3: Conserved water mediated H-bonding interaction and the chemical signaling (Base --- Water --- Acid ---) of the NAD binding region in hIMPDH

References

1. V. Nair and Q. Shu. Antivir Chem Chemother 18 , 245-258 (2007). 2. T. D. Colby, K.Vanderveen, M. D. Stricker, G. D. Markham, and B. M. Goldstein. Proc Nat Acad Sci - Biochemistry 96, 3531-3536 (1999). 3. C. Branden and J. Tooze. Introduction to Protein Structure, Garland Publishing, New York and London (1991). 4. H. R. Bairagya, B. P. Mukhopadhyay, and K. Sekar. J Biomol Struct Dyn 26, 497-508 (2009). 5. H. R. Bairagya, B. P. Mukhopadhyay, and K. Sekar. J Biomol Struct Dyn 27, 149-158 (2009). 6. H. R. Bairagya, B. P. Mukhopadhyay, and Asim K Bera. Journal of Molecular Recognition 24, 35-44 (2011). 7. H. R. Bairagya, B. P. Mukhopadhyay, and S. Bhattacharjee. Journal of Molecular Structure: THEOCHEM 908, 31-39 (2009). Structure and Topology of Phospholamban Monomer 1011 and Pentamer by a Hybrid Solution and Solid-State NMR Method

Phospholamban (PLN) is an integral membrane protein that regulates calcium 2+ 39 homeostasis by inhibiting sarcoplasmic reticulum (SR) Ca -ATPase (SERCA) 1,2 in cardiac muscle. PLN exists in two primary oligomeric forms: (1) a monomer Nathaniel J. Traaseth that directly binds to and inhibits SERCA (1, 2) and (2) a pentamer that indirectly Raffaello Verardi1 influences SERCA activity by regulating the pentamer-monomer equilibrium Lei Shi1 (1, 2). To completely describe the fold space and ultimately the biological function 1 of PLN and other membrane proteins, it is necessary to determine the membrane Gianluigi Veglia protein’s structure and the specific interactions with the lipid bilayer (i.e., topology). In this work, we describe a new hybrid method to calculate the structures of 1Departments of Chemistry and the PLN monomer and pentamer using a combination of solution and solid-state NMR restraints in detergent micelles and lipid bilayers, respectively (3, 4). These Biochemistry, Molecular Biology & high-resolution images of the PLN not only describe the structures, but also the Biophysics, topologies of all the protein domains with respect to the lipid bilayer. University of Minnesota, Minneapolis, To minimize the structure/topology of the monomer and pentamer, we implemented MN 55445 our hybrid objective function into XPLOR-NIH software (5) that utilizes geometri- 2Department of Chemistry, New York cal (E ) and solution (E ) and solid-state NMR (E ) restraints: chem sol-NMR ssNMR University, New York, NY 10003

Etotal 5 Echem 1 Esol–NMR 1 EssNMR

Figure 1: Structure of PLN monomer from Traaseth et al. (Ref. 3). 1012 We obtained short-range distance and angular restraints from solution NMR of PLN reconstituted into DPC detergent micelles and orientation restraints (anisotropic chemical shifts and dipolar couplings) from 2D separated local field experiments such as PISEMA (6, 7) in mechanically aligned lipid bilayers. The final stage of refinement was to incorporate explicit lipids around the protein structure and carry out minimization to reveal the interactions between the lipid and protein domains. In our structures, we find that the N-terminal helical domain Ia (residues 1-16) of monomer and pentamer rests on the surface of the lipid bilayer with the hydrophobic face of domain Ia embedded in the bilayer interior. The helix comprised of domain Ib (residues 23-30) and transmembrane domain II (residues 31-52) tra- verses the bilayer with tilt angles of ~11° (pentamer) and ~24° (monomer; structure in Figure 1). Hybrid methods such as the one presented in this work will be necessary to tackle challenging biophysical problems such as membrane protein structure determination.

References

1. Y. Kimura, K. Kurzydlowski, M. Tada, and D. H. Maclennan. J Biol Chem 272, 15061-15064 (1997). 2. L. G. Reddy, L. R. Jones, and D. D. Thomas. Biochem 38, 3954-3962 (1999). 3. N. J. Traaseth, L. Shi, R. Verardi, D. M. Mullen, G. Barany, and G. Veglia. Proc Natl Acad Sci 106, 10165-10170 (2009). 4. L. Shi, N. J. Traaseth, R. Verardi, A. Cembran, J. Gao, and G. Veglia. J Biomolec NMR 44, 195-205 (2009). 5. C. D. Schwieters, J. J. Kuszewski, N. Tjandra, and G. M. Clore. J Magn Reson 160, 65-73 (2003). 6. C. H. Wu, A. Ramamoorthy, and S. J. Opella. J Magn Reson 109, 270-272 (1994). 7. T. Gopinath and G. Veglia. J Am Chem Soc 131, 5754-5756 (2009).

The Solution and Binding Behavior of the Intrinsically 40 Disordered FG Nups Determined by in cell NMR 1 The nuclear pore complex (NPC) mediates all transport between the nucleus and Loren E. Hough cytoplasm. The channel of the NPC is lined with “FG Nups”, a family of intrin- Kaushik Dutta2 sically disordered proteins characterized by phenylalanine-glycine repeat motifs. Jaclyn Tetenbaum-Novatt1 FG nups form the exquisitely selective filter of the NPC; nonbinding proteins are 1 excluded while the binding of transport factors to the FG nups facilitates their pas- Michael Rout sage through the NPC. Like other intrinsically disordered proteins, the FG nups David Cowburn3* appear to be very sensitive to their environment, showing vastly different behavior in different experimental conditions; in vitro, the observed behavior of the FG varies from rigid gels to flexible random coil polymers. We used stint-NMR to 1Laboratory of Cellular and Structural protein behavior of a model FG not within the living cellular environment. In a Biology, Rockefeller University stint-NMR experiment, NMR observations are directed performed on bacteria, 2New York Structural Biology Center in vivo co expressing an isotopically labeled protein and an unlabeled binding partner. We found that the solution state of the FG nups within living cells is disordered, 3Dept. of Biochemistry while NMR spectra significantly change in vitro buffers, presumably from numer- Albert Einstein College of Medicine ous intra- or inter-molecular contacts. Moreover, the binding interface between of Yeshiva University transport factors in the FG nups differs considerably between solution and cellular conditions. Thus a key determinant to FG nups behavior is the local environment. Bronx, NY 10461 *[email protected] These results indicate that the proper behavior of the FG naps is dependent on the 1013 normal cellular milieu, and is not necessarily represented in vitro; our findings have important implications for the various current models regarding the molecular mechanisms of nuclear cytoplasmic transport and for behavior are weak cellular interactions generally.

Supported by A Charles Revson postdoctoral fellowship, NIH grans GM067854, GM071329 (MR), GM66354 (DC).

A Database/Webserver for Size-Independent Quantification of Ligand Binding Site Burial Depth in Receptor Proteins: Implicationson Protein Dynamics 41 Srujana Cheguri We have developed a database/webserver that implements the two complemen- Vicente M. Reyes* tary methods to quantify the degree of burial of ligand and/or ligand binding site (LBS) in a protein-ligand complex, namely, the ‘secant plane’ (SP) and the ‘tangent sphere’ (TS) methods, which we reported earlier (1). To recapitulate the SP Biological Sciences Dept., Sch. of and TSmethods: the protein molecular centroid (global centroid, GC), and the LBS Medical & Biological Sciences, centroid (local centroid, LC) are first determined. The SP is defined as the plane passing through the LBS centroid (LC) and normal to the line passing through the College of Science, Rochester Institute LC and the protein molecular centroid (GC). The “exterior side” of the SP is the of Technology, side opposite GC. The TS is defined as the sphere with center at GC and tangent to Rochester, NY 14623-5603 the SP at LC. The percentage of protein atoms (a.) inside the TS (a.k.a. ‘TS index’) and (b.) on the exterior side of the SP (a.k.a. ‘SP index’), are two complementary *[email protected] measures of ligand or LBS burial depth since the latter is directly proportional to (b.) and inversely proportional to (a.). We tested the SP and TS methods using a test set of 67 well characterized protein-ligand structures (2), as well as the theoretical case of an artificial protein in the form of a cubic lattice grid of points in the overall shape of a sphere and in which LBS of any depth can be specified. Results from both the SP and TS methods agree very well with reported data (2), and results from the theoretical case further confirm that both methods are suitable measures of ligand or LBS burial. There are two modes by which one can utilize our database/ webserver. In the first mode we term the ‘ligand mode’, the user inputs the PDB structure coordinates of the protein as well as those of its ligand(one ligand at a time if more than one). The second mode - the ‘LBS mode’-is the same as the first except that the ligand coordinates are assumed to be unavailable, hence the user inputs what s/he believes to be the LBS amino acid residues’ coordinates. In both cases, the webserver outputs the SP and TS indices. LBS burial depth is an important parameter as it is usually directly related to the amount of conformational change a protein undergoes upon ligand binding, and ability to quantify it could allow meaningful comparison of protein flexibility and dynamics. The URL of our database/webserver will be http://tortellini.bioinformatics.rit.edu/sxc6274/thesis1. phpand will be made freely available to the community very soon.

References

1. V. M. Reyes. J Biomol Struct & Dyn 26, 875-875 (2009). 2. R. A. Laskowski, N. M. Luscombe, M. B. Swindells, and J. M. Thornton. Protein Sci 5, 2438-2452 (1996). 1014 Using Cylindrical Coordinates to Represent Rod-Shaped and Other Fibrous Protein 3D Structures: Potential Advantages and Applications 42 Based on overall 3D structure, proteins may be grouped into two broad, general categories, namely, globular proteins or ‘spheroproteins’, and elongated or ‘fibrous Srujana Cheguri proteins’, and the former comprises the significant majority. Our research group Vicente M. Reyes* is trying to use alternative representations for proteins structures, and has made progress on representing spheroproteins using spherical coordinates (ρ,φ,θ) (1). This work concerns the second general category of protein structures, namely, the Biological Sciences Dept., Sch. of fibrous or rod-shaped class of proteins. Unlike a spheroprotein, a rod-shaped pro- Medical & Biological Sciences, tein (RSP) possesses a visibly conspicuous axis along its longest dimension. To take advantage of this potential symmetry element, we decided to represent RSPs using College of Science, Rochester Institute cylindrical coordinates, (ρ,θ,z), with the z-axis as the main axis and one ‘tip’ of the of Technology, protein at the origin, with ‘tip’ being defined as one of two points lying along the Rochester, NY 14623-5603 protein axis and defining its longest dimension. To do this, we first visually identify the two tips T and T of the protein using appropriate graphics software, then *[email protected] 1 2 determine their Cartesian coordinates, (h,k,l) and (m,n,o), respectively. Arbitrarily

selecting T1 as the tip to coincide with the origin, we translate the protein by sub- tracting (h,k,l) from all structural coordinates. We then find the angleα (in degrees)

between vectors T1T2 and the positive z-axis by computing the scalar product of

vectors T1T2 and OP where P is an arbitrary point along the positive z-axis, which is typically (0,0,p), where p is the approximate length of the rod-shaped protein under investigation. Then we compute the cross product of the two vectors to determine

the axis about which we should rotate vector T1T2 so it coincides with the positive z-axis.We then use a matrix form of Rodrigues’ formula to perform the actual rotation. Finally we apply the Cartesian to cylindrical coordinate transformation equations to the system. Thus far, we have applied the above transformation to 15 rod-shaped proteins, prominent among which are 1DXX, 2KOL,2KZG, 3LHP and 3MQC. We have also created a database/webserver that can take in the PDB coordinate file of a rod-shaped protein and output its cylindrical coordinates based on the transformation steps described above. We shall implement this process in both all-atom and reduced protein representations (2). The URL will be http://tortellini. bioinformatics.rit.edu/sxc6274/thesis2.php and it will be made freely available to the community very soon.

References

1. V. M. Reyes. Interdiscipl Sci: Comp Life Sci (2011, in press). 2. V. M. Reyes and V. N. Sheth. In: Handbook of Research in Computational and Systems Biology: Interdisciplinary Approaches, L. A. Liu, D. Wei, and Y. Qing (Eds.), Chap. 26 (2011, in press). Towards a Spherical Coordinate System Metric for 1015 Quantitative Comparison of Protein 3D Structures

Although observed protein structures generally represent energetically favorable conformations that may or may not be “functional”, it is also generally agreed that protein structure is closely related to protein function. Given a collection of proteins 43 sharing a common global structure, variations in their local structures at specific, James DeFelice critical locations may result in different biological functions. Structural relationships among proteins are important in the study of the evolution of proteins as well Vicente M. Reyes* as in drug design and development. Biological Sciences Dept., Sch. of Analysis of geometrical 3D protein structure has been shown to be effective with respect to classifying proteins. Prior work has shown that the double-centroid Medical & Biological Sciences reduced representation (DCRR) model (1) is a useful geometric representation for College of Science, Rochester Institute protein structure with respect to visual models, reducing the quantity of modeled of Technology information for each amino acid, yet retaining the most important geometrical and chemical features of each: the centroids of the backbone and of the side-chain. Rochester, NY 14613 Thus far, DCRR has not yet been applied in the calculation of geometric structural *[email protected] similarity.

Meanwhile, multi-dimensional indexing (MDI) of protein structure combines protein structural analysis with distance metrics to facilitate structural similarity queries and is also used for clustering protein structures into related groups. In this respect, the combination of geometric models with MDI has been shown to be effective.

Prior work, notably Distance and Density-based Protein Indexing (DDPIn) (2), applies MDI to protein models based on the geometry of the C backbone. DDPIn’s distance metrics are based on radial and density functions that incorporate spherical-based metrics, and the indices are built from metric tree (M-tree; 3) structures.

This work combines DCRR with DDPIn for the development of new DCRR centroid-based metrics: spherical binning (4) distance and inter-centroid spherical distance. The use of DCRR models will provide additional significant structural information via the inclusion of side-chain centroids. Additionally, the newly developed distance metric functions combined with DCRR and M-tree indexing should improve upon the performance of prior work (DDPIn), given the same data set (5), with respect to both individual k-nearest neighbor search queries as well as clustering all proteins in the index.

References

1. V. M. Reyes and V. N. Sheth. In: Handbook of Research in Computational and Systems Biology: Interdisciplinary Approaches, L. A. Liu, D. Wei and Y. Qing (Eds.), Chap. 26 (2011, in press). 2. D. Hoksza. Proc 6th Ann IEEE Conf Comp Intel Bioinf Comp Biol CIBCB’09, 263-270 (2009). 3. P. Ciaccia, M. Patella, and P. Zezula. Proc 23rd Intl Conf Very Large Data Bases VLDB’97, 426-435 (1997). 4. V. M. Reyes. Interdiscipl Sci: Comp Life Sci (2011, in press). 5. O. Çamoglu, T. Kahveci, and A. K. Singh. Proc IEEE Comp Soc Conf Bioinf CSB’03, 148-158 (2003). 1016 Cancer Meets the “Omics”: A Comprehensive Cancer Biotherapy Database with Links to Multiple Bioinformatics Websites/WebServers – Facilitating 44 the Search for Anticancer Biological Agents PreetyPriya ‘Cancer biotherapy’ – as opposed to ‘cancer chemotherapy’ - is the use of macromolecular biological agents instead of small organic chemicals or ‘drugs’ to treat Vicente M. Reyes* cancer (1, 2). Although there are several important differences between cancer biotherapy and cancer chemotherapy, it suffices to saythat due to the much higher Biological Sciences Dept., selectivity of biological agents than chemical agents for cancer cells over normal cells, there is much less toxic side effects in biotherapy as compared to chemother- Sch. of Medical & Biological Sciences, apy, and as a result, patient survival is usually dramatically enhanced.We have built College of Science, the foundations of acomprehensive cancer biotherapy database for use as a life- Rochester Institute of Technology, saving resource by cancer patients, and as a sounding board for scientific ideas by cancer researchers. The database/webserver will have information about 12 main Rochester, NY 14623-5603 families of cancer biotherapy regimens to date, namely, 1.) Protein Kinase Inhibi- *[email protected] tors, 2.) Ras Pathway Inhibitors, 3.) Cell-Cycle Active Agents, 4.) MAbs (mono- clonal antibodies), 5.) ADEPT (Antibody-Directed Enzyme Pro-Drug Therapy), 6.) Cytokines (interferons, interleukins, TNF, etc.), 7.) Anti-Angiogeneis Agents, 8.) Cancer Vaccines (peptides, proteins,DNA), 9.) Cell-based Immunotherapeutics, 10.) Gene Therapy, 11.) Hematopoietic Growth Factors, and 12.) Retinoids. For each biotherapy regimen, we will extract the following attributes in populating the database: (a.) cancer type, (b.) gene/s and gene product/s involved, (c.) gene sequence (GenBank ID), (d.) organ/s affected, (e.) available chemo treatment, (f.) reference papers, (g.) clinical phase/stage, (h.) survival rate (chemo. vs. biother.), (i.) clinical test center locations, (j.) cost, (k.) patient blog, (l.) researcher blog, etc. The most salient feature of our database/webserver, besides its focus on biological agents, is its multiple links to most, if not all, publicly available databases and webservers, including structural proteomics, metabolomics, glycomics, and lipidomics webservers. It is hoped that these links can provide the researcher with up-to-date knowledge about the structure and function of the specific biomolecules involved with the type of cancer they are studying. Knowledge of these “cancer signatures” or biomarkers is expected to facilitate the work of cancer researchers. As a public resource, on the other hand, the database will include a description attribute that will explain in simple, layman’s language the 12 biotherapy regimens well as other technical items included to ensure public accessibility.The database attributes will be regularly updated for novel attributes as discoveries are made.

References

1. A. Young, L. Rowett, and D. Kerr (Eds.). Cancer Biotherapy: An Introductory Guide, Oxford Univ. Press, Oxford, U.K. (2006). 2. P. T. Rieger. Biotherapy: A Comprehensive Overview, 2nd ed., Jones & Barlett Publ., Sudbury, MA (2001). Using Cartographic Techniques to Project 1017 Protein 3D Surfaces onto the 2D Plane: Potential Applications and Implications Study of protein surfaces is quite important in protein sciencesince the biologi- 45 cal properties of proteins are largely (but not solely) determinedby their surface properties. Meanwhile, it is estimated that a significant majority of all proteins Vicente M. Reyes fold into compact globular structures (‘spheroproteins’), and as such may be lik- ened to the earth. For centuries, the surface of the earth has been represented and Biological Sciences Dept., Sch. of analyzed using cartographic spherical projection methods, such as the Mercator, Eckert, Lambert, Werner and Aitoff projections. We have recently developed a Medical & Biological Sciences program that transforms protein 3D structure coordinates from Cartesian (x,y,z) to College of Science, Rochester Institute spherical (ρ,φ,θ) coordinates, whose origin is the geometric centroid of the protein of Technology (1). In this system, rho (ρ) plays the role of the earth’s radius from its center to an entity in the protein structure, phi (φ), the latitudes, and theta (θ), the longitudes. 2D Rochester, NY 14623-5603 projection of the surface of this sphere, with elevations fromthe “sea level” (ESLs), [email protected] may be achieved using the transformation equations for the particular projection method. The ESLs, on the other hand, can be determined by choosing areference

“sea level” ρ0 (e.g., distance from center of sphere to the smallest nonzero ρvalue) and using the “heights” of surface points above ρ0 as the z-coordinates (elevations along the z-axis). Established methods in geography/cartography may then be used to analyze such projections with elevations. Plots of these 2D raise-relief maps (a.k.a. terrain models) may be easily rendered using MATLAB. These 2D projection methods present a number of benefits for protein surface analysis over 3D-based methods, among which are (a) simplicity of presentation (i.e., 2D vs. 3D), (b) entire protein surface may be viewed all at once, (c) simpler and more direct protein surface comparisons (e.g., between similar but non-identical proteins, or between identical proteins in different conformations or liganded states), and (d) applicability of the myriad well- established cartographic techniques for analysis and comparison of protein surfaces. Thus far, we have written/implemented FORTRAN 77/90 programs for the following map projections: 1.) Mercator, 2.) Miller, 3.) Mollweide, 4.) Robinson and 5.) Gall- Peters. We are currently expanding this list and comparing the methods with each other in both all-atom and reduced protein representations (2) to determine which best represents the proteins’ surface properties by correlation with the proteins’ bioactivity.

References

Self Assembly Study of the Human GPCR Protein b2-Adrenergic Receptor Using Coarse Grained Molecular Dynamics Technique 46 Anirban Ghosh Integral membrane proteins roughly constitutes of 25 to 30% to the human genome, Uddhavesh B. Sonavane of which the G-Protein Coupled Receptors (GPCRs) encode for nearly 3-4% of all the genes, regulating various physiological processes through signal transduc- Rajendra Joshi* tion. As a consequence of this, these receptors have become the targets of several modern day drugs. Most of the studies aimed at designing new drugs for target- Bioinformatics Group, ing GPCRs have assumed that these receptors function in monomeric form. How- ever, this assumption has recently been changed by the description of a number of Centre for Development of Advanced GPCRs that can be found in oligomeric state within the cellular environment (1). Computing, Although many unsolved problems still remain, the idea that GPCRs directly inter- Pune University Campus, act to form oligomers, both homomers as well as heteromers, has been gradually accepted. The mechanism of GPCR dimer or oligomer formation, and its effect on Pune – 411 007, India receptor function, is not currently well understood. *[email protected] 1018 In the present study, coarse grained molecular dynamics (CGMD) approach was adopted for studying the self-assembly process of the human amine GPCR protein β2-adrenergic receptor (β2-AR), for which several experimental evidences of oligomerization process and its effect on its function are available (2, 3). PDB entry 2RH1 was taken as the starting structure. Since 2RH1 lacks ICL3 (residue 231-262), initially the missing loop was modeled in SYBYL and simulated for 10 ns using restrained MD in order to get a stable conformation. The final structure was then used for further studies. To mimic a cellular environment, 16 copies of β2-AR were inserted into DSPC bilayer at a protein to lipid ratio of 1:104 and then solvated with water. The entire system was represented using the MARTINI CG convention (4), resulting in a total system size of 57296 CG beads. The system was then simulated for 3 µs using the GROMACS package with MARTINI force filed parameters. An increased time-step of 30 femto-second was used which resulted in stable integration. At the end of the simulation period, proper dimers and tetramers of β2-AR were found to be formed through the self-assembly mechanism which were further validated through various analysis methods. The gradual decrease in SASA values calculated with a probe radius of 0.52 nm confirmed that the monomers were indeed coming together to form aggregates. The lipid bilayer analysis also helped to quantify the assembly mechanism. In order to identify the exact residues or domains which are responsible for this oligomerization, a conversion of the CG system back to an all-atom model and simulated annealing simulations are being presently carried out.

References

1. S. R. George, B. F. O’Dowd, and S. P. Lee. Nat Rev Drug Discov 1, 808-20 (2002). 2. A. Salahpour, S. Angers, J. F. Mercier, M. Lagacé, S. Marullo, and M. Bouvier. J Bio Chem 279, 33390-7 (2004). 3. T. E. Hebert, S. Moffett, J. P. Morello, T. P. Loisel, D.G. Bichet, C. Barret, and M. Bouvier. J Biol Chem 271, 16384-93 (1996). 4. Luca Monticelli, Senthil K. Kandasamy, Xavier Periole, Ronald G. Larson, D. Peter Tieleman, and Siewert-Jan Marrink. J Chem Theory Comput 4, 819-34 (2008).

Protein Structure Modelling on the Indo-US 47 Cancer Research Grid 1 The importance of protein structures can be understood easily from the fact that the Amit Saxena function of any protein is directly correlated to its structure (1). The three dimen- Anirban Ghosh1 sional structure of a protein directs its function within a cellular environment. Any George A. Komatsoulis2 mutation in the protein sequence leads to changes in its structure which in turn may 1 render the protein non-functional or even attribute some adverse functions (2-6) Hemant Darbari leading to diseases like cancer. Over the decades cancer has become one of the Anil Srivastava2 most prevalent diseases with an estimate of reaching over 12 million deaths in 2030 Ravi Madduri3 according to World Health Organization. Proteins from almost 1% of the human 1 genome have been identified to be involved in oncogenesis (7). In the absence of P.K. Sinha resolved structural data (RCSB database has 65847 resolved protein structures as Rajendra Joshi1* opposed to 525207 sequence entries in UniProtKB) one has to resort to computational techniques to get the 3D structures of proteins in order to properly understand their functions. 1Centre for Development of Advanced Computing, Pune–411007, India The Bioinformatics Group at the Centre for Development of Advanced Computing ® 2Center for Biomedical Informatics and (C-DAC) in collaboration with cancer Biomedical Informatics Grid (caBIG ) has developed a grid-enabled web-based automated pipeline (Figure 1) for ab initio Information Technology, NCI, NIH, USA prediction of protein structures with an emphasis on cancer related proteins. The 3University of Chicago, USA pipeline has been deployed on the Bioinformatics Resources & Applications *[email protected] Facility (BRAF) hosted at C-DAC, Pune India. The upstream component of the pipeline retrieves a protein sequence (according to user input) from the gridPIR 1019

Figure 1: Schematic representation of the protein prediction pipeline. service of caBIG that provides a data resource of high quality annotated information on all protein sequences supported by UniProtKB. The retrieved sequence in a FASTA format is then fed to the prediction pipeline. At its core the pipeline uses the ROSETTA prediction algorithm (8) for determining the 3D structures. The graphical user interface of the pipeline enables the user to choose various control parameters like which secondary structure prediction algorithms to use, number of iterations, number of output structures, uploading NMR constraint files etc. Once submitted, the jobs get distributed over multiple processors in the form of multiple threads on Biogene supercomputing system at BRAF, which highly reduces the prediction time. The resultant output comes in the form of predicted structures in PDB format and parsed energy log files which can be downloaded by the user. All the file transfers are secured over the network by SFTP. JMol has been integrated within the pipeline to provide a visual inspection of the predicted models. Test cases have been run using the pipeline with a few cancer related proteins, whose results will be discussed. This pipeline provides a hassle-free high throughput structure prediction platform. Java has been used for coding the entire pipeline with Struts, AJAX and Hibernate framework. The upstream gridPIR searching module parses XML results using SAX parser while the GUI has been built using JSP.

References

1. H. Hegyi and M. Gerstein. J Mol Biol 288, 147-64 (1999). 2. M. Soskine and D. S. Tawfik. Nat Rev Genet 11, 572-82 (2010) 3. L. Zhong. J Biomol Struct Dyn 28, 355-361 (2010). 4. Y. Yu, Y. Wang, J. He, Y. Liu, H. Li, H. Zhang, and Y. Song. J Biomol Struct Dyn 27, 641-649 (2010). 5. M. S. Achary and H. A. Nagarajaram. J Biomol Struct Dyn 26, 609-623 (2009). 6. A. A. Moosavi-Movahedi, S. J. Mousavy, A. Divsalar, A. Babaahmadi, K. Karimian, A. Shafiee, M. Kamarie, N. Poursasan, B. Farzami, G. H. Riazi, G. H. Hakimelahi, F. Y. Tsai, F. Ahmad, M. Amani, and A. A. Saboury. J Biomol Struct Dyn 27, 319-329 (2009). 7. P. A. Futreal, L. Coin, M. Marshall, T. Down, T. Hubbard, R. Wooster, N. Rahman, and M. R. Stratton. Nat Rev Cancer 4, 177-83 (2004). 8. C. A. Rohl, C. E. Strauss, K. M. Misura, and D. Baker. Methods Enzymol 383, 66-93 (2004). 1020 The Role of Central Pore Residues of p97/VCP on Substrate Unfolding and Translocation: A Computational Model

The p97/VCP nanomachine, a double ring member of the AAA1 superfamily, is 48 involved in substrate protein unfolding within the proteasomal degradation pathway Sam Tonddast-Navaei* (1). Currently, it is unclear how p97/VCP interacts with its substrate. Revealing the George Stan underlying mechanism of p97 could lead to better understanding of its bacterial homologues ClpA and ClpB.

Department of Chemistry, P97/VCP has a homo-hexameric structure that encloses a central pore. Within each University of Cincinnati, subunit, there are two nucleotide binding domains, D1 and D2, and the N domain, which is connected to D1 and is known to interact with p97/VCP’s adaptors. ATP Cincinnati, OH 45221 hydrolysis leads to large scale conformational changes in D2 domain, which affects *[email protected] the topology of its pore (2, 3).

Conserved loops at the entrance of D2 pore are suggested to enable substrate propagation through the pore via ATP-driven paddling motion of Trp551 and Phe552 residues. Two other essential residues inside the D2 pore, Arg586 and Arg599, contribute to the p97 function (4). We propose that the substrate, which enters through the D1 pore, binds to the Arg599 sites on the D2 cavity lining. Repetitive ATP-driven cycles of p97 mediate the complete translocation of the substrate protein into the D2 pore via the paddling motion of the D2 loops (centered onto Trp551 and Phe552).

To test this hypothesis, we perform implicit solvent simulations of the SsrA-SsrA peptide threading through p97/VCP. Our results confirm the role of Arg599 as binding sites. These simulations reveal that these Arginines interact with the substrate primarily via hydrogen-bonds formed with the peptide backbone, indicating a non-specific interaction type.

Using the results from implicit solvent simulations, we develop a coarse-grained model that extends our simulations to biologically relevant timescales. We investigate the unfolding mechanism of a four helix bundle protein fused with the SsrA peptide coupled to ATP-driven conformational changes in the D2 domain of p97 (figure 1). Our simulations show that complete unfolding and translocation is due to the collaboration between Arginine residues and the critical residues at the paddling D2 loop, such that substrate is held by the Arginine residues of adjacent subunits and the force exerted by the D2 loops pull the substrate through the central pore of p97.

Figure 1: A snapshot of unfolding and translocation of the four helix bundle (purple) and the SsrA peptide (orange) by the p97/VCP nanomachine (N-D1 yellow, D2 green). To show the location of the D2 loops (blue) and the Arginines (brown), the two front subunits are not presented in the figure. This research has been supported by a grant from the American Heart Association 1021 and by the National Science Foundation CAREER grant to G. S. and a University Research Council fellowship at the University of Cincinnati to M.J.

References

1. A. Beskow, K. B. Grimberg, L.C. Bott, F. A. Salomons, N. P. Dantuma, and P. Young. J Mol Biol 394, 732-746 (2009). 2. B. DeLaBarre, J. C. Christianson, R. R. Kopito, and A. T. Brunger. J Mol Cell Biol 22, 451-462 (2006). 3. Q. Wang, C. Song, X. Yang, and C. H. Li. J Biol Chem 278, 32784-32793 (2003). 4. J. M. Davies, A. T. Brunger, and W. I. Weis. Struct. 16, 715-726 (2008). 5. A. Kravats, M. Jayasinghe, and G. Stan. Proc Natl Acad Sci USA (in press).

Computer-Aided Pathway to Increasing the Thermostability of Small Proteins 49 A major task of modern bioengineering is the development of molecules with des- ignated properties (1) such as increased stability, including their thermostability. Maxim Kondratyev* The augmented thermostability of proteins allows to increase the speed of enzy- Artem Kabanov** matic catalysis, as well as the duration of their storage. Alexander Samchenko

A few years ago a possible thermostabilization mechanism of small globular pro- Vladislav Komarov teins (2) was developed in the Laboratory of Structure and Dynamics of Biomo- Nikolay Khechinashvili lecular Systems at the Institute of Cell Biophysics (Russian Academy of Sciences). It is based on the alternative hydrogen bonding mechanism between side chains of Institute of Cell Biophysics amino acid residues on protein surface (2). This hypothesis based on experimental data has been essentially supplemented later by modelings of dynamics of for pro- Pushchino, Russia teins from thermophilic and mesophilic organisms (3, 4). *[email protected] **[email protected] Our work uses this theory for improving the thermostability of human Peroxire- doxin 6. This protein (5) is a promising antioxidant for burn treatment. The spatial structure of human Peroxiredoxin 6 was reported previously (6) (Figure 1.) and its

Figure 1: Structure of human Peroxiredoxin 6 (pdb entry: 1PRX) and active site location. 1022 homologs from various organisms have been well characterized, including their thermodynamic properties (7). We propose to predict what point mutaions in Per- oxiredoxin 6 will increase its themostability using our knowledge about alternative hydrogen bonding, as well as the known structure of human Peroxiredoxin 6 and its known homologs.

Data of alignment of Peroxiredoxin 6 homologs (Figure 2) give us information about the variable and stable parts of the amino acid sequence of this protein. The most probable sites of mutations reside only in the evolutionary variable areas of amino acid sequence because changes in stable regions can affect the functional properties of the protein.

It should be noted that certain homologs of Peroxiredoxin 6 possess higher thermostability in native state in comparison to the human protein. It is important to notice that human and rat Peroxiredoxins have the highest homology (91.5%, i.e. 19 residues) (Figure 2.). At the same time, rat protein possesses the greatest thermostability (7) among the homologs studied. Thus, by comparing rat and human Peroxiredoxin 6 amino acid sequences, we can get additional information about the preferred locations of mutations to increase the thermostability of human Peroxiredoxin 6.

The structures of native human Peroxiredoxin 6 protein and it homologs have been studied by molecular dynamics (MD) at various temperatures on GPU NVIDIA (8). The MD provides a powerway to follow formation and destruction of hydrogen bonds in all biological macromolecules (9-12). In the case of Peroxiredoxin 6 protein we tested the amount of hydrogen bonds on the surface of protein globules in each frame of an MD-trajectory on the pairs of studied proteins. Solvent was considered both in explicit and implicit models.

From the data of sequence alignment and our MD calculations, we predict four amino acid substitutions, which we believe will lead to increased thermostability of human Peroxiredoxin 6 protein without violating the spatial structure and functional properties.

The problem of substrate specificity of Peroxiredoxin 6 will also be discussed.

Figure 2: Alignment of Peroxiredoxins 6 from various organisms ([7]). (Note, that active site of all Peroxiredoxin’s 6 (CYS47) is located in stable region of amino acids sequence.) References 1023 1. Donald Lee Wise. Encyclopedic handbook of biomaterials and bioengineering: Applica- tions, vol. 2, New York: Marcel Dekker, 1995. 2. N. N. Khechinashvili, M. V. Fedorov, A. V. Kabanov, S. Monti, C. Ghio, and K. Soda. J Biomol Struct Dyn 24, 255-262 (2006). 3. A. V. Kabanov and N. N. Khechinashvili. J Biomol Struct Dyn 24, 756-756 (2007). 4. N. N. Khechinashvili, S. A. Volchkov, A. V. Kabanov, and G.Barone. Biochim Biophys Acta Proteins & Proteomics, 1784 (11), P.1830 92084). 5. I. V. Peshenko, V. I. Novoselo, V. A. Evdokimov, Y. V. Nikolaev, T. M. Shuvaeva, V. M. Lipkin, and E. E. Fesenko. FEBS Lett 381, 14-19 (1996). 6. H. J. Choi, S. W. Kang , C. H. Yang, S. G. Rhee, and S. E. Ryu. Nat Struct Biol 5, 400-406 (1998). 7. M. G. Sharapov, V. I. Novoselov, and V. K. Ravin. Mol Biol (Mosk) 43, 505-11 (2009). 8. http://www.nvidia.com/object/cuda_home_new.html 9. Z. Gong, Y. Zhao, and Y. Xiao. J Biomol Struct Dyn 28, 431-441 (2010). 10. J. Wiesner, Z. Kriz, K. Kuca, D. Jun, and J. Koca. J Biomol Struct Dyn 28, 393-403 (2010). 11. C. Koshy, M. Parthiban, and R. Sowdhamini. J Biomol Struct Dyn 28, 71-83 (2010). 12. F. Mehrnejad and M. Zarei. J Biomol Struct Dyn 27, 551-559 (2010).

Generating Conformational Ensembles for Flexible Protein-Ligand Docking by Elastic Network Model Guided Molecular Dynamics Simulations: Application 50 to Beta 2 Adrenergic Receptor Basak Isin1* Guillermina Estiu2 Studying the entire motion spectrum of a protein is necessary for a complete under- Olaf Wiest2 standing of its function(s). However, this is not trivial since protein motions are 1 complex ranging from femtosecond scale local atomic vibrations to millisecond Zoltan N. Oltvai scale large motions. We previously developed Anisotropic Network Model (ANM) restrained Molecular dynamics (MD) that takes advantage of these two comple- 1Department of Pathology, and menting methods to sample long time scale biologically relevant global motions of a biomolecular system with realistic deformations favored by a detailed atomic Computational & Systems Biology, force field in the presence of the explicit environment (1). Here, we useANM- University of Pittsburgh, restrained-MD method to generate conformational ensembles of a pharmacologi- Pittsburgh, PA, 15261 cally relevant G-protein Coupled Receptor (GCPR), Beta 2 Adrenergic Receptor 2Department of Chemistry and (β2AR). It has been shown that the binding of GPCRs to structurally diverse ligands and their activation is a complex process that requires these receptors passing through Biochemistry, multiple conformationally distinct states (2, 3). Along with the existing crystal struc- University of Notre Dame, tures, the conformational ensembles are utilized for understanding the dynamics and Notre Dame, IN, 46556 binding modes of β2AR to its known agonists by performing docking against them. We observe that the residues Ser203, Ser204, and Ser207 on H5 become accessible *[email protected], [email protected] at the ligand binding site by the rotation of H5. Additional to Serines on H5, Val114 and Thr118 on H3 and “the rotamer toggle switch”, Phe290, on H6 stabilize the aromatic rings and the catecholamine moieties of the agonists of β2AR by forming hydrogen bonds and phi-stacking interactions. Furthermore, our study also shows that the long acting agonist drug that is currently prescribed for asthma, salmeterol, folds uniquely at the ligand binding pocket of β2AR and stabilizes its extracellular region of forming a “beta sheet-like” structure with the extracellular loop 2.

Authors would like to thank Drs Ivet Bahar, Klaus Schulten and Emad Tajhkorshid for their valuable contributions during the development of ANM-guided-MD algorithm.

References

1. B. Isin, K. Schulten, E. Tajkhorshid, and I. Bahar. Biophys J 95, 789-803 (2008). 2. G. Liapakis, W. C. Chan, M. Papadokosktaki, and J. M. Javitch. Mol Pharmacol 65, 1181- 1190 (2004). 3. B. K. Kobilka and X. Deupi. Trends Pharmacol Sci 28, 397-406 (2007). 1024 Role of M5-M6 Loop in the Biogenesis and Function of the Yeast Pma1 H+-ATPase

Yeast Pma1 H+-ATPase belongs to P2-type ATPases which couple ATP hydrolysis to transport of cations across biomembranes where they are embedded by 10 51 membrane segments. The determinants of cation specificity and stoichiometry lie Valery V. Petrov in M4, M5, M6, and M8 segments; point mutations in these segments affect normal functioning and biogenesis of the enzyme and change both stoichiometry (1-3) and specificity (4). During the reaction cycle P2-ATPases undergo significant confor- Institute of Biochemistry and mational changes: M1-M6 segments bend, unwind partially and even shift normal Physiology of Microorganisms, to the membrane (5). Ala substitutions of the residues in the M6 N-terminal half interfered markedly with the Pma1 functioning and biogenesis, or both (3). The RAS, 142290 Pushchino, Russia results described here extend a systematic study of the yeast H+-ATPase by focus- [email protected] ing on the extracytoplasmic loop between M5 and M6 segments of this enzyme, searching for residues that may play a role in H+ transport or any other aspect of the enzyme functioning and biogenesis. To explore role of this loop in the structure- function relationship of the S. cerevisiae Pma1 ATPase, Ala-scanning mutagenesis was used. The loop consists of 7 residues: 714-DNSLDID. L717 is the most con- servative among them. Accordingly, only L717A led to a complete block in membrane trafficking that prevented the ATPase from reaching secretory vesicles (SV) which points to a severe defect in protein folding, causing the abnormal ATPase to be retained in the endoplasmic reticulum (ER). Mutation D714A was expressed poorly in SV (Figure 1), displaying very low ATPase activity with no detectable H+ pumping. The remaining 5 mutations were expressed at 34 to 94% with ATPase activities of 35 to 101% of the WT level. Given the known contribution of M5 and M6 segments to the transport pathway of P2-ATPases and their mobility during reaction cycle, it was of particular interest to ask whether any of the mutations

Figure 1: Effect of Ala substitutions of the residues in the M5-M6 loop of the Pma1 ATPase on the enzyme expression (left columns) and activity (right columns), %. in the M5-M6 loop affected H+ pumping. For most of the mutants, the coupling 1025 ratio was close to that of the WT (1.00). Only I719A mutant gave the ratio significantly lower (0.29), pointing to a partial uncoupling between ATP hydrolysis and H+ transport. Thus, 3 of 7 residues in M5-M6 loop seem to be important for the proper function and biogenesis of the yeast Pma1 H+-ATPase. L717 is located in the middle of the loop; since the enzyme reaction cycle is accompanied by significant conformational changes, substitution of this residue with a smaller Ala may strongly affect the mobility of M5 and M6 segments causing misfolding and retaining of impaired enzyme in ER. D714 is also important for structure and, especially, functioning of the enzyme; it probably plays a role similar to D739 in M6 which neutralizes neighboring positive charge and, thus, stabilizing the protein (3). Finally, I719 replacement with Ala caused significant uncoupling between ATP hydrolysis and H+ transport.

Acknowledgements

Author is grateful to scientific adviser of this project Prof. C. W. Slayman (Yale School of Medicine).

References

1. V. V. Petrov, K. P. Padmanabha, R. K. Nakamoto, K. E. Allen, and C. W. Slayman. J Biol Chem 275, 15709-15716 (2000). 2. G. Guerra, V. V. Petrov, K. E. Allen, M. Miranda, J. P. Pardo, and C. W. Slayman. Biochim Biophys Acta 1768, 2383-2392 (2007). 3. M. Miranda, J. P. Pardo, and V. V. Petrov. Biochim Biophys Acta (epublished Dec. 13, 2010). 4. D. Mandal, T. B. Woolf, and R. Rao. J Biol Chem 275, 23933-23938 (2000). 5. C. Toyosima and H. Nomura. Nature 418, 605-611 (2002).

Point Mutation in M9-M10 Loop of the Yeast Pma1 H+-ATPase Affects Both ATPase Functioning and Polyphosphate (PolyP) Distribution 52 Alexander A. Tomashevsky Both ATP and linear polymers of inorganic phosphate (P ) PolyP are involved in i Valery V. Petrov* cell energetics and metabolism. Role of ATP and ATPases is well established, while less is known about PolyP. However, it is clear that PolyP are involved in storage of Pi, membrane channel formation, cation binding, regulation of enzyme Institute of Biochemistry and activities, gene expression (1). There are very little data on the interaction between Physiology of Microorganisms, ATP and PolyP metabolism. Yeast plasma membrane (Pma1) H+-ATPase gener- ates electrochemical H+ gradient providing energy for operating the secondary sol- RAS, 142290 Pushchino, Russia ute transport systems. The enzyme is embedded in the membrane by 10 segments *[email protected] (M1-M10) with most of the molecule located in cytosole or in the membrane; only 5% of the molecule face extracellular space. Yeast Pma1 H+-ATPase is regulated by glucose: during glucose consumption ATP is produced, this triggers activation of Pma1 functioning manifested in 3-10-fold increase of Vmax, and decrease of Km and Ki. ATPase activation is structurally accompanied by the enzyme multiple phosphorylation (2); two tandemly positioned sites are located in the enzyme C-terminal tail (3).

Most of the plausible residues for the ATPase phosphorylation (Ser, Thr, Asp, and Glu) are located in the inner parts of the enzyme; however, there are several phos- phorylable residues located in the Pma1 outer parts: D714, S716, D718, and D720 in the M5-M6 loop and S846, E847, T850, and D851 in the M9-M10 loop which is close to the enzyme regulatory C-tail. It seems reasonable that multiple phosphorylation of Pma1 goes subsequently, and first of such sites could be located in 1026

Figure 1: Left: Pma1 ATPase activity in the WT and T850A mutant strains under carbon-starved (CS) and glucose-metabolizing (GM) conditions (%). Right: PolyP3 content in the WT and T850A strains in the logarithmic (log) and stationary (stat) phases (µmol P/g wet weight).

the extracellular part of the enzyme. The M5-M6 loop residues, except D714, were found not to be important for the enzyme structure-function relationship; the D714A mutant activity was unessential to be studied further (4). Therefore, we choose to replace with Ala one of the residues in the M9-M10 loop of the enzyme which could be phosphorylated – T850. When T850 is replaced with Ala, the mutated enzyme activity dropped significantly; at the same time the ability of the mutated enzyme to be activated by glucose was strongly impaired (Figure 1, left). In parallel with ATPase activity assay, distribution of PolyP fractions (PolyP1-PolyP5) was analyzed. No significant changes were found between most PolyP fractions in logarithmic and stationary phases of the wild type (WT) and T850A mutant. However, PolyP3 fraction stood out of the rest displaying almost twofold increase of PolyP amount in the mutant during stationary phase compare with only a quarter in the WT (Figure 1, right). Since ATPase is more active during logarithmic phase (similar to GM and CS conditions in Figure 1, left), it points to a connection between ATP and Poly metabolisms. Significant increase of PolyP3 amount in the T850A mutant in stationary phase may point to the lack of one of the phosphorylation sites. Further study of this and similar mutants, although methodologically challenging, seems certain to yield useful insights into the fundamental mechanisms of ATP and PolyP interactive metabolisms.

References

1. I. S. Kulaev, V. M. Vagabov, and T. V. Kulakovskaya. High-molecular inorganic polyphos- phates: biochemistry, cell biology and biotechnology. Moscow, Scientific World (2005). 2. A. Chang and C. W. Slayman. J Cell Biol 115, 289-295 (1991). 3. S. Lecchi, C. J. Nelson, K. E. Allen, D. L. Swaney, K. L. Thompson, J. J. Coon, M. R. Sussman, and C. W. Slayman. J Biol Chem 282, 35471-35481 (2007). 4. V. V. Petrov. J Biomol Struct Dyn http://www.jbsdonline.com/product-p18051.html (2011). Role of Conserved Water Molecules in Binding 1027 of Thyroxin and Analogs Inhibitors to Human Transthyretin: A Study on Water-Mimic Inhibitor Design 53 Transthyretin (TTR) is a very important protein associated with the transportation Avik Banerjee of thyroxin hormone and vitamin-A in the serum as well as in ceribro-spinal-fluid of human (1). The protein is also responsible to cause amyloid diseases like FAP Hridoy R. Bairagya (Familial Amyloidotic Poly-neuropathy) and SSA (Senile systematic Amyloido- Bishnu P. Mukhopadhyay* sis) due to formation of amyloid fibrils (2, 3) by the protein and their deposition in extra cellular matrixes and tissues in human body (4). Simulation of different Tapas K. Nandi X-ray structures of TTR [available as dimer in Protein Data Bank, Resolution 1.3-2.0Å] and their water dynamics have revealed the presence of six conserved Department of Chemistry, water molecules in the buried core of A-chain, 4- in B-chain and 6- in the interface of the dimer. Among these conserved water molecules, one in each monomer seems National Institute of to play an important role in the thyroxin binding with the protein. Comparative Technology- Durgapur, analysis of unliganded TTR and TTR-Thyroxin complex structures (5) and their West Bengal, Durgapur –713209, India * [email protected]

Figure 1: Interaction of conserved water molecule (W) with Ser 117 and Thr 119 in unliganded form of Protein. Transition of TTR- thyroxin complex to Unliganded TTR. 1028 water molecular dynamics reveal that in the unliganded structures the conserved water molecule forms H-bond with the side-chains of two important residues, Ser-117 and Thr-119, of the thyroxin binding pocket (6). The Ser-Thr bound conserved water molecule seems to migrate when the thyroxin molecule enters in the pocket and forms complex with the protein. Again, during migration of that water molecule, side chains of the respective Ser-117 and Thr-119 adopt a trans like conformation which stereo-chemically assist the thyroxin molecule to occupy the specific binding pocket of TTR [Figure1]. The positional invariance of that water molecule with the 5’-iodine atom of thyroxin molecule seems to be interesting. These results may provide further insight and complementary information into thyroxin/inhibitor (thyroxin analog) binding chemistry, which may be used as a new strategy in search of a new effective TTR-inhibitor design.

References

1. P. A. Peterson. J Biol Chem 246, 44-49 (1971). 2. L. K. Chang, J. H. Zhao, H. L. Liu, J. W. Wu, C. K. Chuang, K. T. Liu, J. T. Chen, W. B. Tsai, and Y. Ho. J Biomol Struct Dyn 28, 39-50 (2010). 3. L. -K. Chang, J. -H. Zhao, H. -L. Liu, K. -T. Liu, J. -T. Chen, W. -B. Tsai, and Y. Ho. J Biomol Struct Dyn 26, 731-740 (2009). 4. X. Hou, M. I. Aguilar, D. H. Small. FEBS J 274, 1637-1650 (2007). 5. A. Wojtczak, V. Cody, J. R. Luft, and W. Pangborn. Acta Crystallogr Sect D 52, 758-765 (1996). 6. A. Banerjee, H. R. Bairagya, B. P. Mukhopadhyay, T. K. Nandi, and A. K. Bera. IJBB 47, 197-202 (2010).

Structures and Dynamics of the Complete Protein 54 Kinase Catalytic Cycle of CDK2/CyclinA Like many protein kinases, CDK2 is known to be a rather flexible enzyme and con- Matthew A. Young* formational transitions and protein dynamics are believed to play important roles Douglas M. Jacobsen in both the catalytic mechanism and the regulation of catalytic activity. We have Zhao-Qin Bao determined high-resolution crystal structures of multiple steps along the complete reaction cycle of CDK2, including a transition-state complex consisting of CDK2/ Department of Biological Chemistry – CyclinA bound to ADP, a substrate peptide and MgF3 , a structural mimic for the and Bioinformatics Program gamma-phosphate of ATP in the transition-state. Compared to structures of active University of Michigan CDK2 bound to its substrates or its products, the catalytic subunit of the kinase in the transition-state adopts a more closed conformation of the active site and, for the Ann Arbor, 48109 first time, a second catalytic Mg ion is observed in the active site. Coupled with a *[email protected] strong [Mg] effect on in vitro kinase activity, this structure suggests that the transient binding of a second Mg ion is necessary to achieve maximum rate-enhancement of the chemical reaction and Mg concentration could represent an important regulator of CDK2 activity in vivo. Molecular dynamics simulations illustrate how the simultaneous binding of substrate peptide, ATP and two Mg2+ ions is able to stabilize the closed and also more rigid organization of the active site that functions to orient the phosphates, stabilize the buildup of negative charge, and shield the subsequently activated gamma-phosphate from solvent. Once the phosphoryl-transfer step is complete, the second Mg is released and the active site returns to the more open conformation to release the products. Protein Flexibility Methods to Compare Protein 1029 Structure Predictions

Geometric measurements such as RMSD or GDT score are routinely used as a preliminary tool to assess the quality of a model to a certain reference structure (usually high resolution X-ray or NMR data). Two problems with these measures 55 are: (1) at a given temperature there is not one single structure, but an ensemble Alberto Perez* of them; (2) geometric analysis gives global information, is highly degenerate and does not take the protein’s topology into consideration. We investigating the utility Justin L. MacCallum of using an energetic measure based on a protein’s intrinsic flexibility pattern to Yang Zhang complement geometry measurements in model quality evaluation. Ivet Bahar To account for flexibility we use the elastic network model (ENM) theory [1-3], Ken A. Dill which is computationally inexpensive and gives an accurate approximation to protein flexibility. In ENM the protein is represented as a series of nodes (Calphas). A Laufer Center for Physical and simple Hamiltonian is derived by: (1) finding all pairs of nodes closer than some cutoff distance; and (2) connecting them via springs. Calculation of the hessian Quantitative Biology, Stony Brook and its diagonalization yields a set of eigenvector-eigenvalue pairs representing the University, Department of Pharmaceutical directions of maximum deformability in the protein system. This information can Chemistry, University of California, be used to calculate deformation energies needed to deform the reference structure into the model one. This energy will rapidly increase when deforming regions of San Francisco, and University of the protein that are not intrinsically flexible. Thus, two structures with the same Pittsburgh RMSD (or same GDT score) might have very different energies. Conversely, two *[email protected] structures that have the same energy value might be far apart in RMSD (see figure below). Having an energetic measure is helpful in identifying structures within thermal noise of each other (e.g. within the native ensemble), meaning that the model is of good enough quality. It might be useful in events such as CASP [4] to quantify how hard refinement of a given template is going to be.

AP acknowledges support from EMBO long-term fellowship.

Figure: Top is a reference crystal structure in CASP9 (TR569). Below are two submitted models. The one on the left would receive a higher score in geometric measurements, but the main difference is in a very flexible area of the protein, resulting in similar deformation energies for both models.

References

1. A. R. Atilgan, et al. Biophys J 80:505-515 (2001). 2. M. Tirion, Phys Rev Lett 77, 1905-1908 (1996). 3. K. Hinsen, Proteins 33, 417-429 (1998). 4. Proteins: Structure, Function, and Genetics 23(3), 295-460 (1995). 1030 Computational Generation of Inhibitor-bound Conformers of p38 MAP Kinase and Comparison with Experiments

We developed an extensible framework, ProDy, for structure-based analysis of protein dynamics (http://www.csb.pitt.edu/ProDy/). ProDy allows for quantita- 56 tive analysis of heterogeneous experimental structural datasets and comparison with theoretically predicted conformational dynamics (1). Datasets include struc- Ahmet Bakan tural ensembles composed of a given family or subfamily members, mutants and Ivet Bahar* sequence homologues, in the presence/absence of their substrates, ligands or inhibitors. We demonstrate the utility of ProDy by way of application to exploring the dynamics of p38 MAP kinases, a family of enzymes which play a critical role in Department of Computational and regulating stress-activated pathways, and serve as molecular targets for controlling Systems Biology, School of Medicine, inflammatory diseases. Computer-aided efforts for developing p38 inhibitors have University of Pittsburgh, 3501 Fifth Ave, been hampered by the necessity to include the enzyme conformational flexibility in ligand docking simulations. A useful strategy in such complicated cases is to perform Suite 3064 BST3, Pittsburgh, PA 15260 ensemble-docking provided that a representative set of conformers is available for *[email protected] the target protein either from computations or experiments. Using ProDy, we explore the abilities of two computational approaches, molecular dynamics (MD) simulations and anisotropic network model (ANM) normal mode analysis, for generating potential ligand-bound conformers starting from the apo state of p38, and benchmark them against the space of conformers inferred from the principal component analysis of 134 experimentally resolved p38 kinase structures (2). The ANM-generated conformations are found to provide a significantly better coverage of the inhibitor-bound conformational space observed in experiments, compared to the conformers generated by MD simulations performed in explicit water. The results suggest that ANM- based sampling of conformations can be advantageously employed for generating structural models to be used as input in docking simulations.

Support from NIH grants 1R01GM086238-01, 5R01GM086238-02 and 5R01LM007994-06 is gratefully acknowledged by I. Bahar.

References

1. A. Bakan and I. Bahar. Proc Natl Acad Sci USA 106, 14349-14354 (2009). 2. A. Bakan and I. Bahar. Pacific Symposium on Biocomputing 16, 181-192 (2011). Dynamics of AMPA-subtype Glutamate Receptor 1031 Using Elastic Network Models

Ionotropic glutamate receptors (iGLURs) are known to mediate several excitatory neurotransmission in the central nervous system. These receptors are ligand gated ion channels that couple agonist binding to a ligand binding core, to open and 57 desensitize the ion channel. Dysfunction of iGLURs due to injury or other stimuli, Indira Shrivastava* results in acute neurological disorders attracting the attention of pharmaceutical industry to iGLURs as potential drug discovery targets. The recent structure of the Anindita Dutta intact α-amino-3-hydroxy-5-methyl-4-isozazole propionic acid (AMPA) receptor Ivet Bahar (1) paved the way for a structural analysis of the dynamics of the receptor. Global, large-scale, co-operative motions of the structure are obtained using the well know Gaussian Network Model (GNM) and Anistropic Network Model (ANM) (2, 3). University of Pittsburgh, Department of The slowmodes within this model, enabled identification of hinge residues at the Computational and Systems Biology, N-terminal domain (NTD), Ligand Binding domain (LBD) and at the Transmem- Fifth Avenue, 3501, Biomedical Science brane Domain (TMD). The Markovian Stochastic Model (MSM) (4), enabled the identification of several key residues with low-commute times. The residues iden- Tower-3, Pittsburgh, PA 15213 tified as hot-spots by MSM and the hinge residues identifed by GNM and ANM *[email protected] although scattered in the different domains, are located at structurally ‘strategic’ places such as NTD/LBD interface, LBD/TMD interface or even the linker region. Several of these residues are also highly conserved among the iGLUR family, and are thus anticipated to play a role in the signal transduction machinery of the AMPA-receptor.

References

1. A. I. Sobolvesky, M. P. Rosconi, and E. Gouaux. Nature 462, 745-756 (2009). 2. A. R. Atilgan, S. R. Durell, R. L. Jernigan, M. C. Demirel, O. Keskin, and I. Bahar. Biophys J 80, 505-515 (2001). 3. I. Bahar, T. R. Lezon, A. Bakan, and I. H. Shrivastava. Chem Rev 110, 1463-1497 (2010). 4. C. Chennubhotla and I. Bahar. PloS Comp Biol 3, e172 (2007). 1032 Protonation of Glutamate-208 Induces the Release of Agmatine in an Outward-Facing Conformation of Arginine/Agmatine Antiporter

Virulent enteric pathogens have developed several systems that maintain intracellular pH in order to survive extreme acidic conditions. One such mechanism is 58 the exchange of arginine (Arg+) from the extracellular region with its intracellular 2+ Elia Zomot decarboxylated form, agmatine (Agm ). The net result of this process is the export of a virtual proton from the cytoplasm per antiport cycle (1). Crystal structures of Ivet Bahar* the arginine/agmatine antiporter from E. coli, AdiC, have been recently resolved in both the apo and Arg+-bound outward-facing conformations (2, 3), which permit us Department of Computational & Systems to assess for the first time the time-resolved mechanisms of interactions that enable the specific antiporter functionality of AdiC. Using data from approximately 1µs Biology, School of Medicine, University of molecular dynamics simulations, we show that the protonation of E208 selec- of Pittsburgh, 3064 BST3, 3501 Fifth tively causes the dissociation and release of Agm2+, but not Arg+, to cell exterior. Avenue, Pittsburgh, PA 15213 The impact of E208 protonation is transmitted to the substrate binding pocket via the reorientation of I205 carbonyl group at the irregular portion of transmembrane *[email protected] (TM) helix 6. This effect, which takes place only in the subunits where Agm2+ is released, invites attention to the functional role of the unwound portion of TM helices (TM6 W202-E208 in AdiC) in facilitating substrate translocation, reminiscent of the behavior observed in structurally similar Na+-coupled transporters.

This research was supported by NIH grants 1R01GM086238-01 and 1U54GM087519-01A1, and by the NSF through TeraGrid resources provided by Kraken (NICS) and Ranger (TACC) under grant number TG-MCB100108.

References

1. R. Iyer, C. Williams, and C. Miller. J Bacteriol 185, 6556-6561 (2003). 2. X. Gao, F. Lu, L. Zhou, S. Dang, L. Sun, X. Li, J. Wang, and Y. Shi. Science 324, 1565-1568 (2009). 3. X. Gao, L. Zhou, X. Jiao, F. Lu, C. Yan, X. Zeng, J. Wang, and Y. Shi. Nature 463, 828-832 (2010).

Transition Pathways of Enzymes Explored by Combining the Anisotropic Network Model, 59 Molecular Dynamics Simulations and a Monte Carlo Mert Gur Sampling of Conformational Space Ivet Bahar The conformational transition between the open and closed forms of Escherichia coli adenylate kinase (AK) is explored using a molecular dynamics (MD) sim- Department of Computational and ulation protocol which is guided by the normal modes derived from the coarse Systems Biology, School of Medicine, grained anisotropic network model (ANM). The methodology applies to the cases e.g., University of Pittsburgh, 3501 Fifth Ave, where the passage from one substate to another ( the open and closed forms of an enzyme) within a global energy minimum (native state) involves relatively Suite 3064 BST3, Pittsburgh, PA 15260 low energy barriers, based on the assumption that low energy barriers may be sur- [email protected] mounted/overlooked by adopting a coarse-grained description of the structure and [email protected] energetics, which smoothes out the energy landscape. The basic approach is to deform the structure along ANM modes, similar to the adaptive ANM (aANM) procedure adopted in our previous work, (1) but with the major improvement that 1033

the intermediate structures are selected by a Monte Carlo scheme and energy minimized by short MD runs. While the detailed energy landscape may usually comprise multiple microstates and multiple barriers/pathways, the coarse-graining of the transition path between the open and closed forms of AK highlights three substates. In agreement with previous work, the conformational change is undergone in two steps: Closing of the LID (shown in red in Figure1) region succeeded by that of the nucleotide binding domain (colored orange). Some residues are observed to experience high internal energies during the simulated transition, this highlight- ing the critical interactions that play a dominant role in destabilizing or stabilizing particular substates.

Support from NIH grants 1R01GM086238-01is gratefully acknowledged by I. Bahar.

Reference

1. Zheng Yang , Peter Májek, and Ivet Bahar. PLoS Comput Biol 5(4): e1000360 (2009).

Intrinsic Dynamics and Allostery: Learning from Theory, Computations and Experiments 60 The significance of protein dynamics in achieving molecular functions in the cell is widely recognized. The old view, ‘a unique structure for each protein’, is now Ivet Bahar replaced by ‘an ensemble of conformers’ accessible near native state conditions. Many studies suggest that fluctuations between the conformers, or transitions Department of Computational and between their representative substates, underlie, if not enable, functional events. Systems Biology, School of Medicine, Elastic network models (ENMs) and spectral graph theoretical analysis methods are broadly used for exploring the collective dynamics intrinsically accessible to University of Pittsburgh, biomolecular systems to elucidate structure-encoded dynamics and function. In Pittsburgh, PA 15260 parallel with the multiplicity of conformers accessible under physiological condi- [email protected] tions, the Protein Data Bank contains multiple structures of the same protein in different forms (e.g., orthologs, mutants, substates visited during an allosteric cycle, or various complexes, multimers or assemblies). These datasets usually convey valuable information on functional changes in structure. Furthermore, they can help benchmark and improve theoretical models, computational methods and software. Our recent work suggests that the information inferred from these experimental datasets and those predicted by theory and computations can be advantageously combined to gain insights into the allosteric mechanisms of activation or inhibition of target proteins. Recent applications will be presented, along with a discussion 1034 of the limits of applicability of ENM-based approaches and molecular dynamics simulations, and future directions to overcome these limitations (1).

Support from NIH 5R01GM086238-02 is gratefully acknowledged.

Reference

1. I. Bahar, T. R. Lezon, L. -W. Yang, and E. Eyal. Annu Rev Biophys 39, 23-42 (2010).

Perturbation Response Scanning Method for Identifying Allosteric Transitions and 61 Utilizing in Flexible Docking Z. N. Gerek A. Bolia We have recently developed coarse-grained method; perturbation response scanning (PRS) that couples elastic network models (1) with linear response theory S. B. Ozkan* (LRT). It computes the response of the protein structure (i.e. displacement vector) upon exerting directed random forces on selected residues. The method has proven successful in reproducing residue displacements for a set of 25 proteins that display Center for Biological Physics, a variety of conformational motions upon ligand binding (2). Using PRS we ana- Department of Physics, lyzed two PDZ domain proteins (PSD95 PDZ3 domain and hPTP1E PDZ2 domain) Arizona State University, whose allosteric behavior play a key role in signaling. By PRS, we first identified the residues that give the highest response upon perturbing the binding sites. Tempe, AZ 85042 Strikingly, we observe that the residues that give the highest response agree with *[email protected] experimentally determined residues involved in allosteric pathways. Second, we constructed the allosteric pathways by clustering the residues giving same type of response upon perturbation of the binding sites. Interestingly our analysis provided molecular understanding of experimentally observed hidden allostery of PSD95. We have shown that removing the distal alpha helix from the binding site alters the allosteric pathway and decreases the binding affinity. Overall, these results indicate that (i) PRS is successful in capturing the conformational changes upon binding (2), (ii) it can identify key residues that mediate long-range communication in PDZ

Figure 1: Allosteric pathways of wild type PSD95 and truncated (distal α-helix) PSD 95. Allosteric pathway upon trun- cation the distal a-helix changes and this also leads a change in binding affinity and confirmed with our flexible docking and experimental analysis. domain proteins (3), (iii) we can construct the allosteric pathways and show how 1035 the allosteric pathway changes upon minor alteration in the fold with PRS (3). Utilizing these exceptional features of PRS, we have recently developed a flexible docking scheme which predicts the peptide-protein interactions accurately.

References

1. E. D. Akten, S. Cansu, and P. Doruker. J Biomol Struct Dyn 27, 13-25 (2009). 2. C. Atilgan, Z. N. Gerek, S. B Ozkan, and A. R. Atilgan. Biophys J 99, 933-943 (2010). 3. Z. N. Gerek and S. B Ozkan. PLoS Comp Biol, submitted (2010).

Linking Allostery in Chaperonins to Protein Folding

Chaperonins consist of two back-to-back stacked oligomeric rings with a cavity at each end in which protein folding can take place under confining conditions (1). 62 They are molecular machines that assist protein folding by undergoing large-scale Amnon Horovitz ATP-driven allosteric transitions between protein substrate binding and release states (1, 2). The intra-ring conformational changes in chaperonins were found to be concerted in the case of the homo-oligomeric prokaryotic chaperonin GroEL Department of Structural Biology, and sequential in the case of the hetero-oligomeric eukaryotic chaperonin CCT Weizmann Institute, (2). Previously (3), we hypothesized that a sequential allosteric mechanism might Rehovot 76100, Israel be more beneficial for eukaryotic proteins that tend to be larger and multi-domain as it may enable one domain to detach from the chaperonin and start folding while [email protected] the other domain(s) is still bound, thereby mimicking co-translational folding. By contrast, we reasoned that a concerted mechanism is likely to be more beneficial for prokaryotic proteins that tend to be smaller and single-domain and, thus, may need to be released in an all-or-none fashion in order to fold efficiently.

Support for this hypothesis was first obtained from lattice model simulations of single- and double-domain protein folding in chaperonin cages that undergo concerted or sequential allosteric transitions (4). We then took advantage of a GroEL mutant (D155A) that undergoes sequential intra-ring allosteric transitions (5) to test our hypothesis experimentally. In one test, we used a chimeric fluorescent protein substrate, CyPet-Ypet, for which it was possible to determine the folding yield of each domain from its intrinsic fluorescence and that of the entire chimera by measuring FRET between the two domains. Hence, it was possible to determine whether release (and thus also folding) of one domain is accompanied by release of the other domain (concerted mechanism) or if their release is not coupled. Our results showed that the chimera’s release tends to be concerted when its folding is assisted by wild-type GroEL but not when it is assisted by the D155A mutant that undergoes a sequential allosteric switch (6). In a second test, we used chimeras in which a substrate whose release requires the co-chaperonin GroES is fused to a GroES-independent substrate (7). In the case of the D155A mutant, release and folding of the GroES-dependent substrate was found to take place in a step-wise fashion upon addition of ATP whereas, in the case of wild-type GroEL, substrate release was found to have a sigmoidal dependence on ATP concentration. Our results, therefore, demonstrate that changes in the allosteric mechanisms of chaperonins can impact their folding function.

References

1. A. L. Horwich, W. A. Fenton, E. Chapman, and G. W. Farr. Annu Rev Cell Dev Biol 23, 115-145 (2007). 2. A. Horovitz and K. R. Willison. Curr Opin Struct Biol 15, 646-651 (2005). 3. D. Rivenzon-Segal, S. G. Wolf, L. Shimon, K. R. Willison, and A. Horovitz. Nat Struct Mol Biol 12, 233-237 (2005). 4. E. Jacob, A. Horovitz, and R. Unger. Bioinformatics 23, i240-i248 (2007). 5. O. Danziger, D. Rivenzon-Segal, S. G. Wolf, and A. Horovitz. Proc Natl Acad Sci USA 100, 13797-13802 (2003). 6. N. Papo, Y. Kipnis, G. Haran, and A. Horovitz. J Mol Biol 380, 717-725 (2008). 7. Y. Kipnis, N. Papo, G. Haran, and A. Horovitz. Proc Natl Acad Sci USA 104, 3119-3126 (2007). 1036 Computational Modeling of Allostery-Driven Unfolding and Translocation of Substrate Proteins

Molecular chaperones employ diverse ATP-dependent mechanisms to effect protein folding and degradation. Chaperonin nanomachines assist protein folding 63 through concerted allostery in bacteria (1, 2) and sequential allostery in eukary- George Stan1* otic and archaeal organisms (3, 4). Clp ATPases, which are hexameric ring-shaped AAA+ nanomachines that perform substrate protein (SP) unfolding and transloca- 1 Andrea Kravats tion for protein degradation, are suggested to undergo sequential allostery(5). We Sam Tonddast-Navaei1 use coarse-grained simulations to study the protein remodeling actions of ClpY, Manori Jayasinghe2 which contains a single ATP-binding domain per subunit, onto a tagged four-helix bundle SP (Figure 1). Our results indicate that unfolding is initiated at the tagged C-terminus via an obligatory intermediate (6). Translocation proceeds on a dif- 1Department of Chemistry, University ferent timescale than unfolding and involves sharp stepped transitions. We find that an ordered sequential mechanism is more effective than random or concerted of Cincinnati, Cincinnati, OH 45221 2Department of Chemistry, Northern Kentucky University, Highland Heights, KY 41099 *[email protected]

Figure 1: Unfolding and translocation of the fusion protein formed by a four-helix bundle protein (purple) and the SsrA peptide (yellow) by the ClpY ATPases (green). For clarity, two of the six ClpY subunits are not shown.

allostery. In the absence of allosteric motions, mechanical unfolding of the SP in atomic force microscopy experiments proceeds via multiple unfolding pathways. SP threading through a non-allosteric ClpY nanopore involves simultaneous unfolding and translocation effected by strong pulling forces.

The p97 nanomachine is a homologue of ClpA and ClpB, which contain two ATP- binding domains per subunit. We use coarse-grained and atomistic simulations to investigate the unfolding mechanism of the four-helix bundle protein coupled with ATP-driven conformational changes in the D2 domain of p97. Our simulations 1037 suggest that SP unfolding and translocation takes place as a result of the collaboration between strongly conserved sites, Arg586 and Arg599 residues and the D2 central pore loop. The mechanism of SP translocation involves a mechanical force exerted by the D2 central pore loop combined with substrate binding at the Arginine sites of adjacent subunits. Unlike ClpY-assisted action, SP unfolding and translocation actions effected by p97 are simultaneous. We find that accumulation of the SP chain within the central cavity of the D2 does not result in significant SP refolding.

This research has been supported by a grant from the American Heart Association and by the National Science Foundation CAREER grant to G. S. and an University Research Council fellowship at the University of Cincinnati to M.J.

References

1. D. Thirumalai and G. H. Lorimer. Annu Rev Biophys Biomol Struct 30, 245-269 (2001). 2. G. Stan, G. H. Lorimer, D. Thirumalai, and B. R. Brooks. Proc Natl Acad Sci USA 104, 8803-8808 (2007). 3. A. Horovitz and K. Willison. Curr Op Struct Biol 15, 646-651 (2005). 4. M. Jayasinghe, C. Tewmey, and G. Stan. Proteins: Structure, Function, and Bioinformatics 78, 1254-1265 (2010). 5. A. Martin, T. A. Baker, and R. T. Sauer. Nature 437, 1115-1120 (2005). 6. A. Kravats, M. Jayasinghe, and G. Stan. Proc Natl Acad Sci USA (in press).

Energetically Favourable Communication Pathways in Pyrrolysyl-tRNA Synthetase 64 Aminoacyl-tRNA synthetases (aaRS) play a pivotal role in the protein biosynthetic machinery, ensuring the correct translation of the genetic code. The response to Moitrayee Bhattacharyya cognate tRNA binding is elicited in the release of the activated amino acid from Saraswathi Vishveshwara* the pre-transfer complex to the 3’ end of the tRNA. Such efficient communication across distant sites (1) underlies allostery, making the aaRS an excellent Molecular Biophysics Unit, Indian model for studying this phenomenon. Several studies at atomistic detail (2, 3), including investigations on aaRS (such as MetRS and TrpRS) from our own lab Institute of Science, Bangalore, India (4, 5), have elucidated allosteric communications. Here we have chosen an atypi- *[email protected] cal aaRS, pyrrolysyl-tRNA synthetase (from D.hafniense [DhPylRS]), in its three different states of ligation (Sys1: native DhPylRS, Sys2: DhPylRS+2YLY, Sys3: DhPylRS+2YLY+2tRNA) for our study. Interestingly, in contrast to canonical aaRS, DhPylRS exhibits a diffused recognition of tRNA bases (not residing in the triplet codon). Recent crystal structure of DhPylRS [dimer] bound to tRNAPyl has given insights into the unique protein-tRNA interactions accounting for the orthog- onality of this aaRS-tRNA pair (6).

In this study, we investigate the interaction energy weighted (7) protein-tRNA network and its dynamical properties to gain insight into the functioning of this non-canonical tRNA-synthetase and understand the mechanism of long-range communications. The concept of pre-existing paths of communication (8) and their manifestations at different liganded forms are probed in this study. Specifically, the ensemble derived interaction energy between residues is a parameter that regulates the transmission of perturbation upon ligand binding between distant functional sites in DhPylRS. Interaction energy based long-range residue coupling from the MD ensembles is found to contribute to global signal transfer. Our analysis reveals 1038 the importance of side-chain interactions while backbone conformational changes are not significant for Sys1-3. The ligand induced changes in conformation and communication pathways are efficiently captured by protein-tRNA energy networks for the three systems. We also probe different weighted network parameters (e.g., betweenness and funneling) to obtain the key residues for signal propagation across distant sites. The cost of communication between distant functional sites and pre-existence of the optimal and sub-optimal pathways in the MD ensemble is also investigated. Furthermore, asymmetry in terms of communication efficiency between the two subunits is clearly evident from all our studies for Sys1-3 to various extents, with Sys3 being maximally asymmetric. Interestingly, the concept of half-sites reactivity discussed in literature agrees well to this asymmetry between the two subunits. Additionally we find that the transfer of activated amino acid to the 3’ end of tRNA is co-ordinated by alternation of global rigidity/flexibility. Our results exhibit good correlation with mutagenesis experiments for DhPylRS. Based on these observations, a general mechanistic insight for allosteric communication and the relevance of asymmetry in the dimeric protein is presented.

Abbreviations: YLY: adenylated pyrrolysine; Pyl: pyrrolysine.

References

1. T. Zhu, B. Wu, B. Wang, and C. Zhu. J Biomol Struct Dyn 27, 573-579 (2009). 2. C. Chennubhotla and I. Bahar. PLoS Comput Biol 3, e172 (2007). 3. A. Sethi, J. Eargle, A. A. Black, and Z. Luthey-Schulten. ProcNatl Acad Sci USA 106, 6620 (2009). 4. A. Ghosh and S. Vishveshwara. Proc Natl Acad Sci USA 104, 15711 (2007). 5. M. Bhattacharyya, A. Ghosh, P. Hansia, and S. Vishveshwara. Proteins: Struct, Funct, and Bioinfo 78, 506 (2010). 6. K. Nozawa, P. O/’Donoghue, S. Gundllapalli, Y. Araiso, R. Ishitani, T. Umehara, D. Soll, and O. Nureki. Nature 457, 1163 (2009). 7. M. Vijayabaskar and S. Vishveshwara. Biophys J (accepted), (2010). 8. A. del Sol, C. -J. Tsai, B. Ma, and R. Nussinov. Structure 17, 1042 (2009). Modeling Three Dimensional Structures of 1039 Complete PKS Modules for Understanding Inter-domain Interactions

Modular polyketide synthases (PKS) utilize multiple copies of distinct sets of catalytic domains, called modules for catalyzing biosynthesis of a variety of phar- 65 maceutically important natural products (1). Even though bioinformatics analysis Swadha Anand* has played a major role in discovery of novel secondary metabolites and rational Debasisa Mohanty design of natural product analogs, most of these computational methods have used sequence information alone. However, recently available crystal structures of mammalian FAS and large polypeptide stretches from modular PKS indicate that, the National Institute of Immunology, Aruna polyketide biosynthesis is brought about by a tightly coupled network of catalytic Asaf Ali Marg, New Delhi-110067, India and structural domains (2). Therefore, it is necessary to model three-dimensional structures of complete PKS modules for understanding role of inter domain interac- *[email protected] tions in substrate channeling.

Structure based sequence analysis on a data set of 662 KS, 541 AT, 308 DH, 99 ER, 450 KR and 562 ACP domains from 55 modular PKS clusters indicate that, except for the structural sub-domain of KR, all other domains show significant sequence similarity with available structural templates. Despite the high sequence divergence, the structural sub-domain of KR can be modeled using threading approach. The dimeric structure of complete PKS module could also be modeled based on the relative orientation of different domains in mechanistically analogous mammalian FAS structure (3). Modeling of a bi-modular PKS protein using this approach has provided valuable clues for recent discovery of a novel modularly iterative mechanism of mycoketide biosynthesis in Mycobacterium tuberculosis (4). Since the mammalian FAS structure lacks ACP domain, we have tried to predict its orientation with respect to other catalytic domains using protein-protein docking and molecular dynamics methods (5, 6). Long molecular dynamics simulations on the KS-AT di-domain structure indicate that, the extent of inter domain movement within a module is not large enough to bring them in proximity for acyl transfer. Thus, intrinsic flexibility of the linker regions preceding ACP might facilitate interaction of ACP with other catalytic domains. These results on inter domain interactions within PKS modules have interesting implications for design of domain swapping experiments for obtaining natural product analogs by biosynthetic engineering.

References

1. B. Shen. Curr Opin Chem Biol 7, 285-95 (2003). 2. R. S. Gokhale, R. Sankaranarayanan, and D. Mohanty. Curr Opin Struct Biol 17, 736-43 (2007). 3. S. Anand, M. V. Prasad, G. Yadav, N. Kumar, J. Shehara, M. Z. Ansari, and D. Mohanty. Nucleic Acids Res 38, W487-96. 4. T. Chopra, S. Banerjee, S. Gupta, G. Yadav, S. Anand, A. Surolia, R. P. Roy, D. Mohanty, and R. S. Gokhale. PLoS Biol 6, e163 (2008). 5. P. Sklenovsky and M. Otyepka. J Biomol Struct Dyn 27, 521-539 (2010). 6. M. J. Aman, H. Karauzum, M. G. Bowden, and T. L. Nguyen. J Biomol Struct Dyn 28, 1-12 (2010). 1040 Molecular Dynamics Simulations of Protein Unfolding and Translocation Resulting from Allosteric Motions of ClpY

Clp ATPases are macromolecular machines which use the energy released from 66 ATP hydrolysis to unfold, translocate and degrade misfolded proteins. ClpY, a bac- Andrea Kravats1* terial unfoldase within this family, assembles into a homohexameric ring structure Manori Jayasinghe2 with a narrow central pore. Flexible diaphragm forming loops within this channel undergo large scale conformational changes driven by ATP binding and hydro- 1 George Stan lysis. The result is unfolding and translocation of a tagged substrate protein. We employ coarse grained molecular dynamics simulations to probe coupling between the allosteric motions of the central pore loops and the unfolding and translocation 1Department of Chemistry, University of a four helix bundle protein (Figure 1). We determine that minimal unfolding of of Cincinnati, Cincinnati, OH 45221 the SP into an obligatory non-native intermediate, a three helix bundle, is required 2Department of Chemistry, Northern for translocation. The pathway for unfolding is unraveling from the C-terminus, which is in agreement with experiments (1). Multiple translocation pathways are Kentucky University, Highland Heights, observed following the initial unfolding event. Weak mechanical forces exerted by KY 41099 the pore loops accompanied by transient SP binding to the I domain effect trans- *[email protected] location (2). We also investigate the ordering of allosteric transitions within individual subunits of the ClpY ring. Experiments suggest non-concerted allostery (3); however, the preference between sequential and random allostery remains unclear. To determine the efficacy of the possible allosteric mechanisms, we perform simulations of concerted, random, and sequential (clockwise and counterclockwise, viewed proximal to the I domain) mechanisms. The concerted simulations do not result in translocation, in accord with experiments (1). Our results indicate that sequential clockwise simulations are the most efficient in the handling and translocation of the substrate protein.

Figure 1: Unfolding and translocation of a substrate protein formed from the four helix bundle protein (magenta) and ssrA degradation tag (yellow). Two subunits of ClpY (green) have been removed. References 1041 1. C. Lee, M. Schwartz, S. Prakash, M. Iwakura, and A. Matouschek. Mol Cell 7, 627-637 (2001). 2. A. Kravats, M. Jayasinghe, and G. Stan. Proc Natl Acad Sci USA 108, 2234-2239 (2011). 3. A. Martin, T. A. Baker, and R. T. Sauer. Nature 437, 1115-1120 (2005).

Evolution of Structure and Dynamics for a Family of Intrinsically Disordered Proteins 67 Intrinsically disordered proteins (IDPs) perform essential functions in organisms 1 from all phyla. IDPs do not form tertiary structures and contain varying amounts of Gary W. Daughdrill * secondary structure. In order to develop general relationships between the structure Wade Borcherds1 and function of IDPs, we are investigating the structure and dynamics of protein Hongwei Wu1 families that are intrinsically disordered. The work is being placed in an evolution- 2 ary context to permit the identification of important structural features by virtue Bin Xue of their conservation and constitutes the first attempt to quantify the relationship Vladimir Uversky2 between sequence identity and structural similarity for IDPs. The intrinsically disordered transactivation domain of the tumor suppressor, p53 (p53TAD) is one 1Department of Cell Biology, model system chosen for study. Significant differences are observed in the secondary structure and dynamics of mammalian homologues of p53TAD. These differ- Microbiology, and Molecular Biology ences are primarily localized to the binding sites for the ubiquitin ligase, MDM2 and Center for Drug Discovery and and the 70 KDa subunit of replication protein A (RPA70) and appear to influence Innovation the kinetics and thermodynamics of binding. We are also investigating the role of prolines in controlling the structure and dynamics of IDPs. Mutating the conserved 2Department of Molecular Medicine, prolines that flank the MDM2 binding site has a striking effect on the structure and University of South Florida, dynamics of this region. An analysis of other IDPs that fold when they bind to their Tampa, Fl 33612 protein partners shows that proline residues are enriched in the regions flanking the binding sites, suggesting the structural and dynamical effects observed for the *[email protected] p53TAD homologues are general.

This work is supported by the American Cancer Society (RSG-07-289-01-GMC) and the National Science Foundation (MCB-0939014). 1042 Disordered Proteins in Parkinson’s and Alzheimer’s Disease: Linking Structural Transformations to Function, Aggregation and Toxicity 68 The proteins tau and alpha-synuclein are linked to neurodegenerative disease both through their appearance within protein-rich deposits in diseased brain and through David Eliezer the identification of mutations in their associated genes which cause hereditary forms of disease. Both protein share the property of being highly disordered when Department of Biochemistry, isolated in solution, but both proteins can undergo disorder-to-order transitions either upon interactions with appropriate binding partners or upon aggregating into Weill Cornell Medical College, fibrillar amyloid-like aggregates similar to those found in brain deposits. Structural New York, NY 10065 studies of these proteins provide the basis for new hypothesis regarding how their [email protected] structural transformations may mediate both the normal function of these proteins, as well as they pathological aggregation.

Tau Change is a Key for Understanding Brain 69 Aging and Alzheimer Disease Neurofibrilarlly tangles (NFTs), which consists fibrillar aggregate of hyperphos- Akihiko Takashima phorylated tau, are commonly seen in aging and Alzheimer’s disease brain. Based on Braak staging of NFTs, NFT first observed in entorhinal cortex. Then, NFTs Laboratory for Alzheimer’s disease, spread from entorhinal cortex to limbic and neocortex. NFTs formation in entorhinal cortex may be correlating with memory loss in brain aging, because entorhinal Brain Science Institute, RIKEN, cortex is involved in memory formation, and NFTs in limbic and neocortex may 2-1 Hirosawa, Wako-shi, cause dementia in AD, because limbic and neocortex serve higher order brain func- Saitama 350-0198 tions. These suggest that regional development of NFTs is correlated with decline of brain functions in aging and AD. Recent reports sugge sted that the process of Japan NFT formation, but not NFT itself is involved in neuronal dysfunction. Normally [email protected] tau binds to microtubules and stabilize them. Once tau receives hyperphosphory- lation by activating tau kinases, tau dislodge from microtubules, and starts tau- tau interaction in cytoplasm, forming tau oligomers. When tau oligomers possess b-sheet structure, tau oligomer forms insoluble granular tau aggregate. Granular tau aggregate sticks together, and form NFT. From analysis of tau Tg mouse, we found that hyperphosphorylated tau is involved in synapse loss, and granular tau aggregate is involved in neuronal loss. Thus, during NFT formation, different tau aggregation induces synapse loss, and neuronal loss, leading to brain dysfunction in brain aging and AD. A process of tau aggregation was analyzed by ThT fluorescence and AFM observation. Role of different tau aggregates was determined by analysis of tau Tg mouse. Before tau fibril formation, tau formed soluble oligomer, insoluble granular tau aggregate. Soluble phosphorylated and oligomer tau may be involved in synapse loss, and insoluble granular tau aggregates may play a role in neuronal death. Inhibition of phosphorylated tau, and granular tau aggregation is expected to block a progression of AD symptom by preventing synapse loss and neuronal loss.

On the next page is illustrated a diagram showing the connection between each tau aggregates on synapse loss and neuron loss. 1043

Connection between each tau aggregates on synapse loss and neuron loss

Understanding The Molecular Basis of Pathogenicity or Lack Thereof of Serum Amyloid a Isoforms 70 Acute-phase protein Serum Amyloid A (SAA) is an important biomarker of inflam- Sai Praveen Srinivasan1,2 mation and the precursor protein responsible for amyloid A (AA) amyloidosis. 1,2 Under normal circumstance, SAA is found associated with high-density lipoproteins Yun Wang (HDL), but during infection or injury, SAA levels can increase as high as 1000-fold Zhuqiu Ye1,2 in ~24hrs. Under chronic inflammatory conditions, persistence of high levels of SAA Marimar Lopez2 leads to amyloid deposits composing of whole length and fragments of SAA leading 1,2 to the systemic AA amyloidosis disease. In mouse models, SAA1.1 predominates in Wilfredo Colón * amyloid deposits both as full length and fragmented forms. However, the CE/J type mouse, which expresses a single isoform (i.e. SAA2.2), was resistant to amyloidosis. 1Department of Chemistry and Chemical SAA1.1 and SAA2.2 differ only by six amino acids, suggesting that factors such as fibrillation kinetics, oligomeric states and ligand interactions might play a critical role Biology in determining their pathogenicity. The present study attempts to understand the oli- 2Center for Biotechnology and gomeric and aggregation mechanism of the pathological and non-pathological forms Interdisciplinary Studies, Rensselaer of SAA, SAA1.1 and SAA2.2 respectively. Our data show that both SAA isoforms exhibit marked differences in oligomeric propensities, fibrillation kinetics and ther- Polytechnic Institute, Troy, mal stabilities suggesting the importance of location of specific amino acid residues. New York, 12180 SAA1.1 was found to be more intrinsically disordered and exhibited low thermal sta- *[email protected] bility when compared to SAA2.2. Furthermore, SAA1.1 showed a longer lag phase (3-4 days) prior to fibrillation, when compared to SAA2.2 (3-6 hrs). To obtain insights on the molecular basis of these observed differences, point mutations were performed on SAA2.2 to make it resemble SAA1.1, one amino acid at a time. Overall, comparison of the stability, oligomeric structure and aggregation properties of SAA2.2 and SAA1.1 provides insight that explains their difference in pathogenicity. Furthermore, the intrinsically disordered structure of SAA2.2 and SAA1.1 and their ability to form a diversity of self-assembled structures at 37°C suggests that the structure of SAA might be modulated in vivo to form different biologically relevant species. 1044 DNA: Not Merely the Secret of Life

Structural DNA nanotechnology is based on using stable branched DNA motifs, like the 4-arm Holliday junction, or related structures, such as double crossover (DX), triple crossover (TX), and paranemic crossover (PX) motifs. The sequence 71 design of stable branched molecules is based on the notion of minimized sequence symmetry. We have been working since the early 1980’s to combine these DNA Nadrian C. Seeman motifs to produce target species. From branched junctions, we have used ligation to construct DNA stick-polyhedra and topological targets, such as Borromean rings. Department of Chemistry, Branched junctions with up to 12 arms have been produced. We have also built DNA nanotubes with lateral interactions. New York University, New York, NY 10003, USA Nanorobotics is a key area of application. PX DNA has been used to produce a [email protected] robust 2-state sequence-dependent device that changes states by varied hybridization topology. We have used this device to make a translational machine that prototypes the simplest features of the ribosome. Two protein-activated devices have been developed that can measure the ability of the protein to do work, and bipedal walkers, both clocked and autonomous have been built. We have also built a robust 3-state device that includes a state corresponding to a contraction.

One of the long-sought goals of nanotechnology has been the construction of molecular assembly lines. We have combined a DNA origami layer with three PX-based devices, so that there are eight different states represented by the arrangements of these 2-state devices; we have programmed a novel DNA walking device to pass these three stations. As a consequence of proximity, the devices add a cargo molecule to the walker. We have demonstrated that all eight products (including the null product) can be built from this system. More extensive origami systems could be used to make even more diverse and complex products. Most recently, we have used DNA origami in a diagnostic tool.

A central goal of DNA nanotechnology is the self-assembly of periodic matter. We have constructed 2-dimensional DNA arrays from many different motifs. We can produce specific designed patterns visible in the AFM. We can change the patterns by changing the components, and by modification after assembly. Recently, we have self-assembled a 3D crystalline array and have solved its crystal structure to 4 Å resolution, using traditional unbiased crystallographic methods. Nine other crystals have been designed following the same principles of sticky-ended cohesion. We can use crystals with two molecules in the crystallographic repeat to control the color of the crystals. Thus, structural DNA nanotechnology has fulfilled its initial goal of controlling the structure of matter in three dimensions. A new era in nanoscale control is beginning.

This research has been supported by grants GM-29554 from the National Institute of General Medical Sciences, CTS-0608889 and CCF-0726378 from the National Science Foundation, 48681-EL and W911NF-07-1-0439 from the Army Research Office, N000140910181 and N000140911118 from the Office of Naval Research and a grant from the W.M. Keck Foundation. The Label-Free Unambiguous Detection and 1045 Symbolic Display of Single Nucleotide Polymorphisms on DNA Origami Single Nucleotide Polymorphisms (SNPs) are the most common genetic variation 72 in the human genome. Kinetic methods based on branch migration have proved successful for detecting SNPs because a mispair will inhibit the progress of branch Hari K. K. Subramanian migration in the direction of the mispair. Biased single-stranded branch migration Banani Chakraborty is used prominently for changing the shapes of DNA nanomachines, because it Ruojie Sha involves the isothermal removal of strands from a DNA machine frame, enabling a change in topology. Here, we have combined the effectiveness of this approach Nadrian C. Seeman with atomic force microscopy (AFM) of DNA origami patterns to produce a direct visual readout of the target nucleotide contained in the probe sequence. The ori- Department of Chemistry, New York gami contains graphical representations of the four possible nucleotide alphabetic characters, A, T, G and C. Each of the components of the letters contains a nucle- University, New York, NY 10003, USA otide at the test site that is the complement of one of the nucleotides in the probe. [email protected] Consequently, the symbol containing the test nucleotide identity vanishes in the [email protected] presence of the probe. Computer processing of a statistically significant group of images produces a direct symbolic readout that directly identifies the nucleotide [email protected] carried by the probe. [email protected]

The figure above shows the schematic diagram of DNA Origami tile used (on the left) and the averaged AFM image of the origami tile (on right), showing the character readouts (the scale bar shows a distance of 50 nm). This method works not only with a single SNP, but also with two different probes, as would be found in the case of a heterozygous diploid organism.

This research has been supported by NIGMS, NSF, ARO, ONR and the W. M. Keck Foundation. 1046 Topological Bonding of DNA Nanostructures

The properties of DNA that allow it to act as the storage medium of genetic information also make it an outstanding molecule for use in nanotechnology. This fact has led to the development of DNA nanotechnology which is based on 73 Watson-Crick base pairing. The backbone structures involved these constructs are 1 complex species, not simple linear duplex molecules. These motifs and various Yoel Ohayon programmable structures in one, two and three dimensions are constructed by using Ruojie Sha1 sticky-ends which are short-linear extensions that hold complementary structures Ortho Flint2 together. The PX (Paranemic Crossover) motif (1) has also been reported as another 1 form of cohesion for large DNA structures. It may be useful in overcoming some Nadrian C. Seeman * of the weaknesses of sticky-ended cohesion (2). Both sticky-ended and PX cohesion are based on hydrogen-bonded interactions that cannot withstand denaturing 1Department of Chemistry, New York conditions (3). In the work presented, we used two approaches to demonstrate that the hydrogen-bonded cohesion of DNA nanostructures can be transformed into University, New York, NY 10003, USA a topological interaction. In the first case, we created a topological interaction 2Department of Mathematics, University between DNA structures that cohere via both PX and sticky-ends. Covalent link- of Western Ontario, London, ages were created between the functionalized 3’ and 5’ ends of the sticky-ends. The torus-like structures were obtained via enzymatic ligation. In the second case, ON N6A 5B7, Canada we converted the PX interaction of DNA circles containing cohesive loops into *[email protected] catenated structures via the use of Topo I enzyme by creating a linkage between the loops. The two methods were used to construct topologically linked one- dimensional DNA arrays assembled from different types of PX cohering tiles. The construction of these poly-catenated scaffolds allowed for a new method to position nano-particles on linear structures. The program Knotilus (4, 5) was used to determine and display the topology of the catenated DNA structures.

This work is supported by the grants from the National Institute of General Medical Sciences, the National Science Foundation, the Army Research Office and the Office of Naval Research.

References

1. Z. Shen, H. Yan, T. Wang, and N. C. Seeman. J Am Chem Soc 126, 1666-1674 (2004). 2. X. Zhang, H. Yan, Z. Shen, and N. C. Seeman. J Am Chem Soc 124, 12940-12941 (2002). 3. C. H. Spink, L. Ding, Q. Yang, R. D. Sheardy, and N. C. Seeman. Biophys J 97, 528-538 (2009). 4. O. Flint. “The Master Array, a complete invariant for prime alternating links” (Thesis, 2007). 5. http://knotilus.math.uwo.ca/ A Controlled DNA Biped Walker on a DX Track 1047

Molecular machines perform elegant work with high efficiency in biological systems. They have inspired attempts to create artificial machines that mimic the ability to produce controlled motion (1, 2). We describe the construction of an extendable DNA walker-track system. A monomer DX track with three foot- holders is constructed, and the biped walker moves along the cylinder side of the 74 track. Therefore the terminus of the monomer track could be used to create sticky Dadong Li ends for track extension and the total steps the walker could take increases as Ruojie Sha more monomer tracks are added. A dimer DX track with six foot-holders demonstrates the feasibility of track extension. By means of sequential addition of James Canary DNA set/unset strands (3, 4), the walker is programmed to move forward and Nadrian C. Seeman* then backward along the track. PAGE analysis demonstrates that the walker-track complex forms well. Psoralen cross-link monitoring is performed with an aliquot of material at each step. The PAGE analysis results further establish the step-wise Department of Chemistry, formation of the expected products. New York University, New York, NY 10003, USA The schematic above shows the attachment of walker to the monomer DX track via a set strand (A), and the attachment of walker to a dimer DX track (B). *[email protected]

References

1. M. Fennimore, T. D. Yuzvinsky, Wei-Qiang Han, M. S. Fuhrer, J. Cumings, and A. Zettl. Nature 424, 408-410 (2003). 2. N. C. Seeman. Nature 421, 427-431 (2003). 3. W. B. Sherman and N. C. Seeman. Nano Lett 4, 1203-1207 (2004). 4. J. Shin and N. A. Pierce. J Am Chem Soc 126, 10834-10835 (2004). 1048 A DNA Crystal Designed to Contain Two Molecules per Asymmetric Unit with Fluorescent Dyes

We have reported the X-ray crystal structure of a designed macroscopic self- 75 assembled 3D crystal based on a robust motif (1). That crystal contained a single Tong Wang1 molecular species, a tensegrity triangle (2), the molecule’s self-assembly into a Ruojie Sha1 crystal was programmed through the sticky ends at the end of each DNA double helix. One of the strengths of using the chemical information contained in sticky 1 Jens J. Birktoft ends to program self-assembly is that one ought to be able to control the number Jianping Zheng1 of species contained within the unit cell. This capability has been demonstrated in Chengde Mao2 very small (∼1-10 µm) two-dimensional crystals (3), but macroscopic 3D crystals provide the most interesting case. Here, we describe the self-assembly of a DNA 1 Nadrian C. Seeman * crystal that contains two tensegrity triangle molecules per asymmetric unit. We have used X-ray crystallography to determine its crystal structure. In addition, we have demonstrated control over the colors of the crystals by attaching either Cy3 1Department of Chemistry, New York dye (pink) or Cy5 dye (blue-green) to the components of the crystal, yielding crys- University, New York, NY 10003, USA tals of corresponding colors. By doing those, we demonstrate that we could use 2Department of Chemistry, more than one component to self-assemble DNA crystal, put the covalently bonded foreign molecules into the DNA crystal while not affecting its self-assembly and Purdue University, West Lafayette, grow the DNA crystal in the less strain condition such as low salt concentration and IN 47907, USA room temperature. *[email protected]

This work is supported by the National Institute of General Medical Sciences, the National Science Foundation, the Army Research Office and the Office of Naval Research.

References

1. J. Zheng, J. J. Birktoft, Y. Chen, T. Wang, R. Sha, P. E. Constantinou, S. L. Ginell, C. Mao, and N. C. Seeman. Nature 461, 74-77 (2009). 2. D. Liu, W. Wang, Z. Deng, R Walulu, and C. Mao. J Am Chem Soc 126, 2324-2325 (2004). 3. E. Winfree, F. Liu, L. A. Wenzler, and N. C. Seeman. Nature 394, 539-544 (1998). A Toolkit for Site-Specific DNA 1049 Interstrand Crosslinks

We report the development of technology that allows interstrand coupling between various positions across minor or major grooves within one turn of DNA duplex. 2’-Modified nucleotides were synthesized as protected phosphoramidites and 76 incorporated into DNA structures (1, 2). The modified nucleotides contained five- Miao Ye atom linkers, with either amine or carboxylic acid functional groups at their termini. Chemical coupling of amine and carboxylic acid groups in designed strands Johan Guillaume resulted in the formation of an amide bond linking two complementary strands. Yu Liu Interstrand crosslinks were formed specifically between nucleotides placed at pre- Ruojie Sha selected positions rather than selective formation between various positions as observed in previous studies. The trajectory lengths of linkers determined if the Risheng Wang termini could reach each other to enable reactions to occur. Modeling (3) and calcu- Nadrian C. Seeman lated crosslink trajectory distances were in agreement our conclusions from experi- James W. Canary* mental data. This technology enables approaches that can control regiospecific and distance dependent linkage formation, offering tools to use site-specific cross-links to probe biological phenomena (4). In addition, this approach can also be applied to Department of Chemistry, DNA nanotechnology to help build nanoscale structures along DNA templates. New York University, New York, NY 10003 *[email protected]

Acknowledgements

This research has been supported by the following grants to JWC and NCS: National Science Foundation (CTS-0608889) and the Office of Naval Research (N000140911118). This work has also been supported by grants to NCS from the National Institute of General Medical Sciences (GM-29544) the National Science Foundation (CCF-0726378), the Army Research Office (48681-EL and W911NF- 07-1-0439), and a grant from the W. M. Keck Foundation. This work was supported partially by the MRSEC Program of the National Science Foundation under Award Number DMR-0820341.

References

1. Y. Liu, A. Kuzuya, R. Sha, J. Guillaume, R. Wang, J. W.Canary, and N. C. Seeman. J Am Chem Soc 130, 10882-10883 (2008). 2. L. Zhu, P. S. Lukeman, J. W. Canary, and N. C. Seeman. J Am Chem Soc 125, 10178-10179 (2003). 3. S. Arnott, D. W. L. Hukins, and S. D. Dover. Biochem Biophys Res Comm 6, 1392 (1972). 4. D. M. Noll, T. M. Mason, and P. S. Miller. Chem Rev 106, 277-301 (2006). 1050 Amyloid Fibrils Captured inside Twenty-Helix DNA Nanotubes

Amyloid fibrils are ordered and insoluble protein aggregates that were originally found associated with neurodegenerative diseases such as Alzheimer’s disease. 77 They share a common core structure, an elongated stack of b-strands, perpendic- Anuttara Udomprasert1 ular to the fibril axis (1). These molecules have rod-like structures with avery Marie Bongiovanni2,3 high persistence length and also exhibit high thermal and chemical stability (2). Short synthetic non-disease-related peptides can induce fibril in vitro (3). There 1 Ruojie Sha are increasing observations on how these fibrils form providing more opportuni- William B. Sherman4 ties to design novel functionalized motifs (2, 4). They offer great potential as self- Monica Menzenski1 assembling materials for nanotechnology and bionanotechnology (5). However, they require the development of new methods to manipulate fibrils into organized 1 Paramjit Arora arrangements. James W. Canary1 Sally L. Gras2,3 DNA nanotubes with a cylindrical structure can be used as sheaths around rod- like molecules in biological systems and nanotechnology (6). In this work, we 1 Nadrian C. Seeman * take advantage of the powerful features of DNA to form nanotubes to capture amyloid fibrils formed from a short peptide fragment of protein transthyretin (TTR ). The scaffolded DNA origami method is used to form DNA nanotubes, 1Department of Chemistry, New York 105-115 using M13mp18 as a scaffold strand with more than 170 staple strands, designed to University, New York, NY 10003, USA have enough space for capturing the fibrils inside. We expect to be able to organize 2Department of Chemical and these bio-inspired materials onto predefined surfaces via DNA-DNA interactions. We report the results of sheathing experiments that are evaluated by atomic force Biomolecular Engineering, microscopy. The University of Melbourne, Parkville, VIC 3010, Australia References 3The Bio21 Molecular Science 1. J. F. Smith, T. P. Knowles, C. M. Dobson, C. E. MacPhee, and M. E. Welland. Proc Natl Acad Sci 103, 15806-15811 (2006). and Biotechnology Institute, 2. C. E. MacPhee and C. M. Dobson. J Am Chem Soc 122, 12707-12713 (2000). The University of Melbourne, Parkville, 3. C. M. Dobson. Trends Biochem Sci 24, 329-332 (1999). 4. S. L. Gras, A. K. Tickler, A. M. Squires, G. L. Devlin, M. A. Horton, C. M. Dobson, and VIC 3010, Australia C. E. MacPhee. Biomaterials 29, 1553-1562 (2008). 4 5. S. L. Gras. Aust J Chem 60, 333-342 (2007). Brookhaven National Laboratory, 6. A. Kuzuya, R. Wang, R. Sha, and N. C. Seeman. Nano Lett 7, 1757-1763 (2007). Upton, NY 11973, USA *[email protected] Crystalline Two-Dimensional DNA-Origami Arrays 1051

Nanotechnology aims to organize matter with the highest possible accuracy and control. Such control will lead to nanoelectronics, nanorobotics, programmable chemical synthesis, scaffolded crystals, and nanoscale systems responsive to their environments. Structural DNA nanotechnology (1) is one of the most powerful routes to this goal. It combines robust branched DNA species with the control of 78 affinity and structure (2) inherent in the programmability of sticky ends. The suc- Wenyan Liu cesses of structural DNA nanotechnology include the formation of objects (3), 2D Hong Zhong crystals (4), 3D crystals (5), nanomechanical devices (6), and various combinations of these species (7). DNA origami (8) is arguably the most effective way of produc- Risheng Wang ing a large addressable area on a 2D DNA surface. This method entails the combi- Nadrian C. Seeman* nation of a long single strand (typically the single-stranded form of the filamentous bacteriophage M13, 7249 nucleotides) with about 250 staple strands to define the shape and patterning of the structure. With a pixelation estimated at about 6 nm (8), Department of Chemistry, it is possible to build patterns with about 100 addressable points within a definable New York University, 2 shape in an area of about 10000 nm . Many investigators have sought unsuccess- New York, NY 10003, USA fully to increase the useful size of 2D origami units by forming crystals of individual origami tiles (9). Herein, we report a double-layer DNA-origami tile with two *[email protected] orthogonal domains underwent self-assembly into well-ordered two-dimensional DNA arrays with edge dimensions of 2–3 μm (see schematic representation and AFM image). This size is likely to be large enough to connect bottom-up methods of patterning with top-down approaches.

Two-dimensional crystals from DNA origami tiles

Acknowledgements

This research has been supported by the following grants to NCS: GM-29544 from the National Institute of General Medical Sciences, CTS-0608889 and CCF- 0726378 from the National Science Foundation, 48681-EL and W911NF-07-1- 0439 from the Army Research Office, N000140910181 and N000140911118 from the Office of Naval Research and a grant from the W.M. Keck Foundation.

References

1. N. C. Seeman. J Theor Biol 99, 237-247 (1982). 2. H. Qiu, J. C. Dewan, and N. C. Seeman. J Mol Biol 267, 881-898 (1997). 3. J. Chen and N. C. Seeman. Nature 350, 631-633 (1991). 4. E. Winfree, F. Liu, L. A. Wenzler, and N. C. Seeman. Nature 394, 539-544 (1998). 5. J. Zheng, J. J. Birktoft, Y. Chen, T. Wang, R. Sha, P. E.Constantinou, S. L. Ginell, C. Mao, and N. C. Seeman. Nature 461, 74-77 (2009). 6. H. Yan, X. Zhang, Z. Shen, and N. C. Seeman. Nature 415, 62-65 (2002). 7. B. Ding and N. C. Seeman. Science 314, 1583-1585 (2006). 8. P. W. K. Rothemund. Nature 440, 297-302 (2006). 9. Z. Li, M. Liu, L. Wang, J. Nangreave, H. Yan, and Y. Liu. J Am Chem Soc 132, 13545- 13552 (2010). 1052 Biosensor Design Using DNA Tile Lattices

Biosensors are of great scientific importance in many fields because of their ability to detect a physiological change or presence of various chemical or biological materials. DNA has become known as an extremely useful building material in 79 nanotechnology, especially in biosensors (1, 2). DNA has the ability to stabilize 1 structure while allowing enough flexibility to achieve the desired shapes (4). Base Lauren Hakker pairing interactions between designed DNA strands are used to construct tiles, Kimberly A. Harris2,3 from which lattice structures are assembled (Figure 1). These lattices are useful for Thom H. LaBean4 diverse molecular scale nanofabrication tasks because of their high thermal stabil- 2 ity and multiple attachment sites within and between tiles (3). These lattice struc- Paul F. Agris tures consist of a central loop, four shell strands, and four arms, totaling from nine strands of DNA (2). The central loop contains 12 unpaired thymine bases. One 1Department of Chemistry, University at goal is to adhere a single-stranded DNA (ssDNA) to the available thymine bases in the central loop, creating a “crown” on top of the lattices. The ssDNA strand will Albany-SUNY, Albany, NY 12222, USA ultimately be designed to contain a biomolecule that will serve as a detector in a 2The RNA Institute, Department of biosensor. Experiments were performed with a fluorescein labeled poly-adenosine Biological Sciences, University at ssDNA binding to the lattice and was detected by polyacrylamide gel electrophoresis in combination with a fluorescence imager. Circular dichroism was also used Albany-SUNY, Albany, NY 12222, USA to detect secondary structures of the interaction. Once preliminary experiments are 3Department of Molecular & Structural completed successfully, other biomolecules will be attached to the ssDNA to create Biochemistry, North Carolina State a tile lattice that will have the ability to function as a biosensor. University, Raleigh, NC 27695, USA 4 Department of Computer Science, Chemistry and Biomedical Engineering, Duke University, Durham, NC 27708, USA

Figure 1: (A) DNA strand structure of tiles (B) schematic drawing of tiles and lattices (C) AFM images of tiles and lattices (Adapted from 2).

References

1. T. H. LaBean and H. Li. Nanotoday 2, 26-35 (2007). 2. S. H. Park, G. Finkelstein, and T. H. LaBean. J Am Chem Soc 130, 40-41 (2007). 3. K. V. Gothelf and T. H. LaBean. Org Biomol Chem 3, 4023-4037 (2005). 4. T. H. LaBean. Nature 459, 331-332 (2009). Deoxyribozyme Sensors for Nucleic Acid Analysis 1053

Deoxyribozyme (DNAzyme) sensors are biocompatible, chemically stabile, and simple in terms of structural prediction and modification. In addition, such sensors have the potential of an improved limit of detection (LOD) due to their ability to catalytically amplify signals. Two types of deoxyribozyme sensors have been developed by us in recent years. The sensor architecture allows for a unique com- 80 bination of high selectivity and the convenience of fluorescent or visible (color Dmitry M. Kolpashchikov* change) signal monitoring in homogeneous solution. The LOD of the sensor was Yulia V. Gerasimova found to vary from 10 nM to 1 pM. Deoxyribozyme sensors are promising tools for the detection of single nucleotide substitutions in DNA and RNA molecules. Evan Cornett

References Department of Chemistry, 1. D. M. Kolpashchikov. ChemBioChem 8, 2039-2042 (2007). University of Central Florida, 2. D. M. Kolpashchikov. J Am Chem Soc 130, 2934-2935 (2008). 3. Y. V. Gerasimova, E. Cornett, and D. M. Kolpashchikov. ChemBioChem 11, 811-817 P.O. Box 162366, (2010). 4000 Central Florida Blvd., 4. D. M. Kolpashchikov. Chem Rev 110, 4709-4723 (2010). Orlando, FL 32816-2366, USA *[email protected]

Molecular Dynamics Simulations of DNA Holliday Junctions: Conformational Stability and Transitions 81 Holliday Junctions (4W-junctions) are highly conserved four-way DNA homologous replication junctions capable of undergoing salt-dependent conformational transi- Elizabeth G. Wheatley* tions between mobile open planar and immobile stacked-X forms (1). This study Susan Pieniazek investigates the effect of salt concentration on Holliday Junction conformation and David L. Beveridge dynamics. All-atom Molecular Dynamics (MD) simulations are reported on 17 base- pair Holliday Junctions with “Junction 3” (J3) cores (2). We carried out the calculations in different ionic concentrations to facilitate the transition between open and Department of Chemistry, stacked-X conformers. The results were analyzed in terms of free energy landscapes Wesleyan University, obtained with Principal Component Analysis. In low salt conditions (electroneutral- Middletown, CT 06459 ity), the open planar form transitioned to the lesser-observed Iso I stacked-X form by passing through a tetrahedral intermediate species capable, in theory, of adopting *[email protected] several stacked conformations (3). The higher salt condition (70 mM KCl) revealed a transition from open to the experimentally observed Iso II stacked form without the presence of an intermediate species. A free energy map of the lower salt condition reveals a multistate model with three distinct minima, two for the open/stacked-X conformations, and a third accounting for the tetrahedral intermediate state. On the other hand, a free energy map of the high salt simulation reveals a distinctly two-state model with two minima and a narrow pathway (energy barrier) connecting the two. The findings imply that there is a unique stacking pathway along the free energy surface for the J3 4W-junction in a high salt condition where phosphate charges are adequately shielded. Above: Holliday Junction open and stacked forms

References

1. F. Hays, J. Watson, and P. Ho. J B C 278, 49663-49666 (2003). 2. D. R. Duckett, A. Murchie, S. Diekmann, E. Kitzing, B. Kemper, and D. Lilley. Cell 58, 79-89 (1988). 3. J. Yu, T. Ha, and K. Schulten. NAR 32, 6683-6695 (2004). 1054 Multi-Step Energy Transfer as a Communication Tool in Nanoscale DNA Assemblies

Bioinspired nanoscale technology requires new means to transfer information and driving forces for reaction of relevant distances. One possible solution can be to use 82 excitation energy transfer in multiple steps to bridge large distances with high pre- Jonas K. Hannestad* cision. This resembles the light harvesting complexes of photosynthetic organisms Bo Albinsson where absorbed energy is transported in multiple steps to the reaction center. We have created photonic systems based on multistep energy transfer capable of trans- ferring excitation energy over long ranges with high specificity. Here, we present Department of Chemical and Biological a self-assembled DNA-based photonic wire where excitation energy is transported over more than 20 nm (1). The wire utilizes the interlcalator YO-PRO to mediate Engineering/Physical Chemistry, end-to-end energy transfer in the photonic wire. We also introduce a photonic net- Chalmers University of Technology, work with selectable outputs incorporated in a self-assembled DNA nano-construct. SE-41296 Gothenburg, Sweden Though the binding of YO-PRO it is possible to direct energy transfer from one output dye to the other. Our work show how self-assembled structures of DNA and *[email protected] fluorophores can function as a basis for communication on the nanometer scale.

Figure: Self-assembled DNA-based photonic wire. End-to-end energy transfer is facilitated by the meditative effect of YO-PRO.

References

1. J. K. Hannestad, P. Sandin, and B. Albinsson. J Am Chem Soc 130, 15889 (2008). Rationalizing the Outcome of One-Pot 1055 DNA Nano-Assemblies

Here we present a fully addressable four-ring DNA nanonetwork composed of tri- podal oligonucleotide building blocks and show that with a set of unique oligonucleotide building blocks, based on orthogonal sequence design, it is possible to 83 assemble non-repetitive networks with high information density. Our DNA based Erik P. Lundberg1* assembly system is focused on building fully addressable networks, using the smallest practical units of DNA, i.e. one turn of the double helix (1, 2). Having address- Calin Plesa1 able structures is a prerequisite for controlled positioning of functional units, being L. Marcus Wilhelmsson1 components for energy transfer or chemical reaction centers. Huge progress has Per Lincoln1 been made in the past decade using DNA as a building block for nanoscale fabrication (3). The nanoscale fabrication relies solely on self-assembly of carefully Tom Brown2 designed DNA molecules with the free energy of hybridization as the underlying Bengt Nordén1 driving force. With extra consideration taken to the design of base sequences, a desired structure can be created in a one-pot, one-step assembly reaction. In our study we address a fundamental problem with DNA nanofabrication based on a 1Department of Chemical and Biological one-step assembly process, i.e. the reaction yield. The total yield of DNA assem- Engineering/Physical Chemistry, blies in one-pot reactions can be described in terms of the yield of one hybridization Chalmers University of Technology, reaction, raised to the power of total number of events. We therefore look for and suggest alternative assembly strategies, one of which being a fixation strategy based SE-41296 Gothenburg, Sweden on click chemistry, creating robust units for a modular build-up approach (4). 2School of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, U.K. *[email protected]

References

1. J. Tumpane, R. Kumar, E. P. Lundberg, P. Sandin, N. Gale, I. S. Nandhakumar, B. Albinsson, P. Lincoln, L. M. Wilhelmsson, T. Brown, and B. Nordén. Nano Lett 7, 3832-3839 (2007). 2. J. Tumpane, P. Sandin, R. Kumar, V. E. C. Powers, E. P. Lundberg, N. Gale, P. Baglioni, J. M. Lehn, B. Albinsson, P. Lincoln, L. M. Wilhelmsson, T. Brown, and B. Nordén. Chem Phys Lett 440, 125-129 (2007). 3. N. C. Seeman. Nano Lett 10, 1971-1978 (2010). 4. E. P. Lundberg, A. H. El-Sagheer, P. Kocalka, L. M. Wilhelmsson, T. Brown, and B. Norden. Chem Commun 46, 3714-3716 (2010). 1056 Programming Curvature in DNA Nanotubes

Organizing materials at the nanoscale with high precision is of great interest for many applications, ranging from electronics to optics and biophysics (1). As one of the most programmable self-assembling materials, DNA is an excellent building block to template this fine organization. We have recently reported the construction 84 of DNA nanotubes with the ability to readily program geometry, length, single- or Graham D. Hamblin double-stranded character, and the ability to encapsulate and selectively release Hanadi F. Sleiman* materials within these structures (2-4). Here, we report our progress towards higher- order control over these nanotubes. They are built in a modular fashion; by making localized structural alterations to individual components, we examine how these Chemistry Department, McGill structural changes can be amplified into long-range motion throughout the final University, 801 Sherbrooke St West, tube. As an example, we present curved DNA nanotubes in which the degree and occurrence of curvature is under external control: pH, light, and externally added Montreal, QC H3A2K6 biomolecules are used as triggers to effect these changes. These assemblies could *[email protected] find applications as biophysical tools, molecular machines, and dynamic templates for nanocircuitry.

References

1. G. Cao. Synthesis, Properties and Applications; Imperial College: London, 110 (2004). 2. P. K. Lo, P. Karam, F. A. Aldaye, C. K. McLaughlin, G. D. Hamblin, G. Cosa, and H. F. Sleiman. Nature Chemistry 2, 319-328 (2010). 3. P. K. Lo, F. Altvater, and H. F. Sleiman. J Am Chem Soc 132, 10212-10214 (2010). 4. F. A. Aldaye, P. K. Lo, P. Karam, C. K. McLaughlin, G. Cosa, and H. F. Sleiman. Nature Nanotechnol 4, 349-352 (2009). DNA Nanoarchitechtures and Mechanical Devices 1057

The idea behind our research is to use DNA as a programmable tool for directing the self-assembly of molecules and materials. The unique specificity of DNA interactions, our ability to code specific DNA sequences and to chemically functional- ize DNA, makes it the ideal material for controlling self-assembly of components 85 attached to DNA sequences. We have explored some new approaches in this area such as the use of DNA for self-assembly of organic molecules and for electro- Kurt V. Gothelf chemical sensors. Centre for DNA Nanotechnology, In this presentation it is demonstrated how DNA origami (1) can be used to assemble organic molecules, study chemical reactions with single molecule resolution iNANO and Department of Chemistry, (2), and position dendrimers and other materials. After initial 2D designs we made Aarhus University, a 3D DNA origami box with a lid that could be controlled and the lid motion was 8000 Aarhus C Denmark monitored by FRET (3). [email protected] Recently, we have designed a new type of DNA actuator that has a sliding type of motion. It can be positioned in 11 discrete positions and be shifted between the positions. The motion was followed by FRET and by performing chemical reactions that are only geometrically possible in certain states of the actuator (4).

References

1. P. K. W. Rothemund. Nature 440, 297 (2006). 2. N. V. Voigt, T. Torring, A. Rotaru, M. F. Jacobsen, J. B. Ravnsbaek, R. Subramani, W. Mamdouh, J. Kjems, A. Mokhir, F. Besenbacher, and K. V. Gothelf. Nature Nanotech 5, 200-203 (2010). 3. E. S. Andersen, M. Dong, M. M. Nielsen, K. Jahn, R. Subramani, W. Mamdouh, M. M. Golas, B. Sander, H. Stark, C. L. Oliveira, J. S. Pedersen, V. Birkedal, F. Besenbacher, K. V. Gothelf, and J. Kjems. Nature 459, 73-76 (2010). 4. Z. Zhang, E. M. Olsen, M. Kryger, N. V. Voigt, T. Tørring, E. Gültekin, M. Nielsen, R. M. Zadegan, E. Andersen, M. M. Nielsen, J. Kjems, V. Birkedal, and K. V. Gothelf. Angew Chem Int Ed (2011), in press.

Nucleic Acid-based Molecular Devices

In recent years, the predictable interactions between complementary sequences of DNA or RNA molecules have been utilized for the construction of a large variety of 86 synthetic biomolecular structures and devices. For instance, the recently developed Friedrich C. Simmel DNA origami technique enables the assembly of two- and even three-dimensional molecular objects with almost arbitrary shape - and with nanoscale precision. These structures can be used to arrange other molecular components, e.g. proteins, into Physics Department, ZNN/WSI well-defined geometries. Researchers envision the utilization of such structures as TU München, molecular assembly lines, or for the arrangement of artificial enzyme cascades. 85748 Garching, Germany

In order to demonstrate the function of molecular-scale structures and devices, appro- [email protected] priate characterization tools are required. Typically, DNA nanostructures are studied using scanning probe or electron microscopy techniques, but in many cases optical characterization methods would be preferable. In the first part of the talk, it will be discussed how modern super-resolution microscopy methods can be applied to study DNA assemblies whose dimensions are well below the classical diffraction limit.

In addition to the realization of static molecular nanostructures one of the visions of molecular nanotechnology is the generation of dynamic molecular assemblies that resemble naturally occurring molecular machines. In fact, DNA and RNA molecules have already been utilized for the construction of a variety of molecular devices that can be switched between several distinct conformational states, that 1058 display nano-scale motion or that bind and release molecules on demand. More- over, DNA recognition reactions have also been employed for the realization of artificial regulatory circuits, which can be used to control the timing of molecular assembly processes, or to direct the operation of nucleic acid-based nanodevices. In the second part of the talk, an example for an artificial RNA-based reaction network will be demonstrated that controls the motion of a DNA nanodevice.

References

1. S. Modi, D. Bhatia, F. C. Simmel, and Y. Krishnan. Journal of Physical Chemistry Letters 1, 1994-2005 (2010). 2. C. Steinhauer, R. Jungmann, T. Sobey, F. C. Simmel, and P. Tinnefeld. Angewandte Chemie 48, 8870-8873 (2009). 3. R. Jungmann, C. Steinhauer, M. Scheible, A. Kuzyk, P. Tinnefeld, F. C. Simmel. Nano Letters 10, 4756-4761 (2010). 4. F. C. Simmel. Nanomedicine 2, 817-830 (2007).

Structure of DNA Four-Way Junctions: Effect 87 of Ions and Proteins Holliday or DNA four-way junctions are important intermediates in recombina- C. Iulia Vitoc tion and repair processes (1-4). These structures can change conformation rapidly Olga Buzovetsky in solution interconverting from an open, four-fold symmetrical structure, with Jacob Litke no central base stacking to one in which coaxial stacking of the helical arms has been observed (5). The open structure, which is capable of branch migration, is Yan Li functionally relevant; however, many proteins have been observed to bind to and Ishita Mukerji* stabilize the stacked form. Our investigations have focused on elucidating the conformational changes induced by the binding of ions and the architectural proteins, HU and IHF, to improve understanding of the stacked form of the junction. Using Molecular Biology and fluorescence spectroscopic methods, we have examined the ion-binding site and Biochemistry Department, have explicitly explored the coordination of ions to the central region of the junc- Molecular Biophysics Program, tion. Förster resonance energy transfer (FRET) experiments have revealed that the degree of stacking or interduplex angle (IDA) of the junction is modulated by ion Wesleyan University, size, where larger ionic radii lead to larger IDAs. The proteins, HU and IHF, which Middletown, CT 06459 are known to bind and bend DNA, stabilize the junction in the stacked conforma- *[email protected] tion and induce a greater degree of stacking upon binding (6). In contrast, the repair protein, Msh2-Msh6, induces the junction to adopt an open conformation. All three proteins recognize and bind to the junction structure with nanomolar affinity and interestingly, Msh2-Msh6 binds the junction with higher affinity than a mismatch site. A novel FRET-mapping approach has been employed to determine the location of these proteins on the junction and indicates that the proteins bind to the central region of the junction.

This research has been supported by grants (MCB-0316625; MCB-0843656) from the NSF awarded to I.M.

References

1. R. Holliday. Genet Res 5, 282-304 (1964). 2. Y. Liu and S. C. West. Nat Rev Mol Cell Biol 5, 937-944 (2004). 3. G. T. Marsischky, S. Lee, J. Griffith, and R. D. Kolodner. J Biol Chem 274, 7200-7206 (1999). 4. T. Snowden, S. Acharya, C. Butz, M. Berardini, and R. Fishel. Molecular Cell 15, 437-451 (2004). 5. S. A. McKinney, A. C. Declais, D. M. Lilley, and T. Ha. Nat Struct Biol 10, 93-97 (2003). 6. C. I. Vitoc and I. Mukerji. Biochemistry 50, 1432-1441 (2011). Towards the Design and Synthesis of an Artificial Cell 1059

The complexity of modern biological life has long made it difficult to understand how life could emerge spontaneously from the chemistry of the early earth. The key to resolving this mystery lies in the simplicity of the earliest living cells. Through our efforts to synthesize extremely simple artificial cells, we hope to discover plau- 88 sible pathways for the transition from chemical evolution to Darwinian evolution. We view the two key components of a primitive cell as a self-replicating nucleic acid Jack W. Szostak genome, and a self-replicating cell membrane. We have recently described a simple and robust pathway for the coupled growth and division of a model primitive cell Department of Molecular Biology, membrane. Much of our current work is focused on the synthesis of self-replicating nucleic acids, including a series of phosphoramidate nucleic acids. While the rate Massachusetts General Hospital, of template copying by the polymerization of amino-sugar nucleotides can be quite Boston, MA 02114 good in such model systems, the fidelity of chemical copying is low. This has led [email protected] us to explore various nucleobase analogs in search of simple ways to enhance the fidelity of chemical replication. I will discuss recent progress towards the realization of an efficient and accurate system for the chemical replication of an informa- tional polymer. I will also describe ways in which a chemically replicating nucleic acid could lead to evolutionary changes in the membrane composition of a simple protocell, as well as ways in which the evolving cell membrane could enhance nucleic acid replication.

This work was supported by the Howard Hughes Medical Institute, the NSF and NASA. 1060 Towards Origin of Life and... What is Life, After All?

Recent literature is full of sensations opening eyes on the origin of life problems, such as creation of artificial bacteria (1), arsenic DNA (2), cyanobacteria in meteorites (3), not mentioning constantly advancing chemical systems aiming at the 89 life origin. Each of the overtures brings us closer to something apparently simple, 1,2 almost trivial, but elusive as well, since nobody knows what we are, actually, get- Edward N. Trifonov ting to. There is no established definition of life to be guided by. In perception of many the phenomenon of life is beyond the perception limits. However, if in the 1Genome Diversity Center, Institute classical Cartesian body/mind dualism of life only material part is taken, one finds himself at the brink of this something simple, almost trivial. Over hundred defini- of Evolution, University of Haifa, tions are suggested by philosophers and scientists of many generations. The fact Mount Carmel, Haifa 31905, Israel that the list continues to grow only attests to actual lack of a convincing consensus. 2Department of Functional Genomics and The author humbly suggests one definition of life that appears to be a “principal component” of all definitions. It is derived by a linguistic (word count) analysis Proteomics, Faculty of Science, Masaryk of the large corpus of the definitions. It turns out identical to the one introduced University, Kotlarska 2, CZ-61137 Brno, earlier (4) on the basis of developing theory of early molecular evolution. And the Czech Republic definition is: [email protected] Life is self-reproduction with variations.

Irrespective of whether it is correct or any close to final, the formula allows one to critically overview all known approaches to the problem of origin of life, and, perhaps, enthuse researchers to further converge on the very point of origin, if only its elusiveness is not due to yet another uncertainty principle.

References

1. D. G. Gibson, et al. Science 329, 52-56 (2010). 2. E. Pennisi and F. Wolfe-Simon. Science 330, 1734-1735 (2010). 3. R. B. Hoover. J Cosmology 13, in press. 4. E. N. Trifonov. Res Microbiol 160, 481-486 (2009).

Formation and Chiroptical Properties of Amino Acids 90 in Interstellar Ice Analogues Comets are accretions of frozen volatiles and rocky debris left over from the for- Uwe J. Meierhenrich* mation of the outer Solar System (1). In order to better understand the chemical Jean-Jacques Filippi and molecular composition of comets their formation was artificially and stepwise Cornelia Meinert reproduced in the laboratory. To this end, representative interstellar molecules containing C1 and N1 units such as 13CO and NH3 were condensed on a solid surface Jan Hendrik Bredehöft at 12 K while being irradiated at Lyman‑α. The obtained interstellar ice analogues Jun-ichi Takahashi were subjected to enantioselective gas chromatographic and mass spectrometric Laurent Nahon analysis allowing us the identification of 16 different amino acids and diamino acids (2). The molecular composition of these ices was found to be similar, but Nykola C. Jones not identical, to the amino acids and diamino acids identified in meteorites (3) Soeren V. Hoffmann and will be discussed in the context of the molecular origin of life on Earth. Ter- restrial life uses homochiral l‑amino acids for the expression of proteins (4). In a follow-up study we investigated whether a chiral symmetry breaking in amino University of Nice-Sophia Antipolis, acids is feasible under simulated interstellar conditions based upon an asymmetric Nice, France photochemical mechanism. In a UHV-chamber we deposited amorphous amino *[email protected] acid films of defined thickness on MgF2 windows (see Figure) and subjected them to circularly polarized light. The differential absorption of circularly polarized light 1061 by individual amino acid enantiomers, which determines speed and intensity of enantioselective photolysis, was recorded in the vacuum-ultraviolet spectral range, where massive circular dichroic transitions were observed (5). We report on the formation of amino acids and diamino acids under interstellar conditions and on chiroptical properties of amino acids in the solid state. These data are of importance for the molecular understanding for both origin and evolution of life on Earth and its molecular asymmetry.

This research has been supported by grants from the ANR and the European Community’s Seventh Framework Program.

References

1. A. Mann. Nature 467, 1013-1014 (2010). 2. G. M. Muñoz Caro et al. Nature 416, 403-406 (2002). 3. U. J. Meierhenrich et al. Proc Natl Acad Sci 101, 9182-9186 (2004). 4. U. J. Meierhenrich. Amino Acids and the Asymmetry of Life, Springer (2008). 5. U. J. Meierhenrich, et al. Angew Chem Int Ed 49, 7799-7802 (2010).

Spontaneous Generation of RNA in Water

Definition of life is an open problem. The largely accepted wording: “Life is a self sustained chemical system capable of undergoing Darwinian evolution” (1) 91 attains a solid operative sense but it is more the description of a process than a Giovanna Costanzo1 formal definition of a system. If we have difficulty in even formulating a rigorous Samanta Pino2 definition of life, certainly we do not know how it started. In recent years, progress 2 has been made in the search for the unitary chemical frame into which the first Fabiana Ciciriello reactions lighted up and started accumulating and evolving chemical information. Ernesto Di Mauro2 In collaboration with R. Saladino group (Università della Tuscia, Italy), we have shown that formamide (HCONH ), one of the simplest molecules grouping the 2 1 four most common elements of the universe H, C, O and N, provides a chemical Istituto di Biologia e Patologia frame potentially affording all the monomeric components necessary for the for- Molecolari, CNR, Roma, Italy mation of nucleic polymers (lastly reviewed in Saladino 2). In the presence of the 2Dip. Biologia e Biotecnologie appropriate catalysts and by moderate heating, formamide yields a complete set of nucleic bases, acyclonucleosides and favours both their phosphorylation and “Charles Darwin”, Università transphosphorylation. “Sapienza”, Roma, Italy *Ernesto. [email protected] Nucleotide phosphorylation and RNA oligomerization take place in water in non- enzymatic abiotic conditions. At moderate temperatures (40-90°C) RNA chains up to 120 nucleotides long may form from 3’, 5’-cAMP and 3’, 5’-cGMP, in the absence of enzymes or inorganic catalysts (3). Mechanisms of abiotic RNA chain extension and ligation based on base-pairing and base-stacking interactions were also observed (4, 5). The enzyme and the template-independent synthesis of long oligomers in water, from prebiotically affordable precursors, approaches the concept of spontaneous generation and evolution of (pre)genetic information.

References

1. J. Joyce, in D. W. Deamer, and G. R. Fleischaker (eds.), the foreword of “Origins of life: the central concepts”, Jones and Bartlett, Boston, 1994. 2. R. Saladino, C. Crestini, F. Ciciriello, F. Pino, G. Costanzo, and E. Di Mauro. Research in Microbiology 160, 441-448 (2009). 3. G. Costanzo, S. Pino, F. Ciciriello, and E. Di Mauro. J Biol Chem 284, 33206-33215. 4. S. Pino, F. Ciciriello, G. Costanzo, and E. Di Mauro. J Biol Chem 283, 36494-36503 (2008). 5. S. Pino, G. Costanzo, A. Giorgi, and E. Di Mauro (2011). Biochemistry, in press. 1062 RNA, a Precursor to the Origin of Life

The RNA world is believed to be the most likely precursor to the origins to life on Earth (1). We observed initially that montmorillonite clay-catalyzed the formation of RNA oligomers from activated nucleotides to yield oligomers as long as 92 10 mers. The reactions were carried out at room temperature in water and High Performance Liquid Chromatography (HPLC) separated the oligomers formed on James P. Ferris* ion exchange and reverse phase HPLC columns. The next stage in the study was Prakash C. Joshi the generation of oligomers by the elongation of decamers to form 30-50 mers. The Michael F. Aldersley third stage in the study was when 1-methyladenine was used to activate the monomer in place of imidazole. The positive charge on the 1-methyladenine resulted in John W. Delano the formation of 40-50 mers in one day rather than the in previous procedure where it took 14 days to generate 40-50 mers. Current studies on our approach to the Department of Chemistry and Chemical RNA world are the investigation of the reactions of racemic activated monomers. Mixtures of D, L-ImpA with D, L-ImpU were reacted on the premise that if RNA Biology, Rensselaer Polytechnic Institute, oligomers were formed on the primitive Earth they would have been formed as Troy, NY 12180 racemic mixtures. Since ImpA and ImpU are complementary we investigated the *[email protected] reaction of D, L-ImpA with D, L-ImpU and then isolated the dimers, trimers and tetramers. The linear dimers, cyclic dimers and trimers were separated by HPLC using ion exchange first and then each fraction was collected and separated by HPLC reverse phase chromatography. The homochirality of the linear dimers and cyclic dimers did not yield noteworthy products but it was impressive that 75% of the trimers were homochiral (2).

References

1. J. P. Ferris, P. C. Joshi, M. F. Aldersley, and J. W. Delano. J Am Chem Soc 13369-13374 (2009). 2. P. C. Joshi, M. F. Aldersley, J. W. Delano, and J. P. Ferris. Orig Life Evol Biosp 40, 000-000.

Understanding the Role of Passenger Mutations 93 in Cancer Progression 1 The development of cancer can be considered an evolutionary process within an Christopher D. McFarland organism: cells acquire mutations, compete for resources, and are subject to natural Shamil Sunyaev1,2 selection. During this transformation, malignant tissues acquire tens of thousands of Leonid Mirny1,3,4 somatic mutations, yet only a handful are believed to be responsible for the cancer phenotype, termed driver mutations (1). The rest, occurring sporadically across cancer genomes, are called passenger mutations. We hypothesize that many passenger muta- 1Harvard University Graduate Biophysics tions may be deleterious to cancer cells, yet occasionally fixate in the population. Program, Cambridge, MA 02138 We developed a stochastic, evolutionary model of cancer progression where cells 2Divison of Genetics may acquire both driver mutations (advantageous to the cells) and passenger muta- Brigham and Women’s Hospital, tions (deleterious to the cells). We found that many passengers fixate in neoplas- Harvard Medical School tic populations, despite purifying selection against them, via a mechanism similar to Muller’s Ratchet (2) and by hitchhiking with driver mutations. An analysis of 3Departmetn of Physics, Massachusetts known somatic mutations in cancer found that many passenger mutations are pre- Institute of Technology dicted to be deleterious to human cells, based on their reduction in protein stability 4Harvard-MIT Division of Health and occurrence at conserved loci—corroborating our model’s findings. We also found that biophysical properties of driver mutations may distinguish oncogenes Sciences and Technology from tumor suppressors.

Through combined analytical and computational analysis, we identified two phases of neoplastic behavior: one where driver mutations dominate dynamics and the population grows exponentially, and another where passenger mutations overwhelm the cells causing prolonged dormancy or regression. We found that the conditions for dormancy and regression may be most exploitable in early metastases and are 1063 currently testing our findings in a mouse model.

References

1. C. C. Maley, P. C. Galipeau, X. Liu, C. A. Sanchez, T. G. Paulson, and B. J. Reid. Cancer Res 64, 3414-3427 (2004). 2. J. Felsenstein. Genetics 78, 737-756 (1974).

Intercalation-Mediated Assembly and the Origin of Nucleic Acids 94 The continued increase of evidence for the central role of RNA in contemporary life seems to provide ever-increasing support for the RNA world hypothesis (1). Aaron E. Engelhart However, it is difficult to imagine how RNA polymers would have appeared Ragan Buckley de novo, as the abiotic formation of long nucleic acid polymers from mononu- Eric D. Horowitz cleotides or short oligonucleotides presents several formidable challenges in the absence of highly evolved enzymes (2). For example, under solution conditions were Nicholas V. Hud* the backbone linkages formed between mononucleotides or oligonucleotides are essentially irreversible very short cyclic products can be kinetically favored, which School of Chemistry and Biochemistry, severely limits polymer growth. We are investigating the hypothesis that reversible Center for Chemical Evolution, non-covalent interactions between small planar molecules, similar to intercalating dye molecules, originally organized and selected the base pairs of nucleic acids (3). Georgia Institute of Technology, Recent experiments in our laboratory involving intercalation-mediated DNA and Atlanta, GA 30332 RNA ligation have confirmed that intercalators, which stabilize and rigidify nucleic *[email protected] acid duplexes, almost totally eliminate strand cyclization, allowing for chemical ligation of tetranucleotides into duplex polymers of up to 100 base pairs in length (4). In contrast, when these reactions are performed in the absence of intercalators, almost exclusively cyclic tetra- and octa- nucleotides are produced. Interca- lator-free polymerization is not observed, even at tetranucleotide concentrations 10 000-fold greater than those at which intercalators enable polymerization. We have also observed that intercalation-mediated polymerization is most favored if the size of the intercalator matches that of the base pair; intercalators that bind to Watson–Crick base pairs promote the polymerization of oligonucleotides that form these base pairs. Additionally, intercalation-mediated polymerization is possible with an alternative, non-Watson–Crick-paired duplexes that selectively bind complementary intercalators. These results support the hypothesis that intercalators (acting as ‘molecular midwives’) could have facilitated the polymerization of the first nucleic acids and possibly helped select the first base pairs, even if only trace amounts of suitable oligomers were available. In another set of experiments, we are exploring the utility of reversible backbone linkages to facilitate nucleic acid polymer growth. Results from these experiments demonstrate how reversible linkages and intercalation can work together to promote the formation of extremely long polymers by the assembly and “recycling” of previously cyclized oligonucleotides.

This research was supported by the NSF and the NASA Astrobiology Center for Chemical Evolution (CHE-1004570) and the NASA Exobiology Program (NNX08A014G).

References

1. R. F. Gesteland, T. R. Cech, J. F. Atkins, Eds., The RNA World, Cold Spring Harbor Labo- ratory Press, 3rd Ed (2006). 2. A. E. Engelhart and N. V. Hud. Primitive genetic polymers, In Origins of Cellular Life, D. Deamer and J. Szostak, Eds., Cold Spring Harbor Laboratory Press (2010). 3. N. V. Hud, S. S. Jain, X. Li, and D. G. Lynn. Chem Biodiver 4, 768-783 (2007). 4. E. D. Horowitz, A. E. Engelhart, M. C. Chen, K. A. Quarles, M. W. Smith, D. G. Lynn, and N. V. Hud. Proc Natl Acad Sci USA 107, 5288-5293 (2010). 1064 Characterization of DNA and RNA Secondary Structures in Anhydrous Media

An aqueous environment has been long been postulated as being necessary for the formation of nucleic acid secondary structures. While there has been consider- 95 able interest and debate regarding the actual number of water molecules required Irena Mamajanov* to maintain a particular nucleic acid structure (1), there are obvious experimental Aaron E. Engelhart challenges to studying nucleic acid structures in the absence of water (e.g. low Heather D. Bean nucleic acid solubility). Thus far, complementary DNA strands have been reported to retain duplex structure only in anhydrous modified polyethylene glycol (2). How- Nicholas V. Hud** ever, DNA duplexes only exhibit marginal stability in other water-free organic solvents (3). An ever expanding set of new polar solvents, such as room temperature School of Chemistry and Biochemistry, ionic liquids (RTIL), provide new opportunities to study nucleic acids in water- free environments. Here, we report that nucleic acids can form duplex, triplex and Parker H. Petit Institute for G-quadruplex secondary structures that undergo reversible thermal denturation in a Bioengineering and Bioscience, water-free solvent (e.g. 0.25% water). This so-called deep eutectic solvent (DES) Georgia Institute of Technology, is comprised of one part choline choride and two parts urea. The choline choloride- urea DES has a melting point of only 12˚C, whereas pure choline chloride melts Atlanta, GA 30332-0400 (USA) at 302˚C and urea at 133˚C (4). We have found that nucleic acid secondary struc- *[email protected] tures exhibit different relative stabilities in the DES, compared to aqueous media **[email protected] (5). Thermal transition midpoints of the duplex structures in the DES are lower than those measured in water solution, whereas triplex and G-quadruplex structures can be more stable in the DES compared to an aqueous solution with the same ionic strength. Deep eutectic solvents and ionic liquids are currently of tremendous interest as nonvolatile media for a wide range of chemical reactions and processes. Given previous reports that these water-free solvents can support protein enzyme catalysis, it is now feasible that catalytic nucleic acids and protein enzyme-nucleic acid complexes could be used in these solvents.

References

1. T. V. Maltseva, P. Agback, and J. Chattopadhyaya. Nucleic Acids Res 21, 4246-4252 (1993). 2. A. M. Leone, S. C. Weatherly, M. E. Williams, H. H. Thorp, and R. W. Murray J Am Chem Soc 123, 218-222 (2003). 3. G. Bonner and A. M. Klibanov. Biotechnol Bioeng 68, 339-344 (2003). 4. A. P. Abbott, G. Capper, D. L. Davies, R. K. Rasheed, and V. Tambyrajah. Chem Comm 70-71 (2003). 5. I. Mamajanov, A. E. Engelhart, H. D. Bean, N. V. Hud. Angew Chem Int Ed Eng 49, 6310–6314 (2010). Emergence and Evolutionary Connections between 1065 Enzymatic Functions via Prototypes of Elementary Functional Loops

Earlier studies of protein structure revealed closed loops with a characteristic size 25-30 residues and ring-like shape as a basic universal structural element of globu- 96 lar proteins (1, 2). Elementary functional loops (EFL) have specific signatures and Alexander Goncearenco provide functional residues important for binding/activation and principal chemi- Igor N. Berezovsky* cal transformation steps of the enzymatic reaction (3). We derive prototypes of the elementary functional loops (EFLs) and use them for dissecting the enzymatic function into its building blocks. As a result, previously uncharted evolutionary Computational Biology connections between seemingly unrelated enzymes become apparent. We show Unit and Department of Informatics, that proteins with different folds and biochemical reactions are built from limited University of Bergen, Bergen, Norway set of common elementary functions (3). *[email protected] We study relations between enzymatic functions in Archaeal superkingdom, using elementary functional loops (EFLs). Connections between different superfamilies and folds indicate events of EFLs’ recombination in the process of emergence of new biochemical functions. We also consider methanogenesis, a metabolic pathway typical for Archaea, as a case study of enzymes with specific biochemical functions and unique cofactors. We show that some methanogenic enzymes reutilize already existing folds with tuned original functions, while other methanogenic functions are apparently built de novo from EFLs. Examples of common elementary functions and role of corresponding EFLs as basic units of enzymes is discussed.

References

1. I. N. Berezovsky, A. Y. Grosberg, and E. N. Trifonov FEBS Letters 466, 283-286 (2000). 2. E. N. Trifonov and I. N. Berezovsky. Curr Opin Struct Biol 13, 110-114 (2003). 3. A. Goncearenco and I. N. Berezovsky. Bioinformatics 26, i497-i503 (2010).

Mechanisms of Protein Oligomerization, the Critical Role of Insertions and Deletions in Maintaining Different Oligomeric States 97 Kosuke Hashimoto* Many proteins form homooligomeric complexes in a cell. Proteins that exist in dif- Anna R. Panchenko ferent oligomeric states are responsible for the diversity and specificity of many pathways, and transitions between different oligomeric states may regulate protein activity. Despite the importance of homooligomers, the mechanisms of oligomeriza- National Center for Biotechnology tion are not very well understood and general principles have not been formulated. Information, National Library In our study, we analyze sequence and structural features of homologous proteins in different oligomeric states, and how they are involved in the interface formation (1). of Medicine, National Institutes of We show that insertions and deletions which differentiate monomers and dimers Health, Bethesda, MD 20894, USA have a significant tendency to be located on the interaction interfaces. We also show *[email protected] that about a quarter of all proteins and forty percent of enzymes in our dataset have regions which mediate or disrupt the formation of homooligomers. These results suggest that relatively small insertions or deletions may have a profound effect on complex stability. Indeed, in many cases removal of enabling regions caused the strong destabilization of the complexes. Moreover, we find that enabling regions contain a larger fraction of hydrophilic residues, glycine and proline compared to conventional interfaces and surfaces, resulting in a lower aggregation propensity. Most likely, these regions may mediate specific interactions, prevent non-specific dysfunctional aggregation and preclude undesired interactions between close paralogs therefore separating their functional pathways. When we closely examine the 1066 glycosyltransferase family, which consists of highly diverged members adopting different oligomeric states, we find that homooligomeric glycosyltransferases appear as ancient as monomeric ones and go back in evolution to the last universal common ancestor and many of them have enabling or disabling features on their interfaces (2).

References

1. K. Hashimoto and A. R. Panchenko. Proc Natl Acad Sci USA 107, 20352-20357 (2010). 2. K. Hashimoto, T. Madej, S. H. Bryant, and A. R. Panchenko. J Mol Biol 28, 196-206 (2010).

An Evolutionary Tree Rooted in Actinobacteria

The three dimensional folds of each of the fifty ribosomal proteins (RibP) are fully 98 conserved in all bacterial species for which complete genomes have been reported William L. Duax* (2300 as of 3/1/11). Over 98% of all members of each bacterial RibP (bRibP) can Robert Huether be accurately aligned with retention of a small number of fully conserved identities commonly including Alanines (Ala), Prolines (Pro), Arginines (Arg), and immu- David Dziak table Glycines (Gly) and precisely located insertion or deletion sites (Indels) of Courtney McEachon restricted size. Accurate full length alignment permits identification of sequence positions where a single residue difference can separate Gram positive from Gram negative bacteria. Co-evolution of different combinations of amino acids in other Department of Structural Biology, positions in the aligned sequence separates bRibP sequences of different phylum, University at Buffalo, classes, orders and genera from one another. Accurate alignment makes it possible Buffalo, NY 14203 to follow sequence divergence through 3 billion years of evolution, reveals that the entire sequences of the ribosomal proteins of individual genera have remained over *[email protected] 95% identical throughout their evolution, detects Gene bank errors in gene length and annotation, and reveals that codon bias and use in ribosomal proteins is not determined by tRNA population. We detect significant sequence homology between all bRibPs and RibPs in the same two dozen chloroplasts (clRibP) and find that the greatest sequence homology is between RibPs of cyanobacteria and these clRibPs. On the basis of our analysis we find less sequence divergence, fewer Indels, and more restricted codon use in RibPs of the small ribosomal subunit than those of the large subunit. Conserved homology between the sequences of the bRibPs and those of archaea, mitochondrial, and chloroplasts is significantly greater for the proteins of the small subunit than the large. Of the RibPs analyzed thus far, the sequence and three dimensional structure of ribosomal protein S19 from the small subunit appears to be the most highly conserved in all species. When we align 3987 sequences of S19 (2353 bacteria, 1528 eukaryotes, and 106 archaea) only two residues are fully conserved (a Gly and an Arg). One or more Gly residues in each RibP family are immutable because the Gly conformations (phi and psi) are in regions of the Ramachandran plot where other amino acids are rarely tolerated. Arg conservation is associated with maintenance of charge balance and direct interaction with specific sites on rRNA and tRNA involved in ribosomal function. Sixteen other sequence positions have 90% conservation of amino acid identities and one sequence position, immediately adjacent to the fully conserved Gly, is occupied by only two amino acids. An Asp in this position isolates 95% of all gram-positive (G1) bacteria (866). An Asn in this position isolates 1487 bacteria, 1528 eukaryotes and 106 archaea. The 1487 bacteria include 95% of all gram-negative (G2) bacteria. This [Asp,Asn] Gly sequence forms a tight turn with an internally hydrogen-bonded network and directly interacts with ribosomal RNA via five additional hydrogen bonds [figure 1]. The amino acid composition of the S19 sequences is found to diverge in the order G1 bacteria, to G2 bacteria, to eukaryotes. In G1 bacteria ten amino acids make up 76% of their total composition, amino acids of the tRNA synthetase II family account for 61% of the composition. All G1 bacteria have 46 residues conserved at 90% identity or greater of which 14 are G, A, R or P (the amino acids encoded by the 8 codons composed of only guanine and cytosine). 1067 Optimizing GARP content of the minimum universal fingerprint of S19 isolates actinobacteria. Twelve species of Actinobacteria are found to have the highest GARP composition. All of the RibPs of the large and small subunits in these species have full length sense/antisense open reading frames, a hallmark of the DNA of genes of ancient species (1, 2). Examination of codon use in the proteins of the 30S subunits of six of these species reveals that 24 codons in which the third nucleotide is A or T are never or rarely found in their DNA. No tRNAs cognate to these “unused” codons are found in the genomes of those six species. These findings reveal that not all species use a 64 letter code, that the earliest peptides and proteins had a bias in amino acid composition favoring amino acids of tRNA synthetase II family, that immutable Gly residues are the key to aligning all members of major protein families, that ribosomal protein S19 is a rosetta stone for accurate determination of a rooted evolutionary tree of all living species and that the last universal common ancestor at the root of the tree is closely related to either K. radiotolerans or C. flavogenans or both. We will trace the evolution of all 50 bRpros in an effort to create a consistent evolutionary tree of all bacteria and unambiguously identify the last universal common ancestor at its root.

Figure 1: The sequences of residues, N53G54K55, in a tight turn in S19 forms four hydrogen bonds with the rRNA in the gram negative bacterium T. thermophilus. The immutably, 100% conserved, G54 has phi/psi values of 96.8o and -15.5o.

Support in part by: Mr Roy Carver, Stafford Graduate Fellowship, Caerus Forum Fund, The East Hill Foundation and the generous help from a number of High School students from the Buffalo NY area.

References

1. W. L. Duax, R. Huether, V. Z. Pletnev, D. Langs, A. Addlagatta A, et al. Proteins 61, 900- 906 (2005). 2. W. W. Carter and W. L. Duax. Mol Cell 10, 705-708 (2002). 3. W. L. Duax, R. Huether, V. Pletnev, and T. C. Umland. Int J Bioinform Res Appl 5, 280-294 (2009). 1068 Recently Duplicated Human Genes: Basics of Evolution

Gene duplications are one of the major sources of new protein functions. We studied the evolution of recently duplicated human genes. A genome-wide procedure 99 was used to find paralogous genes retaining detectable sequence similarity through- Alexander Y. Panchin2,3 out most exons and introns. Similar genes were collected in families. Only families containing three or more genes were analyzed. Elena N. Shustrova3 Irena I. Artamonova1,2,3* A novel method was introduced for calculating the evolutionary rates of individual genes from such families. It shows that negative selection, acting at a duplicated gene and measured by the Kn/Ks test, is relatively weaker immediately after the 1Vavilov Institute of General Genetics duplication event and then increases. Such changes of the negative selection pres- RAS, Gubkina 3, sure seem to be a major trend in the evolution of young human paralogs. 119991 Moscow, Russia Another trend concerns the asymmetry of the evolution of two gene copies result- 2Kharkevich Institute for Information ing from a recent duplication event. In about one fifth of recently duplicated gene Transmission Problems RAS, pairs from young paralogous gene families, the two gene copies accumulate amino Bolshoi Karetny pereulok 19, acid substitutions at significantly different rates. Differences in the gene expression levels do not explain this asymmetry. The asymmetry in the rate of accumulation of 127994 Moscow, Russia synonymous substitutions is much weaker and not significant. In asymmetric gene 3Lomonosov Moscow State University, pairs the number of deleterious mutations is higher in one copy, and lower in the Faculty of Bioengineering and other copy as compared to genes comprising non-asymmetrically evolving pairs. A possible explanation for this trend is the need for one of the two duplicated gene Bioinformatics, Vorobyevy Gory 1-73, copies to retain its initial function, as the other copy rapidly evolves to get a new Moscow, Russia function. *[email protected] We also compared the evolutionary rates for the original and derived copies of recently duplicated paralogous genes. For primate-specific duplications it is possible to distinguish the original copy based on conservation of the gene neighborhood in rodents, although this can be done reliable only for a minor fraction of genes produced by non-tandem duplications. The Kn values and the Kn/Ks ratios were significantly lower for the original copies compared to the derived ones. Both original and derived copies were evolving under negative selection.

This work is partially supported by the Russian Academy of Sciences (programs “Molecular and Cellular Biology” and “Biodiversity”) and the state contract P1376.

Structural and Functional Evolution of Proteins by 100 Conformational Diversity Protein evolution has most commonly been studied either theoretically, by analyz- T. J. Glembo ing the sequence of the protein (1, 2), or experimentally, by resurrecting ancestral S. B. Ozkan* proteins in the lab and performing ligand binding studies to determine function. Thus far, structural and dynamic evolution have largely been left out of molecular evolution studies. Here we incorporate both structure and dynamics to elucidate the Center for Biological Physics, molecular principles behind the divergence in the evolutionary path of the steroid Department of Physics, receptor proteins. We begin by determining the likely structure of three evolution- Arizona State University, ary diverged, ancestral steroid receptor proteins (1) using the Zipping and Assem- bly Method with FRODA (ZAMF) (2). Our predictions are within 1.9Å RMSD Tempe, AZ of the crystal structure of ancestral corticoid steroid receptor. Beyond comparing *[email protected] static structure prediction, the main advantage of ZAMF is that it allows us to observe protein dynamics. Therefore we can investigate differences in the diverged proteins’ available dynamic space. Strikingly, our dynamics analysis of these predicted structures indicates that evolutionary diverged proteins do not share the 1069 same dynamic subspace. Moreover, our dynamic analysis enables us to identify critical mutations that most affect dynamics, therefore it shows the critical mutations leading to a divergence in function, which are verified experimentally.

References

1. P. Anbazhagan, M. Purushottam, H. B. K. Kumar, O. Mukherjee, S. Jain, and R. Sowdhamini. J Biomol Struct Dyn 27, 581-598 (2009). 2. Z. Liu, Y. Zu, L. Wu, and S. Zhang. J Biomol Struct Dyn 28, 97-106 (2010). 3. J. T. Bridgham, E. A. Ortlund, and J. W. Thornton. Nature 461, 515-519 (2009). 4. T. J. Glembo and S. B. Ozkan. Biophys J 98, 1046-1054 (2010).

Structure, Function and Evolutionary Relationship of the Leptin Receptor (OB – Rb) 101 In view of the release of more and more genome sequences, the genome sequence comparisons and phylogenetic tree constructions have become popular (1-3) and R. Malathi* it will be useful to use this methodology to understand body weight regulation R. Raskin Erusan and energy homeostasis. Leptin, a 16 kDa adipocytic protein hormone is known to play a central role in regulation of body weight and energy homeostasis; it is also Department of Genetics, is also known to regulate various physiological functions such as reproduction, hematopoiesis, inflammation etc. (4, 5). Leptin acts via its specific receptor (OB – Dr. A.L.M. Post Graduate Institute of R) or LR. and till date six splice variants of OB – R have been identified and mapped Basic Medical Sciences, to the db gene locus on chromosome 1p in human. Of the six, the longest isoform University of Madras, OB – Rb which is highly expressed in hypothalamus has the signaling capability and the structure-functional relationship of this has been analysed in detail. Chennai- 600 113, India *[email protected] Alignment of the aminoacid sequences of leptin receptor (OB-Rb) from different species such as human, mouse, pig, cattle, bat, turkey, frog, zebra and madaka fish OB- Rb reveals that most of the regions including the N – terminal, transmembrane (TM), disulphide bridges and C – terminal regions are highly conserved in mammalian species with subtle variations is madaka and zebra fish. Phylogenetic analysis is suggestive of evolutionary relationship between human, monkey, cattle etc., pig and sheep, rat and mouse, zebra fish and madaka fish with distinct variation in elelephant, details of which will be discussed.

References

1. V. Sabbia, H. Romero, H. Musto, and H. Naya. J Biomol Struct Dyn 27, 361-369 (2009). 2. P. Anbazhagan, M. Purushottam, H. B. K. Kumar, O. Mukherjee, S. Jain, and R. Sowdhamini. J Biomol Struct Dyn 27, 581-598 (2010). 3. Z. Liu, Y. Xu, L. Wu, and S. Zhang. J Biomol Struct Dyn 28, 97-106 (2010). 4. Y. Zhang, R. Proenca, M. Maffei, M. Barone, L. Leopold, and J. M. Friedman. Nature 372, 425-432 (1994). 5. A. A. Steiner and A. A. Romanovsky. Prog Lipid Res 46(2), 89-107 (2007). 1070 Intrinsically Flexible Ribosomal RNA Segments. Family C 3-Way Junctions and their Possible Role in the Translation 102 We carried out extensive explicit solvent molecular dynamics analysis (1.4 µs) of several RNA three-way junctions (3WJs) from the large ribosomal subunit. The 1 Ivana Besseova * aim was to analyze the intrinsic flexibility of the 3WJs and to consider them in Kamila Reblova1 the ribosomal context in available ribosomal crystal structures. The flexibility of Neocles B. Leontis2 the RNA is inferred from stochastic thermal fluctuations sampled in unrestrained simulations. The simulations identify the intrinsic low-energy deformation modes 1 Jiri Sponer ** of the molecules that can co-operate with the surrounding ribosomal elements to achieve the functional dynamics. 1Institute of Biophysics, Academy of Sci- All studied 3WJs possess significant anisotropic hinge-like flexibility between their ences of the Czech Republic, stacked stems and dynamics within the compact regions of their adjacent stems. Kralovopolska 135, 61265 Brno, The most interesting is the GAC (GTPase associated center) 3WJ which may sup- Czech Republic port large-scale dynamics of the L7/L12-stalk rRNA, i.e. rRNA Helices (H) 42-44 (1). Projection of the observed anisotropic movement into the ribosome shows that 2 Department of Chemistry, Bowling H43/H44 rRNA is flexible in direction towards and away (closing-opening geom- Green State University, Bowling Green, etry path of the GAC 3WJ) of the large ribosomal subunit, see the Figure. When OH 43403, USA the H42-H44 domain is in the overall “closed” conformation, the tip of the hairpin loop of H89 can fit into the groove defined by the docking of the hairpin loops *[email protected], of H43/44. However, such contact is only seen in the 2AW4 crystal structure of **[email protected] vacant E.coli ribosome (2). In other crystal structures the distance between the H89 and GAC RNA varies widely (it even exceeds in some cases 10 Å) (3, 4). The experimental structures show a wide range of positions sampling a set of more inward and more outward structures with respect to the A-site of the large subunit and H89. The range of observed positions agrees with the anisotropic flexibility direction predicted by MD. We conclude that the X-ray observed flexibility of the GAC RNA originates from the 3WJ and includes also the H42 stem region below the 3WJ and above the conserved H42-H97 tertiary interaction. The GAC rRNA could undergo large-scale rapid thermal fluctuations or structural adaptations to facilitate gliding of the tRNA to H89, which leads it into its functional destinations (A/A state) (5), see the Figure. The H42-44 rRNA region is not fully relaxed in

Figure: Left - Large ribosomal subunit (RNA in tan, proteins in cyan, pdb code 2WRO2) from Thermus termophilus including tRNA in the A/T state (in red, 2WRN2). The GAC 3WJ in the X-ray data is in purple, open and closed geometries of the junction occurring in the MD simulations are shown in green and blue, respectively. Right - Detailed view on coordinated hypothetical movement of the tRNA and GAC 3WJ. The closed MD geometry of the GAC 3WJ forms contact with H89 (marked by ellipse), which might be important for guiding the tRNA to H89 (in yellow). the ribosome. It is deformed towards the body of the large subunit by some of the 1071 surrounding elements. The simulations suggest that the L10 protein can regulate or contribute to this deformation. The intrinsic flexibility of the GAC 3WJ will be briefly compared with intrinsic flexibility of RNA from other flexible parts of the large subunit, namely the elbow region at the base of the A-site finger and the bottom part of the L1 stalk.

References

1. I. Besseova, K. Reblova, N. B. Leontis, and J. Sponer. Nucleic Acids Res 38, 6247-6264 (2010). 2. B. S. Schuwirth, M. A. Borovinskaya, C. W. Hau, W. Zhang, A. Villa-Sanjurjo, J. M. Holton, and J. H. D. Cate. Science 310, 827-834 (2005). 3. T. M. Schmeing, R. M. Voorhees, A. C. Kelley, Y. G. Gao, F. V. Murphy, J. R. Weir, and V. Ramakrishnan. Science 326, 688-694 (2009). 4. D. J. Klein, P. B. Moore, and T. A. Steitz. J Mol Biol 340, 141-177 (2004). 5. K. Y. Sanbonmatsu, S. Joseph, and C. S. Tung. Proc Natl Acad Sci USA 102, 15854-15859 (2005).

Ribosomal Paleontology and Resurrection: Molecular Fossils from Before Coded Protein 103 The origins and early development of the translation machinery remain imprinted in the extant ribosome (1-3), in sequences, folding, and function. To mine the Loren Dean Williams* information contained within the ribosome, we are developing new methods of Chiaolong Hsiao molecular paleontology. We are developing experimental and computational tools Jessica C. Bowman Chad R. Bernier Jessica Peters Dana M. Schneider Eric O’Neill

NASA Astrobiology Institute and the Center for Ribosomal Origins and Evolution at the Georgia Institute of Technology, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, 30332-0400 *[email protected]

The large subunit as an onion. 1072 to understand and recapitulate fundamental steps in the origin and evolution of the ribosome, and to estimate the relative ages of ribosomal components. We are biochemically resurrecting working models of ancestral ribosomes. We are developing timelines for critical steps in the evolutionary history of the ribosome. The results of these studies will help provide keys to understanding the origin of proteins and RNA, and the origin of life. We use structure- and sequence-based comparisons of the Large Subunits (LSUs) of Haloarcula marismortui and Thermus thermophilus. These are the highest resolution ribosome structures available, and represent the two primary branches of the evolutionary tree. Using an onion analogy, we have sectioned the superimposed bacterial and archaeal LSUs into concentric shells, using the sites of peptidyl transfer as the centers (4). This spherical approximation allows shell-by-shell dissections and comparisons that clearly capture significant information along the evolutionary timeline. The results support the notion that ever-older molecular fossils are revealed as one bores toward the center of the LSU onion. The conformations and interactions of both RNA and proteins change over time. The frequency with which macromolecules assume regular secondary structural elements, such as A-form helices containing Watson-Crick base pairs (RNA) and α-helices and β-sheets (protein), increases with time. The conformations of the oldest ribosomal protein components suggest they are molecular fossils of the non- coded peptide ancestors of ribosomal proteins. We hypothesize that abbreviated lengths and mixed sequences in the ancestral state proscribed secondary structure, which is indeed nearly absent near the centers of the LSU onions.

References

1. C. R. Woese. RNA 7, 1055-1067 (2001). 2. G. E. Fox. Cold Spring Harb Perspect Biol 2, a003483 (2010). 3. K. Bokov and S. V. Steinberg. Nature 457, 977-980 (2009). 4. C. Hsiao, S. Mohan, B. K. Kalahar, and L. D. Williams. Mol Biol Evol 26, 2415-2425 (2009).

Anticodon Loop Modifications Modulate Structural Flexibility in E. coli tRNAArg1,2 that Lacks a U-turn 104 Conformation in Solution William A. Cantara* Three of the six codons that are decoded by tRNAArg in E. coli are read by the Yann Bilbille Arg1,2 Arg isoacceptors tRNA . The anticodon stem and loop domain (ASL ICG) of these Jia Kim isoacceptors differ only in the identity of the residue at position 32 in the as either 2 Arg1 Arg2 Andrzej Malkiewicz 2-thiocytidine (s C32) or cytidine for tRNA and tRNA , respectively. These isoacceptors also contain important modifications at positions 34 (inosine, 34I ) and 37 Paul F. Agris 2 (2-methyladenosine, m A37). To investigate the roles of the modifications in proper Arg Arg folding of the ASL ICG, six ASL ICG constructs differing in their array of modi- The RNA Institute, fications were analyzed by biophysical spectroscopic methods as well as functional binding assays. Thermal denaturation and circular dichroism spectroscopy showed Department of Biological Sciences, that the modifications contribute competing thermodynamic and base stacking University at Albany, SUNY, Arg properties. Spectroscopic methods indicated that ASL ICG modifications contrib- Albany, NY 12222 uted significant differences in structural properties. However, restrained molecular dynamics calculations of the ASLArg structures from NMR spectroscopy clearly *[email protected] ICG showed that the equilibrium solution conformations of the ASLs are nearly identical, but do not possess the invariant U-turn structure needed for binding to the ribosomal Arg A-site. Yet, all of the ASL constructs were able to bind to the ribosome in the pres- 2 ence of the cognate CGU codon. The m A37 modification restricts binding to CGC, 2 2 while both s C32 and m A37 restrict binding to CGA. Taken together, the results suggest that chemical modifications modulate the flexibility of the loop, allowing induced conformations on the ribosome that can restrict binding to specific codons.

This research is supported by NSF grant number 53855. Structural Characteristics of E. coli YrdC Suggest a 1073 Role in the Enzymatic Biosynthesis of the tRNA Modification N6-Threonylcarbamoyladenosine

Nucleoside modifications are vital for the proper structure and function of tRNA. 6 6 105 The N -threonylcarbamoyladenosine modification at position 37 (t A37), 3’-adjacent to the anticodon, of many tRNA species ensures the accurate recognition of several Kimberly A. Harris1,2 ANN codons by increasing codon affinity and enhancing ribosome binding (1, 2). Victoria Jones1 Considerable data exists on the biophysical aspects of t6A however, the biosynthe- 37, Yann Bilbille1 sis pathway of this hypermodified base is only partially understood (3). This pathway requires ATP, threonine, a carbon source, and possibly the universal protein family Paul F. Agris2 6 YrdC/Sua5, which has been shown to be involved in t A37 biosynthesis (4, 5). To further investigate this possibility, we examined the interaction of E. coli YrdC with 1Department of Molecular & Structural the heptadecamer anticodon stem loop of tRNA lysine (ASLLys). As determined by mass spectrometry analysis, NMR, and quenching of intrinsic fluorescence, YrdC Biochemistry, North Carolina State Lys 6 bound unmodified ASL with high affinity (Kd = 0.27 ± 0.20 µM), t A37-modified University, Raleigh, NC, USA 27695 ASLLys with significantly lower affinity (K = 1.36 ± 0.39 µM), and showed specific- d 2The RNA Institute, ity toward threonine and ATP. YrdC also preferentially binds threonine over other amino acids tested by STD-NMR. Our studies of the YrdC-ASLLys interaction by Department of Biological Sciences, NMR, CD, and fluorescence of 2-aminopuine at position 37 of ASLLys, indicated University at Albany -SUNY, no structural change in the RNA. Therefore, catalytic function appears to be limited 6 Albany, NY, USA 12222 under these in vitro conditions and YrdC is most likely a subunit of a t A37 synthetase complex. Further characterization of the protein-protein and protein-RNA interac- 6 tions will provide information to determine the role of YrdC in t A37 biosynthesis.

References

1. S. Nishimura. Prog Nucleic Acid Res Mol Biol 12, 49-85 (1972). 2. P. F. Agris. Nucleic Acids Res 32, 223-238 (2004). 3. J. W. Stuart, et al. Biochemistry 39, 13396-13404 (2000). 4. A. Körner and D. Söll. FEBS Lett 39, 301-306 (1974). 5. B. El Yacoubi, et al. Nucleic Acids Res 37, 2894-2909 (2009).

A Structural Method with Single Atom Resolution for Investigating the Secondary Structure of RNA: the Deuterium Kinetic Isotope Effect/Hydroxyl 106 Radical Cleavage Experiment Shakti Ingle* Robert Azad The hydroxyl radical is widely used as a high-resolution footprinting agent for Thomas D. Tullius DNA (1) and RNA (2). The hydroxyl radical abstracts a hydrogen atom from the sugar-phosphate backbone, generating a carbon-based radical, which ultimately leads to a strand break. Substituting deuterium for hydrogen on the ribose results in Department of Chemistry, a kinetic isotope effect if abstraction of that hydrogen atom by the hydroxyl radi- Boston University, cal leads to a strand break. The deuterium kinetic isotope effect (KIE) correlates well with the solvent accessibility of the deoxyribose hydrogen atoms in DNA (3). Boston MA 02215 RNA, due to its single-stranded nature, adopts a multitude of secondary and tertiary *[email protected] structures. This results in a non-uniform exposure of its backbone, making it a very attractive system to study using the deuterium KIE/hydroxyl radical experiment. We applied this experiment to the sarcin-ricin loop (SRL) RNA molecule, a structurally well-defined component of the large subunit of ribosomal RNA. SRL plays a critical role in the activation of elongation factor Tu (a GTPase) leading to the hydrolysis of GTP during translation, which is speculated to be the reason for its universal conservation (4).

We utilized a one-pot enzymatic protocol to synthesize specifically deuterated ATP and GTP (5). We then use in vitro transcription to incorporate these deuterated 1074 nucleotides into the 29-mer SRL RNA molecule. We report the observation of a substantial deuterium kinetic isotope effect on hydroxyl radical cleavage of SRL RNA for dideuteration at the C5’ position. This experiment provides direct evidence of abstraction by the hydroxyl radical of hydrogen atoms from the ribose C5’ position in RNA. We also observe isotope effects for deuteration at the C4’ position, although to a lesser extent. Interestingly, we observed different apparent KIEs for the same deuterated nucleotide at different positions in the SRL sequence, suggesting that the extent of reaction with hydroxyl radical depends on the local structural (and perhaps dynamic) environment. We conclude that the deuterium KIE/hydroxyl radical experiment provides a way to investigate the solvent exposure of the ribose backbone of RNA at the level of single hydrogen atoms.

This research is supported by NSF Award MCB-0843265.

References

1. T. D. Tullius and B. A. Dombroski. Science 230, 679-681 (1985). 2. J. A. Latham and T. R. Cech. Science 245, 276-282 (1989). 3. B. Balasubramanian, W. K. Pogozelski, and T. D. Tullius. Proc Natl Acad Sci USA 95, 9738-9743 (1998). 4. R. M. Voorhees, T. M. Schmeing, A. C. Kelley, and V. Ramakrishnan. Science 330, 835-838 (2010). 5. T. J. Tolbert and J. R. Williamson. J Am Chem Soc 118, 7929-7940 (1996).

Insight into Transition States in Protein-RNA 107 Recognition through MD Simulations 1 Protein-RNA recognition plays an important role in many post transcriptional- Lena Dang * gene-expression-regulation, such as RNA modification, transportation, translation Susan Pieniazek1 and degradation. U1A, a member of RNA Recognition Motif (RRM) containing Anne M. Baranger2 protein family, is a component of an RNA-protein complex called U1A snRNP 1 involved in pre-mRNA splicing. RRM proteins’ abilities to recognize and bind to David L. Beveridge their cognate RNA sequence involves various nonbonding forces, such as electrostatic attraction, hydrogen bonding, aromatic stacking and van de Waals interac- 1Department of Chemistry, Wesleyan tions. Results from our Molecular Dynamics (MD) studies on RRM containing proteins (U1A, SXL) and their cognate RNA sequences indicate that significant University, Middletown, Connecticut structural adaptations in both proteins and RNA sequences are required for bind- 2Department of Chemistry, University of ing. Simulations on mutant proteins demonstrate that electrostatics is the dominat- Illinois at Urbana-Champaign, Illinois ing force during the initial steps of the binding event. RMSD and RMSF analyses of the simulations indicate the free RMM containing proteins are more flexible *[email protected] than the protein-RNA complex for both U1A and SXL. Regions of the protein 1075 that undergo the greatest flexibility include N- and C-termini, loops and helixes. Covariance analyses indicate that a strong connectivity in a network of amino acids separated by long distances can be used to explain the mechanism of conformational change upon protein-RNA binding. PCA analyses and 3D-RMSD spaces are constructed to further identify the transition states of the binding events.

RNASTEPS, an Online Database of Sequence-dependent Deformability of RNA Helical Regions 108 Mauricio Esguerra The RNASTEPS database, located at http://rnasteps.rutgers.edu, is a repository of Wilma K. Olson the base-pair steps found in the double-helical regions of 656 RNA crystal structures of 3.5 Å or better resolution. The database includes a variety of structural param- Department of Chemistry & eters and molecular images, computed with the 3DNA software package (1, 2) and Chemical Biology, BioMaPS Institute for known to be useful for characterizing and understanding the sequence-dependent Quantitative Biology, Rutgers, the State spatial arrangements of the DNA and RNA base pairs and base-pair steps. The data can be accessed by searches of Leontis-Westhof base-pair patterns and Protein University of New Jersey, Data Bank and Nucleic Acid Database structure identifiers. The site also includes a Piscataway, NJ 08854, USA repository of the average values and covariance of the rigid-body parameters char- [email protected] acterizing well represented base-pair steps, the knowledge-based elastic potentials derived from these data (3), and other measures of RNA step deformability, such [email protected] as the conformational volume, base-pair overlap, and root-mean-square deviation of atomic coordinates. The collective information provides a useful benchmark for RNA force-field development. The cumulative data account for the measured persistence length of double-stranded RNA (4) in simulations using the approach of Czapla et al. (5). An example of the distributions and ‘energies’ extracted from the roll and twist angles of 1274 GG·CC steps and the corresponding molecular super- position of these steps is shown below: 1076 References 1. X-L. Lu and W. K. Olson. Nucleic Acids Research 31, 5108-5121 (2003). 2. X-L. Lu and W. K. Olson. Nature Protocols 3, 1213-1227 (2008). 3. W. K. Olson, A. A. Gorin, X-J. Lu, and L. M. Hock, and V. B. Zhurkin. Proc Natl Acad Sci, 95, 11163-11168 (1998). 4. J. A. Abels, F. Moreno-Herrero, T. van der Heijden, C. Dekker, and N. H. Dekker. Biophysical Journal 88, 2737-2744 (2005). 5. L. Czapla, D. Swigon, and W. K. Olson. Journal of Chemical Theory and Computation 2, 685-695 (2006)

A-minor I vs A-minor 0 Tertiary Interactions in RNA Kink-turns. Molecular Dynamics and 109 Quantum Chemical Analysis Kamila Reblova* Judit E. Sponer The second tertiary contact in RNA kink-turns is represented by A-minor interaction between adenine of the second A•G base pair in the NC-stem and the first Nada Spackova canonical base pair of the C-stem. Kvnown kink-turn structures possess either Ivana Besseova A-minor I (A-I) or A-minor 0 (A-0) interaction (Figure). Bioinformatics data show Jiri Sponer** that kink-turns with A-I in the available X-ray structures keep primarily G=C pair in the first C-stem position during evolution while the inverted base pair (C=G) is basically not realized. In contrast, kink-turns with A-0 in the observed structures Institute of Biophysics, Academy of alternate G=C and C=G base pairs in sequences. Molecular dynamics simulations of X-ray structures of kink-turns reveal that the A-I interaction with G=C base pair Sciences of the Czech Republic, (A-I/G=C triple) is stable while inversion of the canonical base pair (A-I/C=G) Kralovopolska 135, CZ-61265, leads either to kink-turn disruption or rearrangement to A-0/C=G (1). The A-0/ Brno, Czech Republic G=C initial configuration tends to transform to the A-I/G=C arrangement. Finally, simulations testing the A-0/C=G arrangement lead either to disruption of the struc- *[email protected] ture (after unsuccessful transition to the A-I configuration) or are stable. Thus, the **[email protected] A-I/G=C arrangement appears to be intrinsically preferred by kink-turns while formation of the A-0 interaction is likely related to the context. In addition, as shown for ribosomal Kink-turn 15, A-0 may be supported by additional interactions that do not belong to the kink-turn signature interactions. Quantum-chemical calculations explain how a delicate balance of various intermolecular interactions plays a decisive role in determining the dynamic behavior and stability of the various A-minor patterns in kink-turns.

Figure: Tertiary interactions (A-0 and A-I) stabilizing kink-turn structures.

Reference

1. K. Reblova, J. E. Sponer, N. Spackova, I. Besseova, and J. Sponer. Submitted. On the Geometry, Electronic Structure and 1077 Hydrolytic Stability of the As-DNA Backbone High level quantum chemical calculations have been applied to investigate the 110 geometry and electronic properties of the arsenate analogue of the DNA back- 1 bone (1). The optimized geometries as well as hyperconjugation effects along the Arnost Mladek C3‘-O3‘-X-O5‘-C5‘ linkage (X=P,As) exhibit a remarkable similarity for both Jiri Sponer1 arsenates and phosphates. This suggests that arsenates – if present – might serve as Bobby G. Sumpter2 a potential substitute for phosphates in the DNA-backbone. Miguel Fuentes-Cabrera2* On the other hand, our calculations predict that neither steric hindrance nor less polar Judit E. Sponer1** solvent medium is able to reduce the otherwise high hydrolysis rate of arsenate- esters (2). These results question the stability of As-DNA not only in aqueous but 1 also in non-aqueous environments. Institute of Biophysics, Academy of Sciences of the Czech Republic, References Kralovopolska 135, 1. A. Mladek, J. Sponer, B. G. Sumpter, M. Fuentes-Cabrera, and J. E. Sponer. J Phys Chem CZ-61265, Brno, Czech Republic Lett, In press. http://pubs.acs.org/doi/full/10.1021/jz200015n 2. A. Mladek, J. Sponer, B. G. Sumpter, M. Fuentes-Cabrera, and J. E. Sponer. Submitted. 2Center for Nanophase Materials Sciences, and Computer Sciences and Mathematics Division, Oak Ridge National Laboratory, Oak Ridge, P. O. Box 2008, Oak Ridge, TN 37831-6494, USA *[email protected] **[email protected] 1078 Role of Protonation States of G8 and A38 Nucleobases in Structure Stabilization and Catalysis of the Hairpin Ribozyme

The hairpin ribozyme is a prominent member of the group of small RNA enzymes 111 (ribozymes) because it does not require metal ions to achieve catalysis of the revers- Vojteˇch Mlýnský1 ible, site-specific cleavage of its RNA substrate (1). Guanine 8 (G8) and adenine Pavel Banáš1 38 (A38) were identified by biochemical and structural experiments as the key participants involved in cleavage. Despite broad efforts, their exact roles in cataly- 3 Nils G. Walter sis remains disputed (2). We have carried out classical molecular dynamics (MD) Jirˇí Šponer2* simulations (3, 4) in explicit solvent on 50-150 ns timescales with various protona- Michal Otyepka1** tion states of G8 and A38. The MD simulations reveal that a canonical G8 agrees well with the crystal structures while a deprotonated G8 distorts the active site. The G8 enol tautomer is structurally well tolerated, causing only local rearrangements 1Regional Centre of Advanced in the active site. In most simulations, the canonical A38 departs from the scissile phosphate and substantially perturbs the active site. The Protonated A38H+ is more Technologies and Material, consistent with the crystallography data (5). MD simulations support the idea that Department of Physical Chemistry, A38H+ is the dominant form in the crystals, grown at pH 6. This finding is also in Faculty of Science, Palacky University, agreement with recent NAIM experimental data (6). The MD simulations were also used to find geometries of potential reactive states bearing G8 and A38 of various Olomouc, Czech Republic protonation states in the active site. These geometries were further analyzed by 2 Institute of Biophysics, Academy of hybrid quantum-mechanical/molecular mechanical (QM/MM) methods to evaluate Sciences of the Czech Republic, the energy along the reaction pathway in order to find the most probable reaction mechanism. The DFT MPW1K/6-31+G(d,p) method was used for the QM region Brno, Czech Republic and the parm99 force field for MM region, while the communication between these 3 Department of Chemistry, Single both layers was realized via electronic embedding (7, 8). The quality of our DFT Molecule Analysis Group University of method was tested against reference CCSD(T)/CBS calculations. The mean unas- signed error of the DFT method was found to be below 1.0 kcal·mol-1. The calculated Michigan Ann Arbor, MI, USA activation barriers are in good agreement with experimental data (20-21 kcal·mol-1) *[email protected] for the systems with canonical G8 and both canonical/protonated forms of A38/ **[email protected] A38H+ (19.6 kcal·mol-1 and 20.1 kcal·mol-1, respectively). The initial nucleophile attack of the A-1(2’-OH) group on the scissile phosphate is the rate-limiting step along the reaction path. The G8-enol tautomer increases the overall reaction barrier by 4.7 kcal·mol-1. On the other hand, our preliminary data indicate that the presence of an unprotonated G8- (together with A38H+) seems to significantly decrease the activation barrier. Protonated A38H+ does not significantly affect the overall activation barrier (20.1 kcal·mol-1), but decreases the activation barrier of the exocyclic cleavage step by 7.7 kcal·mol-1. In the reaction path where a protonated A38H+ acts as the general acid, the product is 9.5 kcal·mol-1 higher in energy compared to the product in the reaction where a nonbridging oxygen of the scissile phosphate acts as proton donor and A38H+ is not directly involved in cleavage chemistry.

This work was supported by the Grant Agency of the Academy of Sciences of the Czech Republic (grant IAA400040802), by the Grant Agency of the Czech Repub- lic (grants 203/09/H046, 203/09/1476, P208/11/1822 and P301/11/P558), by Ministry of Youth, Sport and Education of Czech Republic (grants LC512 and MSM6198959216), by Student Project PrF_2010_025 of Palacky University. This work was supported also by the Operational Program Research and Development for Innovations - European Social Fund (CZ.1.05/2.1.00/03.0058) and NIH grant 2R01 GM062357 t o NGW.

References

1. M. J. Fedor. Annu Rev Biophys 38, 271-299 (2009). 2. T. J. Wilson and D. M. Lilley. RNA 17, 213-221 (2011). 3. P. Sklenovský and M. Otyepka. J Biomol Struct Dyn 27, 521-540 (2010). 4. M. A. Ditzler, M. Otyepka, J. Šponer, and N. G. Walter. Acc Chem Res 43, 40-47 (2010). 5. V. Mlýnský, P. Banáš, D. Hollas, K. Réblová, N. G. Walter, J. Šponer, and M. Otyepka. 1079 J Phys Chem B 114, 6642-6652 (2010). 6. I. T. Suydam, S. D. Levandoski, and S. A. Strobel. Biochemistry 49, 3723-3732 (2010). 7. P. Banáš, L. Rulíšek, V. Hánošová, D. Svozil, N.G. Walter, J. Šponer, and M. Otyepka. J Phys Chem B 112, 11177-11187 (2008). 8. P. Banáš, P. Jurecˇka, N. G. Walter, J. Šponer, and M. Otyepka. Methods 49, 202-216 (2009).

Synthesis of a Disubstituted Purine Analog and its Binding Interactions with Xanthine Phosphoribosyl Transferase (xpt) Riboswitch 112 Angela Potenza RNA has the remarkable ability to form 3-dimensional structures that parallel the Rachit Neupane structural complexity of proteins. Highly structured RNA molecules present excellent targets for new classes of antibiotics. Riboswitches are folded regions in the Emily McLaughlin untranslated region of mRNA that control gene expression upon binding to small Swapan S. Jain* ligands (1). Our lab is involved in the synthesis of guanine analogs with modifications at both the C2 and C6 position of the purine ring. These analogs have the potential of tightly binding to the 5’-untranslated region of xanthine phosphoribo- Department of Chemistry, syl transferase (xpt) riboswitch mRNA (2). High-resolution structure of xpt ribo- Bard College, switch shows that there maybe additional binding space (white ovals in the figure Annandale on Hudson, NY 12504 below) where it may be possible to add functional groups without significantly disrupting ligand binding to the existing pocket (3). Modification at the C2 and C6 *[email protected] position of the purine ring can facilitate additional H-bonds with nucleotides near the binding pocket of xpt mRNA potentially yielding stronger ligand-mRNA interactions. Current work in our lab is focused on the synthesis and characterization of 2-acetamido-6-hydrazone-purine. Binding affinity of analogs to riboswitch mRNA is tested using in-line probing with P-32 labeled mRNA and an in vivo GFP-based

reporter system.

References

1. W. C. Winkler and R. R. Breaker. Annu Rev Microbiol 59, 487-517 (2005). 2. J. N. Kim, K. F. Blount, I. Puskarz, J. Lim, K. H. Link, and R. R. Breaker. ACS Chem Biol 4, 915-927 (2009). 3. R. T. Batey, S. D. Gilbert, and R. K. Montange. Nature, 432, 411-415 (2004). 1080 Computer-Aided Design of Novel HIV-1 Entry Inhibitors, Their Synthesis and Trials: Glycolipids Against the Envelope gp120 V3 Loop 113 The HIV-1 V3 loop plays a central role in the biology of the HIV-1 envelope glycoprotein gp120 as a principal target for neutralizing antibodies, and as a major deter- 1 Alexander M. Andrianov * minant in the switch from the non-syncytium-inducing to the syncytium-inducing Ivan V. Anishchenko2 form of HIV-1 that is associated with accelerated disease progression. HIV-1 cell Mikhail A. Kisel1 entry is mediated by the sequential interactions of gp120 with the receptor CD4 and a co-receptor, usually CCR5 or CXCR4, depending on the individual virion. The 1 Vasiliy A. Nikolayevich V3 loop is critically involved in this process. Because of the exceptional role of the Vladimir F. Eremin3 V3 loop in the viral neutralization and cell tropism, one of the actual problems is Alexander V. Tuzikov2 that of identifying chemical compounds able to block this functionally crucial site of gp120. According to empirical observations, glycolipid β-galactosylceramide (β-GalCer) forming on the surface of some susceptible host cells the primary recep- 1Institute of Bioorganic Chemistry, tor for HIV-1 alternative to CD4 exhibits a strong attraction to the V3 loop and, for this reason, may be involved in anti-HIV-1 drug studies. In the light of these obser- National Academy of Sciences of Belarus, vations, the use of bioinformatics tools for imitating the process of making the V3/ Kuprevich Street 5/2, glycolipid complexes may provide a structural rationale for the design of efficient 220141 Minsk, Republic of Belarus blockers of the functionally important V3 sites. 2 United Institute of Informatics Problems, The objects of this study were to generate the 3D structure model for the complex National Academy of Sciences of Belarus, of V3 with β-GalCer and, based on the calculation data, to design its water soluble Surganov Street 6, analogs that could efficiently mask the HIV-1 V3 loop followed by their synthesis and medical trials. To this effect, the following problems were solved: (i) 3D structures for 220012 Minsk, Republic of Belarus, the consensus amino acid sequences of the HIV-1 subtypes A and B V3 loops were 3The Republican Research and Practical computed by homology modeling and simulated annealing; (ii) spatial structures of Center for Epidemiology β-GalCer, as well as of a series of its modified forms were determined by quantum chemistry and molecular dynamics simulations; (iii) supramolecular ensembles of and Microbiology, these glycolipids with V3 were built by molecular docking methodology and energy Filimonova Street 23, characteristics describing their stability were estimated by molecular dynamics com- 220114 Minsk, Republic of Belarus putations; (iv) synthesis of β-GalCer derivatives that, according to the designed data, give rise to the stable complexes with V3 was performed, and (v) testing of these com- *[email protected] pounds for antiviral activity was carried out. From the structural data obtained, the Phe and Arg/Gln amino acids of the gp120 immunogenic crest were revealed to play a key role in forming the complexes of glycolipids with V3 by specific interactions with the galactose residue and sphingosine base respectively. And at the same time, the sugar hydroxyl groups form the H-bonds with the nearby polar atoms of the V3 backbone. Two water soluble analogs of β-GalCer were also found to display a high affinity to V3 close to that of the native glycolipid. This inference results from the values of binding free energy evaluated for the calculated structures and coincides with the experimental data on the complexes of gp120 with β-GalCer. The above theoretical findings are in keeping with those of medical trials of the synthesized molecules, which testify to their anti-HIV-1 activity against the virus subtypes A and B isolates.

As a matter of record, the molecules constructed here are supposed to present the promising basic structures for the rational design of novel potent HIV-1 entry inhibitors that could neutralize the majority of circulating indigenous strains. For some of the details of the methodlogies of current drug designs employed here please consult the full length research articles (1-4).

References

1. A. M. Andrianov and I. V. Anishchenko. J Biomol Struct Dyn 27, 179-193 (2009). 2. A. M. Andrianov. J Biomol Struct Dyn 26, 445-454 (2009). 3. T. T. Chang, H. J. Huang, K. J. Lee, H. W. Yu, H. Y. Chen, F. J. Tsai, M. F. Sun, and C. Y. C. Chen. J Biomol Struct Dyn 28, 309-321 (2010). 4. A. K. Kahlon, S. Roy, and A. Sharma. J Biomol Struct Dyn 28, 201-210 (2010). Disclosure of Conserved Structural Motifs 1081 in the HIV-1 Third Variable (V3) Loop by Comparative Analysis of 3D V3 Structures for Different Virus Subtypes 114 The V3 loop on gp120 from HIV-1 is a focus of many research groups involved Alexander M. Andrianov1* in anti-AIDS drug development because this region of the protein is the princi- 2 pal target for neutralizing antibodies and determines the preference of the virus Ivan V. Anishchenko for T-lymphocytes or primary macrophages. Although the V3 loop is a promising Alexander V. Tuzikov2 target for anti-HIV-1 drug design, its high sequence variability is a major complicating factor. Nevertheless, the occurrence of highly conserved residues within the V3 loop allows one to suggest that they may preserve their conformational states 1Institute of Bioorganic Chemistry, in different HIV-1 strains and, therefore, should be promising targets for designing National Academy of Sciences of Belarus, new anti-HIV drugs. In this connection, the issue of whether these conserved amino Kuprevich Street 5/2, acids may help to keep the local protein structure and form the structurally rigid segments of V3 exhibiting the HIV-1 vulnerable spots is very relevant. One of the 220141 Minsk, Republic of Belarus plausible ways to answer this question consists of examining the V3 structures for 2 United Institute of Informatics Problems , their consensus sequences corresponding to the HIV-1 group M subtypes respon- National Academy of Sciences of Belarus, sible for the AIDS pandemic followed by disclosing the patterns in the 3D arrangement of the variable V3 loops. Because of the deficiency of experimental data on Surganov Street 6, the V3 structures, these studies may be performed by homology modeling using the 220012 Minsk, Republic of Belarus high-resolution X-ray and NMR-based V3 models as the templates. *[email protected] In this work, the 3D structural models for the consensus amino-acid sequences of the V3 loops from the HIV-1 subtypes A, B, C, and D were generated by bioinformatics tools to reveal common structural motifs in this functionally important portion of the gp120 envelope protein. To this effect, the most preferable 3D structures of V3 were computed by homology modeling and simulated annealing methods and compared with each other, as well as with those determined previously by X-ray diffraction and NMR spectroscopy. Besides, the simulated V3 structures were also exposed to molecular dynamics computations, the findings of which were analyzed in conjunction with the data on the conserved elements of V3 that were obtained by collation of its static models.

As a matter of record, despite the high sequence mutability of the V3 loop, its segments 3-7, 15-20 and 28-32 were shown to form the structurally invariant sites, which include amino acids critical for cell tropism. Moreover, the biologically meaningful residues of the identified conserved stretches were also shown to reside in β-turns of the V3 polypeptide chain. In this connection, these structural motifs were suggested to be used by the virus as docking sites for specific and efficacious interactions with receptors of macrophages and T-lymphocytes. Therefore, the structurally invariant V3 sites found here represent potential HIV-1 weak points most suitable for therapeutic intervention.

In the light of the findings obtained, the strategy for anti-HIV-1 drug discovery aimed at the identification of co-receptor antagonists that are able to efficiently mask the structural motifs of the V3 loop, which are conserved in different virus subtypes, is highly challenging. To overcome this problem, an integrated computational approach involving theoretical procedures, such as homology modeling, molecular docking, molecular dynamics, QSAR modeling and free energy calculations, should be of great assistance in the design of novel, potent and broad antiviral agents. For some of the details of the methodlogies of current drug designs employed here please consult the full length research articles (1-4).

Acknowledgment

This study was supported by grants from the Union State of Russia and Belarus (scientific program SKIF-GRID; No 4U-S/07-111), as well as from the Belarusian Foundation for Basic Research (project X10-017). 1082 References 1. A. M. Andrianov and I. V. Anishchenko. J Biomol Struct Dyn 27, 179-193 (2009). 2. A. M. Andrianov. J Biomol Struct Dyn 26, 445-454 (2009). 3. T. T. Chang, H. J. Huang, K. J. Lee, H. W. Yu, H. Y. Chen, F. J. Tsai, M. F. Sun, and C. Y. C. Chen. J Biomol Struct Dyn 28, 309-321 (2010). 4. A. K. Kahlon, S. Roy, and A. Sharma. J Biomol Struct Dyn 28, 201-210 (2010).

Hypothetical Mechanism of the Kissing to Extended 115 Dimer Conversion for the HIV-1 Stem-Loop 1 RNA Two homologous copies of genomic RNA are packaged as a dimer into the HIV-1 Nikolai B. Ulyanov* virions (reviewed in (1-3)). The initiation of the dimer formation, which is believed Richard Tjhen to occur in the cytoplasm of infected cells, has been mapped to a stem-loop SL1 Christophe Guilbert with a palindromic apical loop within the 5’-untranslated region of the HIV-1 RNA. SL1 RNA, its structural features and sequence are strongly conserved among Thomas L. James HIV-1 isolates; deletion of SL1 significantly impairs packaging of RNA into the virions. RNA extracted from immature HIV-1 virions is thermally unstable as a Department of Pharmaceutical Chemistry, dimer; the dimer stability increases with virus maturation, which is a complex process associated with the viral protease activity (4, 5). In vitro, SL1 RNA can form University of California, San Francisco, either a metastable kissing dimer (KD), which is kept together by a 6-bp duplex CA 94158-2517, USA formed by the palindromic loops, or a stable extended dimer (ED). KD SL1 RNA *[email protected] can be converted into ED by the viral nucleocapsid protein NCp7 (6), a proteolytic fragment of the Gag polyprotein. This conversion can occur without disruption of the kissing interaction between the palindromic loops (7), which is present in both KD and ED forms. While it is generally accepted that RNA chaperone properties of NCp7 are responsible for the KD-to-ED conversion, the detailed mechanism of the conversion is not known. Indeed, NCp7 destabilizes duplexes only moderately (8), however, 12 base pairs in each SL1 stem need to be broken and re-arranged during the conversion; further, only two NCp7 molecules per SL1 dimer are suf- ficient for the complete conversion (7). Here, we propose a mechanism for the KD- to-ED conversion that does not require simultaneous dissociation of all base pairs in SL1 stems. This hypothetical mechanism involves formation of an RNA analog of the Holliday junction intermediate between the two stems of the SL1 dimer and a following branch migration towards the palindromic duplex. According to 1083 this model, the torsional stress accumulated due to the stem rotation caused by the branch migration is absorbed by the single-stranded purines flanking the palindromic sequence. The NCp7 role is to bring the two stems together by neutralizing the charges on the phosphate groups and to facilitate formation of the initial cross-over, possibly at the level of the G-rich internal loop. We will present the models of the intermediate structures calculated with the miniCarlo program (9).

This research is supported in part by the California HIV/AIDS Research Program award ID09-SF-030.

References

1. J. C. Paillart, M. Shehu-Xhilaga, R. Marquet, and J. Mak. Nat Rev Microbiol 2, 461-72 (2004). 2. M. D. Moore and W.-S. Hu. AIDS Rev 11, 91-102 (2009). 3. S. F. Johnson and A. Telesnitsky. PLoS Pathog 6, e1001007 (2010). 4. W. Fu, R. J. Gorelick, and A. Rein. J Virol 68, 5013-18 (1994). 5. R. Song, J. Kafaie, L. Yang, and M. Laughrea. J Mol Biol 371, 1084-98 (2007). 6. D. Muriaux, H. De Rocquigny, B.-P. Roques, and J. Paoletti. J Biol Chem 271, 33686-92 (1996). 7. A. Mujeeb, N. B. Ulyanov, S. Georgantis, I. Smirnov, J. Chung, T. G. Parslow, and T. L. James. Nucl Acids Res 35, 2026-34 (2007). 8. M. A. Urbaneja, M. Wu, J. R. Casas-Finet, and R. L. Karpel. J Mol Biol 318, 749-64 (2002). 9. 9. V. B. Zhurkin, N. B. Ulyanov, A. A. Gorin, and R. L. Jernigan. Proc Natl Acad Sci USA 88, 7046-50 (1991).

Probing the Molecular Determinants of HIV Alternative Splicing: NMR and Thermodynamic Studies of UP1/ESS3 116 Blanton S. Tolbert* Alternative splicing of the human immunodeficiency virus type-1 (HIV-1) genomic Clay Mishler RNA is necessary to produce the complete viral protein complement, and aberra- tions in the splicing pattern impairs HIV-1 replication (1-2). The cellular protein, Jeff Levengood hnRNP A1 (A1), regulates splicing activity at several highly conserved 3’ alternative Prashant Rajan splice sites (ssA2, ssA3, and ssA7) by binding 5’-UAG-3’ motifs embedded within regions containing higher-order RNA structure (3). The biophysical determinants of A1/splice site recognition remain poorly defined in HIV-1; thus precluding a detailed Department of Chemistry and understanding of the molecular basis of the splicing pattern. Here, the first 3D struc- Biochemistry, Miami University, ture of an HIV-1 splicing regulatory RNA element, exon splicing silencer 3 (ESS3, Oxford, OH 45056 located at ssA7), has been determined by solution 2H-edited NMR spectroscopy and restrained molecular dynamic simulations (see below). ESS3 adopts a 27-nucleotide *[email protected] hairpin loop where the first 20 base pairs form an A-helical structure. The helix is interrupted by a pH sensitive A+C base pair that is conserved across several HIV-1 isolates. The seven nucleotide hairpin contains the high affinity A1 responsive 5’-UAGU-3’ epitope, and a proximal 5’-GAU-3’ motif. The NMR structure shows that the heptaloop adopts a preformed conformation stabilized by base stacking and non-canonical interactions. Significantly, the apex of the loop is quasi-symmetric where UA dinucleotide steps from the 5’-UAGU-3’ and 5’-GAU-3’ motifs stack on opposite sides of the hairpin - thereby providing a possible A1 binding platform. To further probe the biophysical determinants of high-affinity A1/ESS3 recognition, the thermodynamic profile ∆( G°298K, ∆H°298K, ∆S°298K, and Kd) was measured via ITC for a C-terminal A1 deletion mutant (UP1). UP1 interacts with ESS3 via an enthalpically driven process - giving rise to a complex with nanomolar affinity. The ESS3 binding interface of UP1 was mapped via 15N-1H HSQC titrations. The results show that the UP1/ESS3 binding interface is broad and involves regions not 1084

restricted to the beta-pleated sheet. Taken together, we present the first quantitative study of a host factor/HIV-1 splicing RNA element.

This research has been supported by start up funds from Miami University, College of Arts and Sciences.

References

1. C. M. Stoltzfus. Advances in Virus Research 7 (1), 1-40 (2009). 2. C. M. Stoltzfus and J. M. Madsen. Current HIV Research 4(1), 43-55 (2006). 3. C. Branlant, et al. Frontiers in Bioscience 14, 2714-2729 (2009).

Peptide Nucleic Acid (PNA) – Artificial DNA 117 with Many Faces The pseudo-peptidic DNA mimic PNA (peptide nucleic acid) discovered twenty Peter E. Nielsen years ago is still a subject of considerable interest in a range of scientific disciplines from prebiotic chemistry (origin of life) to drug discovery. With recent examples Department of Cellular and Molecular form these two very different areas, some of the faces of PNA will be illustrated. As both as first step towards a minimal chemical mimic of a peptidyl transferase Medicine, Faculty of Health Sciences translation system as well as in a prebiotic context of the evolution of translational & Department of Medicinal Chemistry, mechanisms we have designed systems that allow PNA directed aminoacyl trans- Faculty of Pharmaceutical Sciences, fer (translation mimic). The properties and characteristic of these systems will be presented. Antisense PNA peptide conjugates targeting essential bacterial genes University of Copenhagen, Denmark (such as acpP and ftsZ) have shown promise as potential antibacterials. However, [email protected] improved bacterial delivery vehicles (which are compatible with safe and effective in vivo administration) are still highly warranted. Progress in discovering novel delivery peptides and in unravelling the mechanism of bacterial uptake of antibac- terial PNA peptide conjugates will be presented. Sequence Specific Targeting Duplex DNA by Artificial 1085 DNA Analogs

Although many natural proteins are capable of targeting duplex DNA (dsDNA) in a sequence-specific manner, our ability to design de novo proteins with desired sequence specificity are very limited, at best. That is why the ability of the Pep- 118 tide Nucleic Acid (PNA) to sequence specifically recognize dsDNA, discovered Maxim D. Frank-Kamenetskii* 20 years ago by Peter Nielsen with colleagues (1), has attracted such considerable interest. During the past years, the basic understanding of the process of dsDNA Irina Smolina invasion by pyrimidine bis-PNAs and various applications of the phenomenon have been elucidated. As a result, novel approaches for detecting short (about 20-bp- Department of Biomedical Engineering, long) signature sites on genomes have been developed in our laboratory. In particular, a PD-loop-based method of pathogen detection has been developed, which Boston University. makes it possible to distinguish not only different bacterial species but also to dis- Boston, MA 02215 criminate drug sensitive versus drug resistant strains (2). The next step would be *[email protected] to extend the approach to human cells, which would open the way for even more promising applications. Some encouraging data in this direction will be presented. Other promising applications of bis-PNAs include their use in DNA detection with solid-state nanopores.

A serious limitation of generic PNAs with respect to targeting duplex DNA consists in requirement for the homopurine nature of the DNA strand, which is captured by bis- PNA via the triplex formation. Therefore, various chemical modifications of the backbone and the bases have been tested in recent years, which could allow to lift this sequence limitation. Two modifications are of greatest interest: pseudo- complementary PNAs and γ-PNAs. Chiral γ-PNAs with some cytosines replaced with G-clamp bases, proposed by Danith Ly with colleagues, have proved to be especially promising. The capability of γ-PNA to invade dsDNA in a sequence- unrestricted manned has been demonstrated. The γ-PNA makes it possible the exceedingly specific DNA capturing in the duplex form (3). This opens up a very interesting opportunity of capturing the chromatin in its native form for further investigation of higher-level chromatin structures.

Over the years, the ability of synthetic DNA analogs to invade dsDNA has been considered as something purely artificial, without any parallels in vivo. The sit- uation has radically changed recently after discovery of the CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeat), a bacterial immune system. It has been shown that in the CRISPR pathway small (about 40-nt-long) single- stranded RNAs recognize the complementary DNA strand within dsDNA via a still unknown mechanism with the help of special proteins of the CRISPR system (4). Advancements in the field of dsDNA recognition by artificial DNA analogs not only provides with new tools for DNA analysis but also can prove to be of great help in understanding new important biological mechanisms.

References

1. P. E. Nielsen, M. Egholm, R. H. Berg, and O. Buchardt. Science 254, 1497-500 (1991). 2. I. Smolina, N. Miller, and M. Frank-Kamenetskii. Artificial DNA ,1 76-82 (2010). 3. H. Kuhn, B. Sahu, D. H. Ly, and M. D. Frank-Kamenetskii. Artificial DNA ,1 45-53 (2010) 4. J. E.Garneau et al. Nature 468, 67-71 (2010). 1086 Development and Applications of the Fluorescent Tricyclic Cytosine Family – The First Nucleic Acid Base Analogue FRET–pair

O The tricyclic cytosine family, tC, tC , and tCnitro, has been increasingly used in 119 biophysical and biochemical applications (1). The two fluorescent members of 1 Karl Börjesson this family, tC and tCO, both have unique properties among fluorescent base ana- Søren Preus2 logues (2, 3). Furthermore, the three tricyclic base analogues all form stable base Kristine Kilså2 pairs with guanine and give minimal perturbations to the native structure of DNA. Importantly, we have recently utilized tCO as a donor and developed tC as an 3,4 nitro Afaf H. El-Sagheer acceptor and, thus, established the first nucleic acid base analogue FRET-pair (4). Tom Brown3 This pair enables accurate distinction between distance- and orientation-changes in Bo Albinsson1 nucleic acid systems using FRET (see Figure). In combination with the favorable base pairing properties this will facilitate detailed studies of nucleic acid structures 1 L. Marcus Wilhelmsson * and structural changes. Moreover, the placement of the FRET-pair inside the base stack will be a great advantage in studies where other (biomacro)molecules interact with the nucleic acid. 1Department of Chemical and Biological Engineering/Physical Chemistry, Chalmers University of Technology, SE-41296 Gothenburg, Sweden 2Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark 3School of Chemistry, University of Southampton, Highfield, Southampton, SO17 1BJ, United Kingdom 4Chemistry Branch, Department of Science and Mathematics, Faculty of Petroleum and Mining Engineering,

Suez Canal University, Suez, Egypt O Figure: Efficiency of energy transfer for the base analogue FRET-pair tC -tCnitro estimated using O O *[email protected] decreases in tC emission (green circles) and tC average lifetimes (purple squares) as the two analogues are separated by 2-13 bases in a DNA duplex.

This research is funded by the Swedish Research Council and Olle Engkvist Byggmästare Foundation.

References

1. L. M. Wilhelmsson. Q Rev Biophys 43(2), 159-183 (2010). 2. P. Sandin, L. M. Wilhelmsson, P. Lincoln, V. E. C. Powers, T. Brown, and B. Albinsson. Nucleic Acids Res 33, 5019-5025 (2005). 3. P. Sandin, K. Börjesson, H. Li, J. Mårtensson, T. Brown, L. M. Wilhelmsson, and B. Albinsson. Nucleic Acids Res 36, 157-167 (2008). 4. K. Börjesson, S. Preus, A. H. El-Sagheer, T. Brown, B. Albinsson, L. M. Wilhelmsson. J Am Chem Soc 131, 4288-4293 (2009). Two-step Construction of Base-modified Nucleic Acids 1087 for Electrochemical DNA Sensing

Electrochemical DNA sensing strategies, oriented towards development of novel biosensors and bioassays, use various label-free (based on intrinsic DNA electro- activity) as well as label-based detection principles (1). The latter appear better 120 suited for sequence-specific DNA analysis when reliable recognition of a specific Miroslav Fojta1* nucleotide sequence among others, or when a nucleotide at a specific position (as in 1 single nucloeotide polymorphism typing), is desirable. Labeled nucleic acids have Petra Horakova usually been prepared via multistep chemical synthesis of oligonucleotides (ON). Hana Pivonkova1 We have introduced a novel two-step construction of modified ON, relying in direct Ludek Havran1 aqueous cross-coupling of halogenated deoxynucleoside triphosphates (dNTP) with 1 various functional groups and enzymatic incorporation of the nucleotide conjugates Peter Sebest into ON by DNA polymerases (2-6) or by terminal nucleotidyl transferase (TdT) Jan Spacek1 (7). Compared to the fully chemical ON synthesis, this approach is more versatile, Petr Orsag1 allowing – once a set of modified dNTPs is available – an easy and instant construc- 2 tion of labeled ON bearing various tags (or their combinations) at specific positions Milan Vrabel in the nucloeotide sequence using standard molecular biological tools. Hana Macickova-Cahova2 Lubica Kalachova2 Modified dNTPs, applicable as substrates for DNA polymerases or the TdT enzyme, 2 were substituted with electrochemically active labels at 7-position of 7-deazapurines Jan Riedl or 5-position of pyrimidines (8). The base-coupled tags include ferrocene (2), Veronika Raindlova2 2+ 2 3-nitro- or 3-aminophenyl groups (3) [M(bpy)3] (M = Ru or Os) complexes (4), Michal Hocek tetrathiafulvalene (5) as well as alkylsulfanylphenyl moieties possessing protected mercaptogroup (6). The correponding nucleotides were successfully incorporated into ONs, resuling in ON probes with distinct electrochemical properties owing to 1Institute of Biophysics, Academy of redox processes of the labels occuring at diverse potentials. Moreover, in some cases Sciences of the Czech Republic, the electronic communication between the tags and nucleobases made their redox potentials responding to the conjugate nucleobase and to incorporation into DNA, CZ-61265 Brno, Czech Republic thus offering another type of information to gain. We demonstrated applications of 2Institute of Organic Chemistry and the modified dNTPs and ONs in sensing DNA hybridization, SNP typing and moni- Biochemistry, Academy of Sciences of toring DNA-protein interactions. Further applications of the proposed strategy in nanotechnology, ON self-assembly, chemical biology and biosensing is anticipated. the Czech Republic, Gilead Sciences & IOCB Research Center, This work was supported by the ASCR (Z4 055 0506, Z5 004 0507 and Z5 004 CZ-16610 Prague 6, Czech Republic 0702) GA ASCR (IAA400040901), GACR (203/09/0317, P301/11/2076) and MEYS CR (LC06035, LC512). Department of Chemistry, University of Pittsburgh, References Pittsburgh, PA 15260 1. Labuda, A. M. O. Brett, G. Evtugyn, M. Fojta, M. Mascini, M. Ozsoz, I. Palchetti, E. Palecek, *[email protected] and J. Wang. Pure Appl Chem 82, 1161-1187 (2010). 2. P. Brazdilova, M. Vrabel, R. Pohl, H. Pivonkova, L. Havran, M. Hocek, and M. Fojta. Chem Eur J 13, 9527-9533 (2007). 3. H. Cahova, L. Havran, P. Brazdilova, H. Pivonkova, R. Pohl, M. Fojta, and M. Hocek. Angew Chem Int Ed 47, 2059-2062 (2008). 4. M. Vrabel, P. Horakova, H. Pivonkova, L. Kalachova, H. Cernocka, H. Cahova, R. Pohl, P. Sebest, L. Havran, M. Hocek, and M. Fojta. Chem Eur J 15, 1144-1154 (2009). 5. J. Riedl, P. Horakova, P. Sebest, R. Pohl, L. Havran, M. Fojta, and M. Hocek. Eur J Org Chem 3519-3525 (2009). 6. H. Macickova-Cahova, R. Pohl, P. Horakova, L. Havran, J. Spacek, M. Fojta, and M. Hocek. Chem Eur J in press (2011). 7. P. Horakova, H. Macicková-Cahova, H. Pivonkova, J. Spacek, L. Havran, M. Hocek, and M. Fojta. Org Biomol Chem 9, 1366-1371 (2011). 8. M. Hocek and M. Fojta. Org Biomol Chem 6, 2233-2241 (2008). 1088 Roles of Z-DNA Binding Proteins in Immunity & Infection

Four different Z-DNA binding proteins have been identified and characterized.

Two are normally present in higher eukaryotes. This includes the editing enzyme double-stranded RNA adenosine deaminase (ADAR1) and the double-stranded 121 DNA cytoplasmic receptor of the innate immune system which is active in stimu- Alexander Rich lating interferon production. Each of these proteins has Z-DNA binding domains Ky Lowenhaupt that are important for certain aspects of their activity. Jin-Ah Kwon Two other Z-DNA binding proteins have been identified. One of these is the E3L protein found in pox viruses. In particular vaccinia E3L has been studied, and it plays an active role in the pathogenesis of pox virus infections. Another protein Department of Biology, identified in zebra fish and gold fish is a component of the detection system for Massachusetts Institute of Technology, alerting the cell that a virus has infected it. Cambridge, MA 02139 All four of these proteins have a similar protein fold. The possibility of other classes of Z-DNA binding proteins will be discussed.

Role of Z-DNA Forming Silencer in the Regulation 122 of Human ADAM-12 Gene Expression ADAM-12 is a member of novel multifunctional ADAM family of proteins. Expres- Alpana Ray* sion of ADAM-12 is upregulated in many human cancers, arthritis and cardiac Srijita Dhar hypertrophy. The multidomain structure composed of a prodomain, a metallopro- Arvind Shakya teinase, disintegrin-like, epidermal growth factor-like, cysteine-rich and transmembrane domains and a cytoplasmic tail allows ADAM-12 to promote matrix Bimal K. Ray degradation, cell-cell adhesion and intracellular signaling capacities (1). Basal expression of ADAM-12 is very low in adult tissues but rises markedly in response Department of Veterinary Pathobiology, to certain physiological cues, such as during pregnancy in the placenta, during development in neonatal skeletal muscle and bone and in regenerating muscle University of Missouri, (2, 3). We have identified a highly conserved negative regulatory element (NRE) at Columbia, MO 65211 the 5′-UTR of human ADAM-12 gene, which acts as a transcriptional repressor (4). *[email protected] The NRE contains a stretch of dinucleotide-repeat sequence that is able to adopt a Z-DNA conformation both in vitro and in vivo. Substitution of the dinucleotide- repeat-element with a non-Z-DNA-forming sequence inhibits NRE function. We have detected Z-DNA-binding protein activity in several tissues where ADAM-12 expression is low while no such activity was seen in the placenta where ADAM-12 expression is high. These observations suggest that interaction of Z-DNA-binding proteins with ADAM-12 NRE is critical for transcriptional repression of ADAM-12. We further show that the Z-DNA forming transcriptional repressor element, by interacting with Z-DNA-binding proteins, is involved in the maintenance of consti- tutive low-level expression of human ADAM-12. Together these results provide a new insight on the regulatory role of Z-DNA-binding proteins which could be used as therapeutic targets for down-regulation of ADAM-12.

This research has been supported by grants from U.S. Army Medical research and Material Command and University of Missouri.

References

1. J. M. White. Current Opinion in Cell Biology 15, 598-606 (2003). 2. B. J. Gilpin, F. Loechel, M. G. Mattei, E. Engvall, R. Albrechtsen, and U. M. Wewer. Journal of Biological Chemistry 273, 157-166 (1998). 3. N. Kawaguchi et al. Journal of Cell Science 116, 3893-3904 (2003). 4. B. K. Ray, S. Dhar, A. Shakya, and A. Ray. Proceedings of the National Academy of Sciences of the United States of America 108, 103-108 (2011). Efficient B-Z DNA Conformational Transition 1089 Induced by Ru(II) Complexes

The left-handed Z-DNA plays important role in biological processes, it can regulate gene expression and involve in DNA processing event such as genetic instability (1). This may provides target for the prevention and treatment of some human dis- 123 eases (2). However, Z-DNA is a higher energy conformation than B-former, it is a Lv-Ying Li transient structure and intrinsically instable (3). Z-DNA is rarely formed without Hui Chao the help of high salt concentrations or negative supercoiling. It was found in d(CG)n polymer at high salt concentrations (2.5 M NaCl) (4). Recently, the designs of Li-Ping Weng molecules that can induce transition from B to Z conformations and stabilize the Liang-Nian Ji* Z-DNA have received considerable attention (5).

In the present work, two new MOE Key Laboratory of ruthenium complexes [Ru(tpy) Bioinorganic and Synthetic (pnt)]2+ (1) and Ru(dmtpy)(pnt)]2+ Chemistry, State Key (2) have been synthesized (tpy = 2,2’:6’,2”-terpyridine, dmtpy = Laboratory of Optoelectronic Materials 5,5''-dimethyl-2,2':6',2"-terpyridine, and Technologies, MOE Key Laboratory pnt = 2-(1,10-phenanthrolin-2-yl)- of Gene Engineering, School of naphtho[1,2-e][1,2,4] triazine). Two ruthenium complexes interact with Chemistry and Chemical Engineering, DNA by an intercalation binding Sun Yat-Sen University, Guangzhou mode (6-8). More interesting, they 510275, P. R. China can efficiently convert the B-form of DNA into the Z conformation under *[email protected] physiological salt condition in micro- mole concentration. This induced left-handed helix is extraordinary stable against high temperature. The mechanism is proposed that complexes insert to DNA base pairs and distort DNA helix structure. Mean- while, the Ru(II) center reduces electrostatic repulsion of phosphate backbone in the zigzag path of Z-DNA and locks the left-handed conformer irreversible to right-handed helix.

Acknowledgements

We gratefully acknowledge the supports of the 973 Program, the National Natu- ral Science Foundation of China, the Program for New Century Excellent Talents in University, the Guangdong Provincial Natural Science Foundation and Sun Yat-Sen University.

References

1. J. Zhao, A. Bacolla, G. Wang, and K. M. Vasquez. Cell Mol Life Sci 67, 43-62 (2010). 2. A. Rich and S. Zhang. Nat Rev Genet 4, 566-572 (2003). 3. J. Geng, C. Zhao, J. Ren, and X. Qu. Chem Commun 46, 7187-7189 (2010). 4. F. M. Pohl and T. M. Jovin. J Mol Biol 67, 375-396 (1972). 5. I. Doi, G. Tsuji, K. Kawakami, O. Nakagawa, Y. Taniguchi, and S. Sasaki. Chem Eur J 16, 11993-11999 (2010). 6. Y. Dalyan, I. Vardanyan, A. Chavushyan, and G. Balayan. J Biomol Struct Dyn 28, 123-131 (2010). 7. B. Jin, H. M. Lee, and S. K. Kim. J Biomol Struct Dyn 27, 457-464 (2010). 8. H. M. Lee, B. Jin, S. W. Han, and S. K. Kim. J Biomol Struct Dyn 28, 421-430 (2010). 1090 Platinum(II) Phenanthroimidazoles for G-quadruplex Targeting: The Effect of Structure on Binding Affinity, Selectivity and Telomerase Inhibition

G-quadruplexes have gained recognition as viable targets for chemotherapeutic 124 drug design, based on their ability to interfere with cancer cell proliferation (1, 2). Katherine Castor These higher order DNA structures, held together by Hoogsteen hydrogen bonds, Roxanne Kieltyka result from the folding of a guanine (G) rich DNA sequence in the presence of potassium or sodium cations (3). G-quadruplex forming sequences have been iden- Pablo Englebienne tified throughout the human genome in telomeres, promoter regions of oncogenes, Nathanael Weill nuclease hypersensitivity regions and untranslated regions of RNA (4, 5). From Johans Fakhoury this list, one highly investigated G-quadruplex target for drug design is the telomere. Small molecules that promote the folding of the human telomeric sequence, Johanna Mancini (TTAGGG)n, into a G-quadruplex structure can result in biologically relevant phe- Nicole Avakyan nomena, such as the loss of telomere integrity through disruption of the shelterin Anthony Mittermaier complex of proteins (6, 7) or the prevention of telomere elongation by the reverse transcriptase enzyme telomerase (8). Both of these events have profound effects on Chantal Autexier cancer cell proliferation thereby accomplishing one of the main goals of chemo- Nicolas Moitessier therapy, to halt tumor growth. Hanadi F. Sleiman* We have previously shown that platinum phenanthroimidazole-based binders are

good stabilizers of the intermolecular T4G4T4 G-quadruplex motif (9). These com- Department of Chemistry, plexes possess optimal geometries for targeting this structure and are substitutionally McGill University, Montreal, inert. Their synthesis is facile and highly modular, lending itself to the ready generation of compound libraries to maximize affinity and selectivity. However, upon ini- Quebec, Canada H3A2K6 tial studies involving the binding of the phenyl [1] and naphthyl [2] derivatives to the *[email protected] intramolecular motif based on the human telomere, we discovered that a significant twist within the ligand itself may prevent favorable π-stacking interactions of the naphthyl [2] ligand with the G-quadruplex. We hypothesized that incorporating an internal hydrogen bond within our phenanthroimidazole ligands may reduce the twist, resulting in a planar ligand surface for optimal overlap with the G-quadruplex.

Through the use of molecular modeling, circular dichroism, and fluorescence displacement assays, we have shown that phenanthroimidazole platinum(II) complexes template and stabilize G-quadruplex forming sequences based on the human telo-

meric repeat, (TTAGGG)n with the greatest stabilization from the indoyl [3] deriva- G4 tive ( DC50 = 0.53 µM). However, while the incorporation of the internal hydrogen bond does increase binding strength to the quadruplex motif, it also tends to reduce selectivity between quadruplex and duplex DNA. We found that the introduction of a chloride modification to the phenanthroimidazole core, as well as a sidearm with protonable sites restores selectivity while also increasing binding strength to G4 the quadruplex motif ( DC50 = 0.28 µM for 6). We also show complexes 1-4 are able to inhibit telomerase through the TRAP-LIG assay, thus verifying that phenanthroimidazole-based platinum(II) complexes can elicit antiproliferative effects and act as telomere disruption agents. 1091

References

1. A. De Cian, L. Lacroix, C. Douarre, N. Temime-Smaali, C. Trentesaux, J. F. Riou, and J. L. Mergny. Biochimie 90, 131-155 (2008). 2. Y. Qin and L. H. Hurley. Biochimie 90, 1149-1171 (2008). 3. A. N. Lane, J. B. Chaires, R. D. Gray, and J. O. Trent. Nucleic acids research 36, 5482-5515 (2008). 4. S. Balasubramanian and S. Neidle. Current Opinion in Chemical Biology 13, 345-353 (2009). 5. J. L. Huppert. Biochimie 90, 1140-1148 (2008). 6. H. Tahara, K. Shin-ya, H. Seimiya, H. Yamada, T. Tsuruo, and T. Ide. Oncogene 25, 1955-1966 (2006). 7. R. Rodriguez, S. Müller, J. A. Yeoman, C. Trentesaux, J. F. Riou, and S. Balasubramanian. Journal of the American Chemical Society 130, 15758-15759 (2008). 8. T. Tauchi, K. Shin-Ya, G. Sashida, M. Sumi, S. Okabe, J. H. Ohyashiki, and K. Ohyashiki. Oncogene 25, 5719-5725 (2006). 9. R. Kieltyka, J. Fakhoury, N. Moitessier, and H. F. Sleiman. Chemistry-A European Journal 14, 1145-1154 (2008).

G-Quadruplex, Telomere and Telomerase

Telomeres serve as protective caps at the ends of linear eukaryotic chromosomes, playing a crucial role in cell survival and proliferation. Tandem repeats of the 125 sequence TTAGGG constitute the human telomeres, with pendent G-rich single Lim Kah Wai strands of 100–200 nt at the 3’ ends. The propensity of these G-rich overhangs to Phan Anh Tuân form G-quadruplexes, and the inhibitory effects of such structures on the catalytic activity of the enzyme telomerase, have led to a growing interest in the study of telomeric G-quadruplexes and the development of specific telomeric quadruplex- School of Physical and Mathematical stabilizing ligands as anticancer drugs. Previously, it has been reported that human Sciences and School of Biological telomeric DNA sequences could adopt in different experimental conditions four different intramolecular G-quadruplexes each involving three G-tetrad layers, Sciences, Nanyang Technological namely, Na+ solution antiparallel-stranded basket form (1), K+ crystal parallel- University, Singapore + + stranded propeller form (2), K solution (3 + 1) Form 1 (3-5), and K solution [email protected] (3 + 1) Form 2 (6). Here we report novel intramolecular G-quadruplex structures adopted by canonical (TTAGGG) (7) and variant (CTAGGG, TAGGG) (8, 9) four- [email protected] repeat human telomeric sequences in K+ solution, which surprisingly utilize only two of the three contiguous guanines from successive G-tracts for G-tetrad core formation. Structural elucidation of these oligonucleotides revealed extensive base 1092 pairing and stacking interactions in the loops, indicating that the overall G-quadruplex topology of a G-rich sequence is defined not only by maximizing the number of G-tetrads but also by maximizing all possible interactions in the loops. On the other hand, promoter G-quadruplex formation represents an alternative approach of selective gene regulation at the transcriptional level. The promoter for the catalytic subunit of human telomerase, hTERT, contains many guanine-rich stretches on the same DNA strand suitable for targeting (10-12). We show here that one particular G-rich sequence in this region coexists in two G-quadruplex conformations (11), each of which comprises several robust structural motifs. Recurrence of structural elements in the structures presented suggests a “cut-and-paste” principle for the design and prediction of G-quadruplex topologies, for which different elements could be extracted from one G-quadruplex and inserted into another.

This research was supported by grants from Singapore Biomedical Research Council, Singapore Ministry of Education, and Nanyang Technological University.

References

1. Y. Wang and D. J. Patel. Structure 1, 263-282 (1993). 2. G. N. Parkinson, M. P. Lee, and S. Neidle. Nature 417, 876-880 (2002). 3. Y. Xu, Y. Noguchi, and H. Sugiyama. Bioorg Med Chem 14, 5584-5591 (2006). 4. A. Ambrus, D. Chen, J. Dai, T. Bialis, R. A. Jones, and D. Yang. Nucleic Acids Res 34, 2723-2735 (2006). 5. K. N. Luu, A. T. Phan, V. Kuryavyi, L. Lacroix, and D. J. Patel. J Am Chem Soc 128, 9963-9970 (2006). 6. A. T. Phan, K. N. Luu, and D. J. Patel. Nucleic Acids Res 34, 5715-5719 (2006). 7. K. W. Lim, S. Amrane, S. Bouaziz, W. Xu, Y. Mu, D. J. Patel, K. N. Luu, and A. T. Phan. J Am Chem Soc 131, 4301-4309 (2009). 8. K. W. Lim, P. Alberti, A. Guedin, L. Lacroix, J. F. Riou, N. J. Royle, J. L. Mergny, and A. T. Phan. Nucleic Acids Res 37, 6239-6248 (2009). 9. L. Hu, K. W. Lim, S. Bouaziz, and A. T. Phan. J Am Chem Soc 131, 16824-16831 (2009). 10. S. L. Palumbo, S. W. Ebbinghaus, and L. H. Hurley. J Am Chem Soc 131, 10878-10891 (2009). 11. K. W. Lim, L. Lacroix, D. J. Yue, J. K. Lim, J. M. Lim, and A. T. Phan. J Am Chem Soc 132, 12331-12342 (2010). 12. E. Micheli, M. Martufi, S. Cacchione, P. De Santis, and M. Savino. Biophys Chem 153, 43-53 (2010).

Structure of Human Telomeric RNA G-quadruplexes

Telomeres, which are located at the chromosomal ends, act as protective caps that 126 prevent chromosome loss and degradation. Telomeres had always been thought to Herry Martadinata be transcriptionally silent until the recent finding that they could be transcribed into Anh Tuan Phan RNA molecules with lengths ranging from 100 to 9000 nt (1, 2). It has further been shown that telomeric-repeat-containing RNA (TERRA) perform various cellular regulatory functions, such as regulation of telomere length, inhibition of telomerase, School of Physical and Mathematical telomeric heterochromatin formation, and telomere protection (3-7). Sciences, School of Biological Sciences, Our structural studies showed that human TERRA sequences formed propeller-type Nanyang Technological University, parallel-stranded RNA G-quadruplexes. We have determined the NMR-based solu- Singapore 637371 tion structure of a dimeric propeller-type RNA G-quadruplex formed by the 12-nt [email protected] human TERRA sequence r(UAGGGUUAGGGU). We also observed the stacking of two such propeller-type G-quadruplex blocks for the 10-nt human TERRA [email protected] sequence r(GGGUUAGGGU) and a higher-order G-quadruplex structure for the 9-nt human TERRA sequence r(GGGUUAGGG).

Ribonuclease protection assay was used to investigate the structures formed by long human TERRA (9,10). We found that G-quadruplexes comprising four and eight UUAGGG repeats were most resistant to RNase T1 digestion, presumably with the former adopting an all-parallel-stranded conformation and the latter forming a structure with two tandemly stacked G-quadruplex subunits each containing three 1093 G-tetrad layers. Molecular dynamics simulations of eight-repeat human TERRA sequences consisting of different stacking interfaces between the two G-quadruplex subunits, i.e. 5’-5’, 3’-3’, 3’-5’, and 5’-3’, demonstrated stacking feasibility for all but the 5’-3’ arrangement. A continuous stacking of the loop bases from one G-quadruplex subunit to the next was observed for the 5’-5’ stacking conformation. Based on the results, we propose a “beads-on-a-string”-like arrangement along human TERRA (11), whereby each bead is made up of either four or eight UUAGGG repeats in a one- or two-block G-quadruplex arrangement, respectively (10).

References

1. C. M. Azzalin, P. Reichenbach, L. Khoriauli, E. Giulotto, and J. Lingner. Science 318, 798-801 (2007). 2. S. Schoeftner and M. A. Blasco. Nat Cell Biol 10, 228-236 (2008). 3. B. Horard and E. Gilson. Nat Cell Biol 10, 113-115 (2008). 4. B. Luke and J. Lingner. EMBO J 28, 2503-2510 (2009). 5. S. Feuerhan, N. Iglesias, A. Panza, A. Porro, and J. Linger. FEBS Letter 584, 3812-3818 (2010). 6. Z. Deng, A. E. Campbell, and P. M. Lieberman. Cell Cycle 9, 69-74 (2010). 7. I. L. de Silanes, M. S. d’Alcontres, and M. A. Blasco. Nat Comm 1, 1-9 (2010). 8. H. Martadinata and A.T. Phan. J Am Chem Soc 131, 2570-2578 (2009). 9. A. Randall, J. D. Griffith. J Biol Chem 284, 13980-13986 (2009). 10. H. Martadinata, B. Heddi, K. W. Lim, and A. T. Phan (2011) submitted. 11. H. Yu, D. Miyoshi, and N. Sugimoto. J Am Chem Soc 128, 15461-15468 (2006).

Structural Transition in the Human Telomeric DNA Sequence d[(TTAGGG)4] upon interaction With Putative Anticancer Agents, Sanguinarine 127 And Ellipticine Saptaparni Ghosh1* Suman Kalyan Pradhan1,2 Even though there have been several studies of interaction between DNA and ant- Gautam Basu3 cancer agents (1-4), not very much is known about their interactions with the telo- 1 meric regions of chromosomes. Guanine rich sequences fold into a non-canonical Dipak Dasgupta * structure known as G-quartet in the presence of appropriate salt concentration. G-quartets stack on one another to form G-quadruplex. G-rich sequences are found 1Biophysics Division, Saha Institute in the telomeric regions of chromosomes and play an important role in chromosome duplication. They are potential targets for anticancer drugs (5). Here we have of Nuclear Physics, Block-AF, Sector-I, reported the structural transition of G-quadruplex induced by two putative antican- Bidhannagar, Kolkata 700064, India cer agents, Sanguinarine (SGR) and Ellipticine (ELP), from plant sources. 2Section of Molecular Biology, UC San Diego, 9500 Gilman, SGR binds with the human telomeric DNA sequence d[(TTAGGG)4] (H24) in the presence of K+ with a 2:1 binding stoichiometry possibly via the end stacking Dr. La Jolla, CA 92093 (present address) mechanism. Studies based on CD spectroscopy have indicated that at higher con- 3Department of Biophysics, Bose centration (above 20 µM) SGR induces a structural alteration in H24. The structure of H24 changes from the mixed Type-I conformation to the Na+ -conformation Institute, P-1/12 CIT Scheme VIIM, (6). ELP also binds with H24. At lower concentrations (100 nM) it binds with 3:2 Kolkata 700054, India * [email protected] * [email protected] 1094 stoichiometry (ELP : H24) as suggested from Jobs’ Plot based on fluorescence measurements. But at higher ellipticine concentration (8 µM), it binds with a stoichiometry of 2:1 (ELP : H24) and a relatively lower affinity. CD spectroscopic studies suggest that at higher concentrations of drug, H24 undergoes a structural change from the mixed Type-I conformation to the Na+ -conformation. Similar studies from other group (7) along with our results suggest that G-quadruplex interacting drugs probably induce a structural change at higher drug concentration. It could be a plausible molecular mechanism of the action of G-quadruplex binding drugs that interferes with normal biological activities.

References

Dynamic XPD & Mre11-Rad50-Nbs1 DNA Repair Complexes: Disease-causing Mutations and 128 Biological Outcomes John A. Tainer1,2 DNA ends at breaks, replication forks and telomeres are paradoxically often critically controlled for repair and integrity by a single trimeric complex of Mre11-Rad50- 1Lawrence Berkeley National Nbs1 (MRN) dimers. MRN heterohexamer acts in key sensing, signaling, regula- Laboratory, Berkeley, CA, USA tion, and effector responses to DNA double-strand breaks including ATM activation, homologous recombinational repair, microhomology-mediated end joining and, in 2Department of Molecular Biology, some organisms, non-homologous end joining. Our results suggest that this is pos- The Skaggs Institute for Chemical sible because each MRN subunit can exist in three or more distinct states; thus, the Biology, The Scripps Research Institute, trimer of MRN dimers can exist in a stunningly large number of states. MRN can therefore act as a molecular machine that effectively assesses optimal responses and La Jolla, CA 92037, USA signals pathway choice based upon its states as set by cell status and the nature of the [email protected] DNA damage. Diverse bulky lesions that distort double helical DNA are repaired by nucleotide excision repair (NER) orchestrated by the TFIIH complex enzymes: the XPB and XPD helicases and CAK kinase. Combined with mapping of XP patient mutations, detailed structural analyses provide a framework for integrating and unifying the rich biochemical and cellular information that has accumulated on NER over nearly forty years of study. This integration resolves puzzles regarding XP helicase functions and indicates that XP helicase positions and activities within TFIIH detect and verify damage, select damaged strand for incision, and coordinate repair with transcription and cell cycle through CAK signaling. Overall this concept that allosteric changes coordinate repair with replication, transcription, and cell cycle by coupling conformations to kinase activities provides opportunities to develop ligand master keys to cell biology and improved therapeutic interventions (1-4).

References

1. R. S. Williams, G. Moncalian, J. S. Williams, Y. Yamada, O. Limbo, D. S. Shin, L. M. Groocock, D. Cahill, C. Hitomi, G. Guenther, D. Moiani, J. P. Carney, P. Russell, and J. A. Tainer. Cell 135, 97-109 (2008). 2. L. Fan, J. O. Fuss, Q. J. Cheng, A. S. Arvai, M. Hammel, V. A. Roberts, P. K. Cooper, and J. A. Tainer. Cell 133, 789-800 (2008). 3. R. S. Williams, G. E. Dodson, O. Limbo, Y. Yamada, J. S. Williams, G. Guenther, S. Classen, J. N. M. Glover, H. Iwasaki, P. Russell, and J. A. Tainer. Cell 139, 87-99 (2009). 4. E. A. Rahal, L. A. Henricksen, Y. Li, R. S. Williams, J. A. Tainer, and K. Dixon. Cell Cycle 9, 2866-2877 (2010). Search of Damaged Bases by DNA Repair Enzymes: 1095 Random Walks in One and Three Dimensions

Many DNA-dependent proteins, such as restriction endonucleases, transcription factors, or DNA repair enzymes face the challenge of finding rare specific sequences or structural elements of DNA in a vast excess of competing non-specific DNA (1, 2). 129 Proteins may locate targets in DNA using either diffusion in three dimensions of Dmitry O. Zharkov1* movement along the DNA contour. The proteins that move along DNA use two Grigory V. Mechetin1 fundamentally different movement mechanisms: directed movement coupled with 2 ATP hydrolysis and random one-dimensional diffusion driven by Brownian fluc- Boris A. Veytsman tuations. We have developed a new approach to quantitatively analyze the latter mechanism (3) and used it to study the process of lesion search by several DNA 1SB RAS Institute of Chemical Biology repair enzymes: Escherichia coli and human uracil DNA glycosylases, 8-oxoguanine-DNA glycosylases, and AP endonucleases. All these enzymes were able to and Fundamental Medicine, move along DNA by one-dimensional diffusion over distances up to 80 base pairs, Novosibirsk, Russia 630090 with the probability of passage decreasing with the increasing travel distance. The 2Computational Materials Science Center, average travel distance was significantly influenced by ionic strength, Mg2+ ions, and competing non-specific DNA-binding molecules but was barely affected by George Mason University, crowding agents. Nicks and short gaps in DNA, as well as specifically bound small Fairfax, VA 22030, USA ligands, were efficiently overpassed, and DNA strands could be switched during the *[email protected] search, indicating that the enzymes are able to use hopping, a mode of movement involving dissociation of the protein–DNA complex and immediate reassociation of the protein with DNA in the close vicinity of its previous position. Differences in the behavior of uracil-DNA glycosylase on blunt and hairpin DNA ends was observed, suggesting that the ends serve neither as points of irreversible loss nor total reflection of the moving protein. An analytical model has been developed that describes the one-dimensional random walk of proteins along DNA in terms of probabilities of the enzyme to move or dissociate at each step.

This research has been supported by RFBR (11-04-00807-a) and Presidium of RAS “Molecular and Cell Biology” program (6.14).

References

1. O. G. Berg, R. B. Winter, and P. H. von Hippel. Biochemistry 27, 6929-6948 (1981). 2. D. O. Zharkov, and A. P. Grollman. Mutat Res 577, 24-54 (2005). 3. V. S. Sidorenko, G. V. Mechetin, G. A. Nevinsky, and D. O. Zharkov. FEBS Lett 582, 410-414 (2008).

Kinetic Characterization of the Repair Enzyme OGG1 acting on Triplet Repeat DNA 130 Triplet repeat sequences, such as CAG/CTG, expand in the human genome to cause several neurological disorders. Work from other laboratories has implicated Daniel A. Jarem the DNA repair enzyme 8-oxo-7,8-dihydroguanine glycosylase (OGG1) in triplet Kelly M. Schermerhorn repeat expansion (1, 2). OGG1 initiates the base excision repair (BER) process in Nicole R. Wilson mammalian cells by excising the oxidatively damaged nucleobase 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Motivated by the demonstrated involvement of Sarah Delaney* OGG1 in triplet repeat expansion we initiated a comprehensive kinetic analysis of the activity of OGG1 on triplet repeat oligonucleotide substrates. These substrates Department of Chemistry, include canonical CAG/CTG duplexes and also the non-canonical stem-loop hair- Bown University, pins that are proposed to contribute to repeat expansion. We determined the binding affinity, the rate of cleavage of the N-glycosidic bond of 8-oxoG, and the rate 324 Brook St, Box H, of product release for human OGG1 acting on TNR sequences. Comparison of the Providen RI 02912, results obtained for the triplet repeats sequences to those obtained for a mixed- *[email protected] sequence duplex allowed us to define the contribution of sequence context of the damage to enzyme activity. We demonstrate that the structure of the DNA substrate 1096 modulates OGG1 activity whereas changes to the sequence composition are well tolerated. Taken together, these results contribute to our knowledge of the sequence and structural specificity of OGG1 and, furthermore, define the kinetic parameters of the event that initiates triplet repeat expansion via BER.

References

1. I. V. Kovtun, Y. Liu, M. Bjoras, A. Klungland, S. H. Wilson, and C. T. McMurray. Nature 447, 447-452 (2007). 2. Y. Liu, R. Prasad, W. A. Beard, E. W. Hou, J. K. Horton, C. T. McMurray, and S. H. Wilson. J Biol Chem 284, 28352-28366 (2009).

Enzyme-catalyzed DNA Cytidine Deamination in 131 Human Biology Over the past decade, we have learned that humans have ten DNA cytidine deami- Reuben S. Harris nases that catalyze a variety of physiological functions from triggering antibody gene diversification to providing innate immunity to a broad number of foreign Department of Biochemistry, DNA elements such as viruses and transposons. I will review the biology of these enzymes and discuss recent biochemical and structural advances that help us to Molecular Biology & Biophysics, better appreciate their integral cellular functions (1-4). University of Minnesota – Twin Cities, Minneapolis & Saint Paul, MN 55455 This research has been supported by NIH grants R01 GM080437, R01 AI064046, R03 MH089432 and R21/R33 AI073167. [email protected] References

1. S. M. D. Shandilya, M. N. L. Nalam, E. A. Nalivaika, P. J. Gross, J. C. Valesano, K. Shindo, M. Li, M. Munson, E. Harjes, T. Kouno, H. Matsuo, R. S. Harris, M. Somasundaran, and C. A. Schiffer. Structure 18, 28-38 (2010). 2. M. D. Stenglein, M. B. Burns, M. Li, J. Lengye, and R. S. Harris. Nature Structural and Molecular Biology 17, 222-229 (2010). 3. E. W. Refsland, M. D. Stenglein, K. Shindo, J. S. Albin, W. L. Brown, and R. S. Harris. Nucleic Acids Research 38, 4274-84 (2010). 4. L. S. Shlyakhtenko, A. Y. Lushnikov, M. Li, L. Lackey, R. S. Harris, and Y. L. Lyubchenko. Journal of Biological Chemistry 286, 3387-95 (2011).

The Good and the Bad of DNA Damage DNA Damage and Epigenetic Marks Drive 132 Transcription and Repair Enrico V. Avvedimento Methylation is an epigenetic modification of the DNA, which can silence genes. By using a sophisticated genetic system, we have induced a break in the double helix of Dept Biologia e Patologia Molecolare DNA at a single site in mouse or human cells. This important lesion is repaired by e Cellulare, Facolta’ di Medicina, a precise mechanism: the damaged chromosome pairs and retrieves genetic information from the homologous chromosome partner. The repair of the double strand Universita’ “Federico II”, break leaves a series of methyl groups on the DNA base C (methylation) in a frac- Naples, Italy tion of the repaired molecules flanking the break and, as consequence, the underlying genetic information may be packed and silenced. DNA methylation represents a scar marking damaged and repaired DNA. This processi s a strong evolutionary adaptive response used by the cell to protect the genome. Packed chromatin DNA is more resistant to damage, and silencing protects the cell from expressing a damaged gene, since the repair may introduce some changes in the code.

During these experiments we found that selective and local DNA damage is an essential step governing transcription of genes. Estrogens are hormones that associated to the specific receptor penetrate into chromatin-DNA and bind a specific DNA sequence 1097 present in several places in the chromosomes. We have found that the receptor bound to the hormone induces oxidation and single strand breaks in the DNA, where the receptor binds. The relaxed DNA chromatin bends and loops out bringing in close proximity non-contiguous regions of chromosomes. The loops of DNA promote the association of the receptor-hormone and RNA polymerase II. This enzyme normally is poised for activation of transcription at 5’ end of genes across the genome. As soon as the RNA polymerase touches the receptor, the enzyme receives directions and starts transcription.

We found that DNA untwisting, which renders the strands flexible, is caused by nicks in the DNA induced by the receptor. These “holes” open an entry site for RNA polymerase, which now can find the bound receptor and eventually are sealed rapidly by repair enzymes. At the end of transcription, the DNA is packed back into condensed chromatin. This mechanism was unexpected, since “nicking” of DNA is rather a dangerous event, which shall be quickly repaired. However, the price to pay for estrogen, sex differentiation, and maintenance of the gnetic information may be high, because repeated nicks and sealing events may result in imperfect repair and change of the genetic code. This mechanism may explain the occurrence of breast cancer, which is considered strictly dependent on estrogens. Also, it suggests that transcription is a costly process that deteriorate the whole genomic machine in the long run.

Substrate Interactions of a Human DNA Alkyltransferase 133 Human cells contain DNA alkyltransferases that protect genomic integrity under normal conditions but also defend tumor cells against chemotherapeutic alkylat- Manana Melikishvili ing agents. Here we explore how structural features of the DNA substrate affect Lance Hellman the binding and repair activities of the human O6-alkylguanine-DNA alkyltrans- Michael G. Fried* ferase (AGT).

To perform its repair functions, AGT partitions between adduct-containing sites Department of Molecular and Cellular and the large excess of adduct-free genomic DNA. Cooperative binding results in Biochemistry, University of Kentucky, an all-or-nothing association pattern on short templates. The apparent binding site 741 S. Limestone St., Lexington, size Sapp (mean = 4.39 ± 0.02 bp) oscillates with template length. Oscillations in cooperativity factor ω have the same frequency but are of opposite phase to Sapp so KY 40536 the most stable complexes occur at the highest packing densities. At high binding *[email protected] densities the site size (~4 bp/protein) is much smaller than the contour length (~8 bp) occupied in crystalline 1:1 complexes. A model in which protein molecules overlap along the DNA contour has been proposed; this model predicts that optimal protein- protein contacts will occur when the DNA is torsionally relaxed. Supporting this prediction, competition assays show that AGT binds relaxed DNAs in preference to negatively-supercoiled forms and topoisomerase assays show that AGT binding is accompanied by a small-but-measurable net unwinding (7.14 ± 0.33 deg/protein). These results predict that AGT will partition in favor of torsionally-relaxed, relatively protein-free DNA structures like those near replication forks.

AGT must also function at telomeres, where G-rich sequences have the potential to form quadruplex structures and where methylation at the O6 position of guanines interferes with quadruplex formation. AGT interactions with small unimolecular quadruplexes are characterized by reduced binding stoichiometries, affinities and O6-methyl G repair activities when compared to those with linear DNAs. Thus, AGT may function best at telomeres when quadruplex formation is inhibited by helicases or other telomere-binding proteins. 1098

This work was supported by NIH Grant GM070662 to MGF.

Model of AGT-DNA complex with double-stranded DNA. The repeating unit of this model is one molecule of AGT (colors) plus 4 base-pairs of DNA (black); the coordinates were derived from PDB file 1T38. Repeating units were juxtaposed with preservation of B-DNA helical parameters (separation = 3.4 Å, twist = 34.6 degrees) between base-pairs of adjacent units. The result is a 3-start helical array (left) with important contacts between proteins n and n + 3 (right).

Terminalia chebula Extract Enhances Fenton-reaction 134 Mediated Nucleoside Damage Antioxidant and reactive oxygen species scavenging properties of 70% methanol- Indrani Kar in-water extracts of fruits of the plants Terminalia chebula, Terminalia bellirica and Rajagopal Chattopadhyaya* Emblica officinalis (respectively Harra, Bahera and Amala in Hindi) were reported by another group in our institute (1). The efficacy of these and other plant extracts in prolonging lives of mice suffering from laboratory-induced cancer (ascitis) Department of Biochemistry, were tested by the same group, though hitherto unpublished (2). These plants are Bose Institute, already documented in India for their medicinal use for several other diseases (3), P-1/12, C.I.T. Scheme VIIM, and popular as an ayurvedic tonic called ‘trifala’. We have investigated the effect of including these extracts in concentrations 30 µg/ml, 60 µg/ml and 90 µg/ml Calcutta 700054, India along with 1 mM deoxynucleoside concentrations taken as standard Fenton-reaction *[email protected] mediated in vitro damage assays (4-6). For all four deoxynucleosides, it is found that the fruit extract enhances the amount of Fenton-reaction mediated damage, amount of enhancement increasing with amount of plant extract added. It is likely that these plants prove beneficial in cancer due to this enhancement of nucleoside damage rather than its reduction (as scavengers).

References

1. B. Hazra, R. Sarkar, S. Biswas, and N. Mandal. B M C Complementary and Alternative Medicine 10, 20 (2010). 2. S. Biswas and N. Mandal. private communication to R.C. (2008). 3. P. Kaushik and A. K. Dhiman. Medicinal Plants and Raw Drugs of India, 255-257, 254-255 and 484-486, Dehradun, Bishen Singh and Mahendra Pal Singh (1999). 4. Y. Luo, E. S. Henle, and S. Linn. J Biol Chem 271, 21167-21176 (1996). 5. E. S. Henle, Y. Luo, W. Gassmann, and S. Linn. J Biol Chem 271, 21177-21186 (1996). 6. Y. Luo, E. Henle, R. Chattopadhyaya, R. Jin, and S. Linn. Methods in Enzymology 234, 51-59 (1994). Characterization of erp Operator DNA Binding by 1099 Borrelia burgdorferi Protein BpaB

BpaB is an erp Operator DNA binding protein expressed by Borrelia burgdorferi, the causative agent of Lyme disease. BpaB and 2 other transcription regulatory proteins, EbfC and BpuR proteins control erp transcription levels. Erp proteins bind 135 host factor H, a regulator of complement activation. By doing so they help protect the bacterium from the alternative pathway that involves complement-mediated Claire Adams* killing. Transcription and expression of Erp proteins occurs only upon association Manana Melikishvili with mammalian host blood or tissue, when multiple environmental signals, includ- Mike Fried ing temperature induce expression. Brian Stevenson BpaB has recently been identified as an erp transcription repressor and competes for binding to erp Operator DNA with a second B. burgdorferi DNA-binding pro- Department of Microbiology, tein, EbfC (1). Mutagenesis of erp Operator DNA and EMSA binding specificity measurements show that BpaB binds within a 15 bp region just 5’ of the -35 Immunology and Molecular sequence on erp operator 2 DNA. DNA Footprinting experiments also indicate that Genetics, University of Kentucky, BpaB binds initially within the 15 bp erp DNA region after which it then initiates College of Medicine, Lexington, nonspecific binding to flanking regions. BpaB binds initially at a stoichiometry of 1, to a region of erp Operator DNA that contains the 15 bp binding region. Further Kentucky 40536-0298 complex formations observed on EMSA gels indicated that 1 protein was being *[email protected] added in a step wise fashion. Ultracentrifugation equilibrium analysis indicated that up to 13 proteins can attach to the same 23 bp erp DNA oligomer thereby supporting footprinting results. Similar analysis also showed that 24 proteins are capable of binding to a 50 bp erp dsDNA oligomer. EMSA binding assays indicate a

Kd/dissociation constant of ~0.25 µM for binding to 50 bp erp dsDNA. Experiments are under way to determine if BpaB binding to erp operator DNA is cooperative.

Reference

1. L. H. Burns, C. A. Adams, S. P. Riley, B. L. Jutras, A. Bowman, A. M. Chenail, A. E. Cooley, L. A. Haselhorst, A. M. Moore, K. Babb, M. G. Fried, and B. Stevenson. Nucleic Acids Research, 38, 1-13 (2010).

Characterization of Fpg Protein Binding to DNA Lesions using Pyrrolocytosine Fluorescence 136 Reactive oxygen species damage DNA to produce a variety of genotoxic lesions. In particular, 7,8-dihydro-8-oxoguanine (oxoG) is one of the most common pre-mu- Nikita A. Kuznetsov tagenic products of base oxidation in DNA. OxoG is repaired (1) through excision Olga S. Fedorova* by formamidopyrimidine-DNA glycosylase (Fpg) in bacteria or 8-oxoguanine- DNA glycosylase (OGG1) in eukaryotes. In addition to its glycosylase activity, Institute of Chemical Biology and Fpg possesses an AP-lyase activity, which catalyzes sequential elimination of the 3’-phosphate (β-elimination) and the 5’-phosphate (δ-elimination) at the nascent Fundamental Medicine, Novosibirsk or pre-formed abasic (AP) site, producing a one-nucleotide gap flanked by two 630090, Russia phosphates (2). The glycosidic bond breakage is initiated by a nucleophilic attack *[email protected] at C1’ by the Pro-1 residue, resulting in a covalent enzyme–DNA Schiff base intermediate, which then rearranges and undergoes elimination. The three-dimensional structure of E. coli Fpg shows that DNA binding is accompanied with drastic conformational changes, including DNA bending, eversion of oxoG from DNA, and insertion of Met-73, Arg-108 and Phe-110 residues into DNA (3, 4).

In our previous studies we have used quench-flow technique to show that the kinetics of processing of oxoG and AP site lesions by Fpg from E. coli involves a burst and a stationary phases. The data from stopped-flow kinetics with tryptophan and 2-aminopurine fluorescence detection revealed that both the protein and the C H 1100 (A) 3 H (B) N oxoG/pyrC N

N O F/pyrC O O AP/pyrC Fluorescence pyrC

O 0.1 110 100 Time, s Figure 1: Structure of pyrC (A) and its fluorescence traces in the Fpg catalytic cycles with oxoG-, AP- and tetrahydrofurane (F) containing DNA-substrates. Stopped-flow fluorescence kinetics demonstrated the multi-step character of lesion recognition (Figure 1B). Thermodynamic parameters of each recognition step were found by analysis of fluorescence traces at different temperatures.

damaged DNA undergo extensive conformational changes in the course of DNA substrate binding and cleavage (5, 6). It was concluded that the cleaved product formation is initially reversible. We have also applied mass spectrometry with elec- trospray ionization to follow appearance and disappearance of transient covalent intermediates between Fpg and the substrate DNA (6, 7). The overall rate-limiting step of the enzymatic reaction seemed to be the release of Fpg from its adduct with the 4-oxo-2-pentenal remnant of the deoxyribose moiety formed as a result of DNA strand cleavage by β,δ-elmination.

To gain a deeper insight into mechanism by which Fpg protein recognizes DNA lesions we have studied the changes both in fluorescence of pyrrolocytosine (pyrC) (Fig. 1A) and in FRET of Trp/pyrC donor/acceptor pair. PyrC was placed opposite damaged nucleotides in DNA strand.

Acknowledgements

This work was supported by Grants from the RFBR (10-04-00070), Siberian Divi- sion of the Russian Academy of Sciences (28, 48), Russian Ministry of Education and Sciences (02.740.11.0079, NS-3185.2010.04, MK-1304.2010.04).

References

1. N. A. Timofeyeva, V. V. Koval, D. G. Knorre, D. O. Zharkov, M. K. Saparbaev, A. A. Ishchenko, and O. S. Fedorova. J Biomol Struct Dyn 26, 637-652 (2009). 2. D. O. Zharkov, G. Shoham, and A. P. Grollman. DNA Repair 2, 839-862 (2003). 3. R. Gilboa, D. O. Zharkov, G. Golan, A. S. Fernandes, S. E. Gerchman, E. Matz, J. H. Kycia, A. P. Grollman, and G. Shoham. J Biol Chem 277, 19811-19816 (2002). 4. V. V. Koval, N. A. Kuznetsov, D. O. Zharkov, A. A. Ishchenko, K. T. Douglas, G. A. Nevinsky, and O. S. Fedorova. Nucleic Acids Res 32, 926-935 (2004). 5. N. A. Kuznetsov, V. V. Koval, D. O. Zharkov, Y. N. Vorobjev, G. A. Nevinsky, K. T. Douglas, and O. S. Fedorova. Biochemistry 46, 424-435 (2007). 6. N. A. Kuznetsov, D. O. Zharkov, V. V. Koval, M. Buckle, and O. S. Fedorova. Biochemistry 48, 11335-11343 (2009). 7. V. V. Koval, N. A. Kuznetsov, A. A. Ishchenko, M. K. Saparbaev, O. S. Fedorova. Mutation Res 685, 3-10 (2010). Dynamical Allosterism in the Mechanism of Action 1101 of DNA Mismatch Repair Protein MutS

The multidomain protein T. aquaticus MutS and its prokaryotic and eukaryotic homologs recognize DNA replication errors and initiate mismatch repair (MMR). MutS actions are fueled by ATP binding and hydrolysis, which modulate its inter- 137 actions with DNA and other proteins in the MMR pathway. The DNA binding Susan N. Pieniazek* and ATPase activities are allosterically coupled over a distance of ~70 Å, and the Manju M. Hingorani molecular mechanism of coupling has not been clarified. To address this problem, all-atom molecular dynamics (MD) simulations of ~150 ns including explicit D. L. Beveridge solvent were performed on two key complexes—ATP-bound and ATP-free MutS•DNA(+T bulge). We used principal component analysis (PCA) in fluctua- Department of Chemistry, tion space to assess ATP ligand-induced changes in MutS structure and dynamics. The MD calculated ensembles of thermally accessible structures showed markedly Wesleyan University, small differences between the two complexes. However, analysis of the covariance Middletown, CT 06459 of dynamical fluctuations revealed a number of potentially significant inter-residue *[email protected] and inter-domain couplings. Moreover, PCA analysis revealed clusters of correlated atomic fluctuations linking the DNA and nucleotide binding sites, especially in the ATP-bound MutS•DNA(+T) complex. These results support the idea that allosterism between the nucleotide and DNA binding sites in MutS can occur via ligand-induced changes in motion, i.e., dynamical allosterism.

This work was supported by the NSF (MCB-1022203 to M.M.H) and the NIH (GM-076490 to D.L.B). S.N.P. was supported by a NIH NRSA Postdoctoral Fellowship (F32-GM-87101).

Kinetics of the Initial Step of Base Excision Repair in Trinucleotide Repeats 138 Dynamic mutations arising from trinucleotide repeat expansion cause a number of grave hereditary neurodegenerative diseases and possibly contribute to neurode- A. V. Endutkin* generation during normal aging. In the case of terminally differentiated neurons A. G. Derevyanko such expansion can be caused by DNA base excision repair (BER). This hypothesis D. O. Zharkov was confirmed by experiments in which expansion of CAG triplets characteristic of Huntington disease was initiated in the course of normal repair of the damaged base 8-oxoguanine (8-oxoG) (1). Yet it is unclear how such a DNA base lesion and SB RAS Institute of Chemical Biology its repair might cause the expansion. Besides, there is extremely little information and Fundamental Medicine, is available on efficiency of BER enzyme and their substrate specificity when DNA Novosibirsk, Russia substrate contains trinucleotide repeats. Thus it would be very useful to gain kinetic parameters of reaction involving DNA glycosylase, the main participants of BER, *[email protected] and such DNA substrates. First of all, we were interested in how the position of 8-oxoG in a substrate containing a run of CAG repeats can influence on its excision rate by human 8-oxoguanine glycosylase (OGG1). For this purpose we have determined rate constants of 8-oxoG excision (k2) by OGG1 enzyme from CAG- substrate depending on the position of the damaged triplet. Also for these substrates we have determined the rate constants of DNA product release from a complex with

OGG1 (k3). We have then compared the rates of 8-oxoG between purified enzyme and nuclear extract of the human hepatoma cell line (LICH). We have observed that k2 of purified OGG1 and the rate of excision in the extracts exhibited a similar dependence on the position of the lesion in the substrate. Therefore we suggest that the reaction rate in the cell extract is limited not by the product release stage, as in the case of pure protein, but by the catalytic step of reaction, as shown previously for the activity of OGG1 in the presence of AP-endonuclease APEX1 (2). 1102

Figure 1: Dependence of the excision rate constant (k2) of oxoG from CAG substrates by purified OGG1 (black bars) and the excision rate (v) of oxoG in LICH extracts (gray bars) on the position of oxoG in the substrate containing seven CAG trinucleotides. The number in the name of the substrate corresponds to the damaged trinucleotide.

In the case of substrates containing 8-oxoG in CGG repeats, typical for fragile X syndrome, the kinetic parameters were comparable with those for the CAG- substrates. Given that potentially not only repair of 8-oxoG can lead to expansion of triplets, we have obtained the values of Michaelis constant and catalytic constants for the reaction of uracil excision by human uracil-DNA glycosylase (UNG) from CAG-run substrates containing a single uracil, the product of cytosine deamination. Remarkably, despite the strict dependence of both constants on the position

of the damaged triplet in the substrate, the specificity constant (kcat/KM) did not significantly change.

This work was supported in part by Russian Foundation for Basic Research (10-04- 91058-PICS_a).

References

1. I. V. Kovtun, Y. Liu, M. Bjoras, A. Klungland, S. H. Wilson, and C. T. McMurray. Nature 447, 447-452 (2007). 2. V. S. Sidorenko, G. A. Nevinsky, and D. O. Zharkov. DNA Repair 6(3), 317-328 (2007). Post-synthetic Generation of Fapy-dG Lesion within 1103 Oligodeoxynucleotides: Differential Anomeric Impacts on DNA Duplex Properties

Cellular DNA is a target for oxidative stress arising from exposure to environmental and endogenous sources that account for thousands of oxidatively damaged bases 6 139 per day. The prominent oxidative lesion, imidazole ring opened N -(2-Deoxy- 1, a,b-D-erythro-pentafuranosyl)-2,6-diamino-4-hydroxy-5-formylamidopyrimidine Mark Lukin * (Fapy-dG) is one of the most common lesions of this type (1). Its formation has Conceição A. S. A. Minetti2 been reported under various experimental conditions, and its accumulation is asso- David P. Remeta2 ciated with progression of many age-related diseases and cancer (2, 3). Structural 1 and thermodynamic studies of this lesion require development of universal and Sivaprasad Attaluri reliable synthetic strategy to incorporate cognate Fapy-dG site-specifically within Francis Johnson1 any oligodeoxynucleotide sequence. We elaborated the scheme that consists of a Kenneth J. Breslauer2 two-step post-synthetic treatment of the modified nitropyrimidine oligonucleotide 1 and does not require purification of the intermediate product (4). This approach Carlos de los Santos has been successfully applied to the preparation of isotopically labeled Fapy-dG in DNA for subsequent solution state NMR studies. We demonstrated that the lesion 1Department of Pharmacological exists in DNA in a several slowly interconverting anomeric and rotameric forms (4). The anomeric forms of the Fapy-dG containing synthetic oligonucleotides have Sciences, School of Medicine, Stony been successfully separated using ion-exchange HPLC. The resultant anomeric Brook University, Stony Brook, duplexes exhibit distinct thermal and thermodynamic profiles that are characteris- New York 11794-8651 tic of α- and β-anomers. 2Department of Chemistry and Chemical References Biology, Rutgers – The State University 1. M. Dizdaroglu, G. Kirkali, and P. Jaruga. Free Radical Biol Med 45, 1610-1621 (2008). of New Jersey, Piscataway, New Jersey 2. J. Q. Wang, W. R. Markesbery, and M. A. Lovell. J Neurochem 96, 825-832 (2006). 3. D. C. Malins, N. L. Polissar, and S. J. Gunselman. Proc Natl Acad Sci USA 93, 2557-2563 08854, USA (1996). *[email protected] 4. M. Lukin, C. A. S. A. Minetti, D. P. Remeta, S. Attaluri, F. Johnson, K. J. Breslauer, and C. de los Santos. Nucl Acids Res 2011 (in press).

Base Excision Repair of Trinucleotide Repeat DNA

The expansion of trinucleotide repeat DNA leads to a multitude of neurodegenerative disorders, where CAG/CTG expansion leads to the onset of Huntington’s Disease 140

(1). Interestingly, in mouse model studies, 8-oxo-7,8-dihydroguanine glycosylase Kelly Schermerhorn* (OGG1) has been implicated in the expansion mechanism of CAG/CTG repeats in Sarah Delaney the huntingtin gene (2). OGG1, a glycosylase enzyme, is responsible for initiating the base excision repair (BER) pathway by removal of an 8-oxo-7,8-dihydroguanine (8-oxoG) lesion. AP endonuclease (APE1) processes the abasic site created Department of Chemistry, by OGG1 to cleave the DNA backbone and further the BER pathway; polymerase Brown University, β and DNA ligase then complete the repair event. In vitro, it has been shown that DNA containing CAG or CTG trinucleotide repeats has the ability to form non-B Providence, RI 02912 type conformations; more specifically they have been shown to adopt hairpin confor- *[email protected] mations (3). Recent work done in our laboratory has shown that the guanine located in the loop region of these DNA hairpins is highly susceptible to oxidation (4). Given these observations, the aim of this work is to characterize the BER pathway on trinucleotide repeat hairpin and duplex DNA constructs. Here we have examined the activity of OGG1 and APE1 on trinucleotide repeat substrates containing an 8-oxoG lesion.

References

1. Huntington Disease Collaborative Research Group, Cell 72, 971-983, (1993). 2. I. V. Kovtun, Y. Liu, M. Bjoras, A. Klungland, S. H. Wilson, and C. T. McMurray. Nature 447, 447-452, (2007). 3. A. M. Gacy, G. Goellner, N. Juranic, S. Macura, C. T. McMurray. Cell 81, 533-540 (1995). 4. D. Jarem, N. Wilson, and S. Delaney. Biochemistry 48, 6655-6663, (2009). 1104 A Prebiotic Role for Oxidized Purine Nucleotides as Mimics of Flavins in Cyclobutane Pyrimidine Dimer Repair

The “RNA World” hypothesis suggests that ancient life evolved from the catalytic 141 chemistry of RNA oligomers. Though RNA has been found to catalyze a wide Khiem V. Nguyen range of chemical reactions including ligation, hydrolysis, and replication (1), our Cynthia J. Burrows* understanding of RNA catalysis of electron transfer processes is still limited. Today, protein enzymes require functional molecules such as flavin, nicotinamide or pter- ins to promote redox reactions. How can RNA catalyze redox reactions while these Department of Chemistry, complex molecules were not likely present in the primitive world? University of Utah, Salt Lake City, We hypothesize that oxidized purine nucleotides may predate flavin as redox UT 84112 cofactors that assisted RNA in redox reactions. Using a model of electron transfer *[email protected] through double-stranded oligonucleotides, we investigated redox-active purines as mimics of flavin in repairing the photochemical lesion cyclobutane pyrimidine dimer (CPD). The repair efficiency was studied in duplexes with different positions of oxidized purines relative to a thymine dimer. We found that photorepair of the CPD was dependent upon the 5’ vs. 3’ orientation as well as the strand location and base pair context. Compared with previous studies (2-4), this data is consistent with a reductive repair mechanism in which thymine dimer repair is triggered by accepting one electron from the photoexcited state of the purine. Similarly, oxidized purines were able to photorepair the uracil CPD in both a DNA/RNA hybrid duplex and a RNA/RNA duplex, though the efficiency was less than the repair of thymine dimer in the DNA/DNA duplex.

The similarity of purine and flavin chemistries suggests that nature may have adopted these simple nucleotide derivatives as redox cofactors prior to the evolution of modern enzyme cofactors.

References

1. X. Chen, N. Li, and A.D. Ellington. Chem Biodiversity 4, 633-655 (2007). 2. D. J-F. Chinnapen and D. Sen. Proc Natl Acad Sci USA 101, 65-69 (2004). 3. M. R. Holman, T. Ito, and S. E. Rokita. J Am Chem Soc 129, 6-7 (2006). 4. M. A. O’Neill and J. K. Barton. Proc Natl Acad Sci USA 99, 16543-16550 (2002).

Incorporation of Oxidized Guanine 142 Nucleotides into DNA An oxidized product of guanine that is detected following exposure of DNA to one Craig J. Yennie* of several oxidizing agents is 8-oxo-7,8-dihydro-guanine (8-oxoGua). 8-oxoGua is Sarah Delaney present in genomic DNA at steady-state levels of ~1-10 per 107 bases and is a biomarker for several diseases (1). In order to prevent the genetic effects caused by the presence of 8-oxoGua, cells are equipped with several repair enzymes. Mammalian Department of Chemistry, cells have a glycosylase/AP lyase, OGG1 (MutM in E. coli), that excise 8-oxoGua Brown University, from duplex DNA when it is paired with cytosine. A second glycosylase, MYH Providence, RI 02912 (MutY in E. coli), removes adenine from an OG:A mispair. Cells are also equipped with a phosphatase, MTH1 (MutT in E. coli), that can convert 8-oxodGuoTP to *[email protected] 8-oxodGuoMP; this action removes 8-oxodGuoTP from the nucleotide pool in order to prevent incorporation during DNA replication. The importance of removing 8-oxodGuoTP from the nucleotide pool is underscored by the fact that E. coli lacking MutT, have a 100-10,000-fold higher mutation rate compared to wild type E. coli (2, 3). This dramatic increase in mutation rate in the absence of MutT indicates that the nucleotide pool represents a biologically significant source of 8-oxoGua. Indeed, the incorporation of 8-oxodGuoTP by several bacterial and mammalian polymerases has been examined (4, 5). Not only has 8-oxoGua been shown to be 1105 mutagenic when replicated in DNA, it has also been shown to be chemically labile towards further oxidation (6, 7). Several oxidized 8-oxoGua lesions have been identified such as spiroiminodihydantoin and guanidinohydantoin. Many of these oxidized lesions have been shown to be potently toxic and mutagenic when replicated in vitro and in vivo. The aim of this investigation is to determine the extent to which the nucleotide pool serves as a source of these oxidized lesions and their ability to be substrates for DNA polymerases.

References

1. H. J. Helbock, K.B. Beckman, M. K. Shigenaga, P. Walter, A. A. Woodall, H. C. Yeo, and B. N. Ames. Proc Natl Acad Sci USA, 95, 288-293 (1998). 2. H. Maki and M. Sekiguchi. Nature, 355, 273-275 (1992). 3. M. L. Michaels, C. Cruz, A. P. Grollmam, and J. H. Miller. Proc Natl Acad Sci USA 89, 7022-7025 (1992). 4. H. J. Einolf, N. Schnetz-Boutaud, and P. F. Geungerich. Biochemistry 37, 13300-13312 (1998). 5. J. W. Hanes, D. M. Thal, and K. A. Johnson. J Biol Chem 281, 36241-36248 (2006). 6. W. L. Neeley and J. M. Essigmann. Chem Res Toxicol 19, 491-505 (2006). 7. J. C. Niles, J. S. Wishnok, and S. R. Tannenbaum. Nitric Oxide 14, 109-121(2006).

Investigation of Trinucleotide Repeats in Nucleosome Core Particles

Both prokaryotic and eukaryotic genomes have incredible potential to grow, shrink, 143 and change. One such mechanism of change is through the expansion of trinucleotide Catherine Burke Volle repeats (TNRs). TNRs occur throughout the genome and their expansion can affect Sarah Delaney* varied cellular processes such as gene expression, mRNA processing, and protein folding. In particular, TNR expansion has been linked to several neurodegenerative diseases, such as Huntington’s Disease, Myotonic Dystrophy (both caused by Department of Molecular and Cellular expansion of CAG•CTG repeats), and Fragile X Mental Retardation (caused by Biology and Biochemistry and expansion of CGG•CCG repeats) (1). These expansions are ascribed to formation of non-canonical structures in the repeat region, leading to DNA polymerase slip- Department of Chemistry, page during replication. DNA repair also appears to play a role in expansion (2). Brown University, Providence, RI 02912 TNR structure and expansion has been studied both in vivo and in vitro. Work with both oligonucleotides and TNRs incorporated into plasmids indicates the *[email protected] potential to form several stable non-canonical secondary structures (e.g. hairpins) (1). However, genomic DNA is packaged into chromatin. Previous studies have shown that disease-length, expanded CAG•CTG repeats readily incorporate into the basic unit of chromatin packing, the nucleosome core particle (NCP), composed of 146 base pairs of DNA wrapped around a core of eight histone proteins (3). However, long CGG•CCG repeats exclude NCP formation. Here, we seek to investigate the properties associated with shorter repeats (those that have not expanded) incorporated into NCPs. To assess the global interactions involved in interaction between the TNRs and the histone core, we performed competi- tive nucleosome exchanges to determine the efficiency with which various TNR sequences incorporate into NCPs. It is not yet known what structure TNRs will adopt in this context but understanding that structure is important to understanding how TNRs expand in the genome.

This research has been supported by National Institute of Environmental Health Sciences (NIEHS) award ES019296. C.B.V. is supported by a NDSEG fellowship.

References

1. R. R. Sinden. Am J Hum Genet 64, 346-353 (1999). 2. C. T. McMurray. Nat Rev Genet 11, 786-799 (2010). 3. Y.-H. Wang. Cancer Lett 232, 70-78 (2006). 1106 Structure and Reactivity of Triplet Repeat Sequences Associated with Neurodegenerative Disorders

Expansion of triplet repeat DNA is implicated in several neurodegenerative disorders, including Huntington’s Disease (HD). Expansion of the triplet repeat polymorphism is 144 strongly dependent on repeat length, with longer repeats being more likely to expand Amalia Ávila-Figueroa across generations. While the mechanism of expansion for HD remains unknown, Douglas Cattie formation of non-B DNA structures by a repetitive (CAG)/(CTG) motif has been proposed to facilitate expansion. Persistence of the aforementioned non-B DNA structures Sarah Delaney* during events such as DNA replication and/or repair could influence the likelihood of expansion to occur (1-5). Department of Chemistry, We studied the structural properties as well as the reactivity of a (CAG)10 non-B Brown University, hairpin construct and a series of complementary (CTG)n strands of variable length Providence, RI 02906, USA and sequence composition. A molecular beacon methodology was employed to monitor the behavior of the (CAG) hairpin by labeling with a 5’-fluorophore and *[email protected] 10 a 3’-quencher functionalities (6). Modifications in the structure and base composition for the series of complementary hairpins have a profound effect on the stability

of the (CAG)10 hairpin as seen by fluorescence and UV-Vis optical melting assays. Time-resolved electrophoretic assays also revealed that structural differences can alter the kinetics of hairpin-duplex conversion. These studies show that structure and base composition at distinct sites within these stem-loop DNA conformations influences molecular recognition between hairpins and modulates conversion to duplex.

This work was supported by National Institute of Environmental Health Sciences (R01ES019296). A.A.F. was supported by a National Science Foundation Gradu- ate Research Fellowship.

References

1. S. M. Mirkin. Nature 447, 932-940 (2007). 2. I. V. Kovtun and C. T. McMurray. DNA Repair 6, 517-529 (2007). 3. C. T. McMurray. Nat Rev Genet 11, 786-799 (2010). 4. A. L. Castel, J. D. Cleary, and C. E. Pearson. Nat Rev Mol Cell Bio 11, 165-170 (2010). 5. J. Zhao, A. Bacolla, G. Wang, and K. M. Vasquez. Cell Mol Life Sci 67, 43-62 (2010). 6. A. A. Figueroa and S. Delaney. J Biol Chem 285, 14648-14657 (2010).

Substrate Binding, Catalytic Activity and Product Release by Human 8-Oxo-7,8-dihydroguanine 145 Glycosylase (hOGG1) are Modulated by the Daniel A. Jarem* Structural Context of 8-Oxo-7,8-dihydroguanine in a Nicole R. Wilson CAG Trinucleotide Repeat Sequence Kelly M. Schermerhorn Sarah Delaney The DNA repair protein human 8-oxo-7,8-dihydroguanine glycosylase (hOGG1) initiates base excision repair (BER) in mammalian cells by removing the oxidized guanine base 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA (1). Interest- Department of Chemistry, ingly, OGG1 has been implicated in expansion of the trinucleotide repeat (TNR) 324 Brook Street, Box H, sequence CAG/CTG and this expansion represents the molecular basis of several neurodegenerative disorders (2). Furthermore, in addition to the duplex conforma- Brown University, Providence, tion, CAG/CTG sequences have been shown to adopt non-B conformations such RI 02912 as stem-loop hairpins (3). Via a long-patch BER (LP-BER) pathway it has been *[email protected] shown in vitro that the ability of these repeat regions to form hairpins during BER can result in a flap that is refractory to flap endonuclease 1 (FEN1). Expansion of the TNR DNA is a consequence of the persistence of a trapped hairpin that can be 1107 ligated into the duplex (4, 5).

We reported previously that hairpins that may form during this BER expansion mechanism contain hot spots for oxidative damage when treated with peroxynitrite (ONOO−) (6). Therefore, TNR substrates containing site-specifically incorporated 8-oxoG were then synthesized to define the kinetic parameters of hOGG1 activity on duplex and hairpin structures. In this work we first used an electrophoretic mobility shift assay to determine the KD for hOGG1 binding to hairpin and duplex substrates in which the position of the 8-oxoG was varied. Second, the rate at which hOGG1 catalyzes excision of 8-oxoG was quantified by performing single-turnover experiments. Third, multiple-turnover experiments were used to define the rate of product release for hOGG1 acting on the hairpin and duplex substrates. As a benchmark for hOGG1 activity, the data obtained for the TNR substrates were compared to those obtained for a mixed-sequence duplex.

We find that hOGG1 binding, activity and product release for TNR duplexes is indistinguishable from the mixed-sequence control, indicating the BER can be initiated by hOGG1 just as efficiently in TNR regions of DNA as elsewhere in the genome. Interestingly, the activity of hOGG1 is modulated by the structure of the DNA substrate. For the hairpin substrates, hOGG1 has a reduced affinity, excises 8-oxoG at a slower rate, and releases the DNA product faster as compared to the corresponding TNR duplex substrates.

References

1. S. David, V. O’Shea, and S. Kundu. Nature 447, 941-950 (2007). 2. A. López Castel, J. D. Cleary, and C. E. Pearson. Nat Rev Mol Cell Biol 11, 165-170 (2010). 3. A. Gacy, G. Goellner, N. Juranic´, S. Macura, and C. McMurray. Cell 81, 533-540 (1995). 4. I. V. Kovtun, Y. Liu, M. Bjoras, A. Klungland, S. H. Wilson, and C. T. McMurray. Nature 447, 447-452 (2007). 5. Y. Liu, R. Prasad, W. Beard, E. Hou, J. Horton, C. McMurray, and S. Wilson. J Biol Chem 284, 28352-28366 (2009). 6. D. Jarem, N. Wilson, and S. Delaney. Biochemistry 48, 6655-6663 (2009). 1108 Functional Nuclear Architecture Studied by High-resolution Microscopy

Recent developments of 4D (space-time) live cell microscopy (1), 3D light optical nanoscopy with resolution beyond the classical Abbe limit (2-6) and advanced 146 electron microscopic approaches (7, 8) complement each other in studies of the Thomas Cremer1* functional nuclear architecture (for review see 9). The results of these studies 1 support the chromosome territory-interchromatin compartment model of nuclear Marion Cremer architecture (10-13). Yolanda Markaki1 Barbara Hübner1 Chromosome territories (CTs) occupy distinct regions of the nuclear space. They 1 are built up from a network of interconnected chromatin domains with a DNA- Katharina Austen content in the order of 1 Mb, termed ~1Mb CDs. The structure of ~1Mb CDs has Daniel Smets1 not yet been resolved, but we argue that each ~1Mb CD is built up from a series of Jacques Rouquette1 more or less compacted chromatin loop domains with a DNA content in the order 2 of 100 kb, termed ~100 kb CDs. Moreover, several ~1Mb CDs can cluster together Manuel Gunkel and form larger CDs. During S-phase replicating ~1Mb CDs act as replication Sven Beichmanis2 foci. Direct contacts between neighboring ~1Mb CDs provide ample opportunities Rainer Kaufmann2 for interactions in cis (within a given CT) or trans (between neighboring CTs), 1 including the formation of intra- and interchromosomal rearrangements. Beyond Heinrich Leonhardt the nucleosome and the 10 nm thick, ‘beads on a string’ chromatin fiber, we lack Lothar Schermelleh1 indisputable, quantitative evidence for possible hierarchies of higher order chro- Stanislav Fakan1 matin fibers and loops. In particular, we do not know the extent of quantitative 2 variation possible with regard to the size distributions and extent of intermingling Christoph Cremer ** of fibers and loops at different hierarchical levels in different cell types of different species exposed to different internal or external stimuli. To which extent 1LMU Biocenter, Department of interactions between giant chromatin loops expanding from widely separated CTs are involved in the relocalization of genes to specialized subnuclear comparte- Biology II, Ludwig Maximilians ments (“gene kissing”), is still controversially discussed. Based on our current University (LMU), knowledge, we expect that such events are mostly driven by locally constrained 82152 Martinsried, Germany Brownian movements. 2 Kirchhoff-Institute for Physics and The perichromatin region (PR) represents a 100-200 nm thick layer of decon- BioQuant Center, University of densed chromatin, located at the periphery of CDs. It constitutes the nuclear Heidelberg, 69120 Heidelberg, Germany compartment for transcription, splicing, DNA-replication and possibly also DNA-repair (6 - 8, 9). The PR is in direct contact with the interchromatin- *[email protected] compartment (IC), which forms an interconnected 3D system of IC-channels **[email protected] (width <400 nm) and IC-lacunas (width >400 nm). It starts/ends with small channels at the nuclear pores and expands both between CTs and throughout the interior of CTs. Accordingly the 3D organization of a CT can be compared with a sponge built up from a 3D chromatin network permeated by the IC (Figure 1) (6, 13). It should be noted that constrained Brownian movements of CDs in the nucleus of living cells (1) result in continuous changes of the width of IC channels providing dynamic opportunities for normal or pathological interactions. The interior of IC lacunas is free of chromatin and harbors nuclear bodies and splicing speckles (6, 7). This structural organization allows direct functional interactions between the IC and the PR, such as the delivery of splicing components from splicing speckles to sites of co-transcriptional splicing (Figure 1). Although individual proteins may be able to diffuse through the whole nuclear space, including heterochromatic domains, it still seems to be a valid possibility that the IC and the PR represent specific nuclear compartments for the intra- nuclear traffic of protein complexes involved in nuclear functions and the export ribonucleoprotein complexes. 1109

Figure 1: This 2D cartoon shows a simplified scheme of the more complex 3D nuclear architecture. CDs are not drawn to scale (adapted from Figure 4 in T. Cremer and M. Cremer, 2010).

References

1. H. Strickfaden. Nucleus 1, 284-297 (2010). 2. L. Schermelleh, et al. Science 320, 1332-1336 (2008). 3. M. Gunkel, et al. Biotechnol J 4, 927-938 (2009). 4. D. Baddeley, et al. Nucleic Acids Res 38, e8 1-11 (2010). 5. C. Cremer, et al. In: Nanoscopy and Multidimensional Optical Fluorescence Microscopy (A. Diaspro, Edit.) Taylor & Francis, pp. 3/1 - 3/35 (2010). 6. Y. Markaki, et al. Cold Spring Harb Sym Quant Biol 75, in press (2011). 7. J. Rouquette, et al. Chromosome Res 17, 801-810 (2009). 8. J. Niedojadlo, et al. Exp Cell Res 317 433-444 (2011). 9. J. Rouquette, et al. Int Rev Cell Mol Biol 282, 1-90 (2010). 10. T. Cremer, et al. Crit Rev Eukar Gene 10, 179-212 (2000). 11. T. Cremer, and C. Cremer. Nat Rev Genet 2, 292-301 (2001). 12. C. Lanctôt, et al. Nat Rev Genet 8, 104-115 (2007). 13. T. Cremer and M. Cremer Cold Spring Harb Perspect Biol 2 a003889 (2010).

How is a Long Strand of DNA Compacted into a Chromosome? 147 Mitotic chromosomes are essential structures for the faithful transmission of duplicated genomic DNA into two daughter cells during cell division (1). A long strand of Kazuhiro Maeshima DNA is wrapped around the core histone and forms a nucleosome. The nucleosome has long been assumed to be folded into 30-nm chromatin fibers (Figure A) (1). Biological Macromolecules Laboratory However, it remains unclear how the nucleosome or 30-nm chromatin fiber is organized into mitotic chromosomes, although it is well known that condensins and Structural Biology Center, topoisomerase IIα are implicated in this process (2-4). When we observed frozen National Institute of Genetics, hydrated (vitrified) human mitotic cells using cryo-electron microscopy, which Mishima, Japan enables direct high-resolution imaging of the cellular structures in a close-to-native state, we did not find any higher order structures, or even 30-nm chromatin [email protected] 1110

fibers, but just a uniform disordered texture of the chromosome (Figure B) (5, 6). To further investigate the structure of mitotic chromosome, we performed small angle x-ray scattering or SAXS, which can detect regular internal structures in non- crystalline materials in solution. Mitotic chromosomes purified from HeLa cells were exposed to the synchrotron radiation beam at SPring-8 in Japan. Again, the results were striking: no structural peaks larger than 11-nm were detected. Therefore, we propose that the nucleosome fibers exist in a highly disordered, interdigitated state like a “polymer melt” that undergoes local dynamic movement (Figure B) (5, 6). We also postulate that a similar state exists in active interphase nuclei, resulting in several advantages in the transcription and DNA replication processes (6, 7). The possible genomic organization in the mitotic chromosomes and nuclei is discussed.

References

1. B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter. Molecular Biology of the Cell, Garland, NY. (2007). 2. J. R. Swedlow and T. Hirano. Mol Cell 11, 557-569 (2003). 3. S. Ohta, L. Wood, J. C. Bukowski-Wills, J. Rappsilber, and W. C. Earnshaw. Curr Opin Cell Biol 23, 114-121 (2010). 4. K. Maeshima, S. Hihara, and H. Takata. Cold Spring Harb Symp Quant Biol 75 (2011), in press. 5. M. Eltsov, K. M. Maclellan, K. Maeshima, A. S. Frangakis, and J. Dubochet. Proc Natl Acad Sci USA 105, 19732-19737 (2008). 6. K. Maeshima, S. Hihara, and M. Eltsov. Curr Opin Cell Biol 22, 291-297 (2010). 7. E. Fussner, R. W. Ching, and D. P. Bazett-Jones. Trends Biochem Sci 36, 1-6 (2011). A Genomic Code for Nucleosome Positioning from 1111 Archaebacteria to Man

Eukaryotic genomes are packaged into nucleosome particles that occlude the DNA from interacting with most DNA binding proteins. We have discovered that genomes care where their nucleosomes are located on average, and that genomes manifest 148 this care by encoding an additional layer of genetic information, superimposed on Jonathan Widom top of other kinds of regulatory and coding information that were previously recognized. The physical basis of the nucleosome DNA sequences preferences lies in the sequence-dependent mechanics of DNA itself. We have an increasingly good Department of Molecular Biosciences ability to read this nucleosome positioning information and predict the in vivo loca- and Department of Chemistry, tions of nucleosomes. Our results suggest that genomes utilize this nucleosome positioning code to facilitate specific chromosome functions, including to define Northwestern University, the next higher level of chromosome structure itself. Comparisons across diverse Evanston, IL 60208-3500 organisms suggests that basic aspects of this nucleosome positioning code are con- [email protected] served from archaebacteria to man. While we now have a good theoretical and experimental understanding of the approximate locations of nucleosomes in vivo, many aspects of chromosome structure and function hinge on knowing nucleosome locations to basepair resolution; and current experimental mapping methods do not come close to achieving such accuracy. I will discuss a new experimental approach to obtaining nucleosome maps with true basepair resolution, and novel discoveries resulting from the use of this methodology.

Computational Approach to Unravel Molecular Events During Vitiligo 149 Vitiligo is an acquired depigmenting autoimmune disorder characterized by the loss of functional melanocytes from the epidermis. The pathomechanism of the Khushhali Menaria disease is unpredictable because of multifactorial and overlapping mechanisms. Vitiligo involves complex interaction of environmental and genetic factors that Department of Bioinformatics, Maulana ultimately contribute to melanocyte destruction, resulting in the characteristic Azad National Institute of Technology, depigmented lesions. There is no agreement about the pathomechanisms involved in the disappearance of melanocytes to form the characteristic achromic lesions. Bhopal (M.P.) Several hypothesis have been developed for pathogenesis, but none of the hypothe- [email protected] sis enlighten the path followed during the disorder. Therefore to construct a map of [email protected] molecular interactions of the vitiligo disease is a valuable resource for research in this area. In this paper, we present a comprehensive pathway map of vitiligo, based on the protein-protein interaction network using String 8.0. The map reveals that the overall architecture of the pathway is a bow-tie structure with several feedback loops. The map is created using CellDesigner software that enables us to graphi- cally represent interactions using a well defined and consistent graphical notation, and to store it in Systems Biology Markup Language (SBML).

Key words: Vitiligo; CellDesigner; String; Protein-Protein Interaction Network. 1112 The Impact of Interchromosomal Associations on the Functional State of the Human Genome

The chromatin state is one of the main determinants of transcription rate in eukaryotes. The chromatin is described as a fiber made up of an array of nucleosomes 150 which consist of core histone proteins wrapped around by DNA double helix Ekaterina E. Khrameeva* (1). Dynamic chromatin movements and interactions play a crucial role in gene Andrey A. Mironov regulation. Adjacent genes can be packed together in distinct chromatin domains that ensure coordinated gene expression. Evidence is emerging that distant genes Mikhail S. Gelfand located on different chromosomes also can interact, and their proximity might be essential for coordinated regulation. However, it has been unclear whether these interchromosomal associations are exceptional, or occur frequently. Institute for Information Transmission Problems, Russian Academy of Sciences, We systematically analyzed genome-wide interchromosomal interactions in the Bolshoy Karetny per. 19, Moscow, nuclei of human cells. 3D data from (2) were associated with the results of several high-throughput studies of the chromatin functional state (3). All pairs of regions 127994, Russia from different chromosomes were divided into groups according to their proximity, *[email protected] and the distribution of various chromatin marks was calculated within these groups and then compared between the groups.

The results show that, indeed, gene regions that are spatially close tend to have similar patterns of histone modifications, methylation state, open or closed chromatin state, and expression level. Spatially close genome domains tend to have similar chromatin state and to be coregulated and coexpressed.

Moreover, we found that interacting domains may produce transcripts composed of sequence segments coming from two different chromosomes. We analyzed chimeric transcripts as determined by genome mapping of paired-read RNA-Seq data (4, 5) and observed that the frequency of pairs mapping to two different genome loci is higher among spatially proximal regions. We suggest that these transcripts might be formed by trans-splicing.

In summary, interchromosomal associations seem to be much more common than previously believed, and they likely play important roles in the regulation of gene expression.

This research has been supported by State contracts, grants of the Russian Foundation of Basic Research, programs “Molecular and Cellular Biology” and “Basic Science for Medicine” of the Russian Academy of Sciences.

References

1. A. Travers, E. Hiriart, M. Churcher, M. Caserta, and E. Di Mauro. 3. J Biomol Struct Dyn 27, 713-724 (2010). 2. Lieberman-Aiden, N. L. van Berkum, L. Williams, M. Imakaev, T. Ragoczy, et al. Science 326, 289-293 (2009). 3. ENCODE Project Consortium Science 306, 636-640 (2004). 4. A. G. Xu, L. He, Z. Li, Y. Xu, M. Li, et al. PLoS Comp Biol 6(7), e1000843 (2010). 5. M. F. Berger, J. Z. Levin, K. Vijayendran, A. Sivachenko, X. Adiconis, et al. Genome Res. 20, 413-427 (2010). Mapping Oxidative Damage at High Resolution 1113 Throughout an Entire Genome

Oxidative damage to DNA has been proposed to be a major contributing factor to the process of ageing. Accumulation of oxidative damage has been associated with many degenerative diseases. Determining the locations of vulnerability to oxida- 151 tive damage on a genome-wide scale will be pivotal to understanding the ageing Sarah Bernard1 process. One type of oxidative damage is produced by Reactive Oxygen species Cheryl Chiang1 (ROS), primarily in the form of hydroxyl radicals, which abstract deoxyribose 2 hydrogens along the DNA backbone, resulting in strand breaks. Regions of chro- Stephen C. J. Parker matin that encode essential genes that are highly expressed often are found in areas Elliott H. Margulies2 of relatively low chromatin condensation, and so may be more susceptible to ROS Thomas D. Tullius1,3* attack. These genomic regions are bound by numerous proteins, including transcription factors, and their accessibility is governed by nucleosome positioning. 1Department of Chemistry, We are developing a new method to biochemically process oxidatively damaged Boston University, genomic DNA to make it suitable for high-throughput sequencing, in order to map oxidative damage throughout a genome at single-nucleotide resolution. Our initial Boston, MA 02215 experiments on a well-studied model system (1) demonstrate that this new method 2Genome Informatics Section, successfully identifies sites of oxidative damage. We will use these quantitative Genome Technology Branch, maps to determine which regions of a genome are particularly susceptible to oxidative damage, and which are resistant, and relate these damage maps to the underly- National Human Genome Research Insti- ing genes and functional regions of the genome. These studies will provide a view tute, National Institutes of Health, at unprecedented resolution of the spectrum of oxidative damage in the genome, to Bethesda, MD 20892 facilitate a deeper understanding of the relationship of DNA damage to ageing. 3Program in Bioinformatics, Reference Boston University, 1. L. M. Ottinger and T. D. Tullius. J Am Chem Soc 122, 5901-5902 (2000). Boston, MA 02215 *[email protected]

Three-Dimensional Genome Architecture Predicts the Distribution of Chromosomal Alterations in Human Cancers 152 Geoffrey Fudenberg1* Over the last decade, novel technologies have exposed how cancer genomes are 1,2,3 riddled with mutations, including everything from single nucleotide substitutions Leonid Mirny through large-scale copy-number alterations. Progress towards deciphering highly contorted cancer genomes lies in a better understanding of the forces and muta- 1Harvard University Graduate tional mechanisms behind patterns of genomic alterations in cancer. Biophysics Program, Unequivocally establishing a connection between genomic alterations and three- Cambridge, MA 02138 dimensional genome structure in cancer has up to this point been limited by our abil- 2Departmetn of Physics, Massachusetts ity to measure three-dimensional structure of DNA, and the resolution with which we are able to observe genomic alterations in cancer. Fortunately, both of these Institute of Technology have become available in the last two years. Array-based technology now allows 3Harvard-MIT Division of Health determination of somatic copy number alterations (SCNAs) in cancer at much Sciences and Technology higher resolutions and throughput than microscopy-based methods (1). Similarly doing away with microscopy, the 3C technique and its descendants (2), use a bio- *[email protected] chemical approach for high resolution determination of three-dimensional genome 1114 architecture across a population of cells. In 3C, DNA is crosslinked, digested, and ligated; ligation products are then read using sequencing to determine pairs of DNA fragments which are close in space. Pairing SCNA data with HiC data allows us to examine the role of three-dimensional chromosomal architecture in the formation of somatic structural alterations.

By constructing a heatmap (or matrix) of Somatic Copy-Number Alterations (SCNAs) from (1), we demonstrate the influence of three-dimensional genome structure as determined by Hi-C (2) on the distribution of SCNAs. Towards this end, we developed a Maximum Likelihood framework and permutation procedures for statistically justified comparisons of heatmaps. These statistical techniques establish a connection between intra-chromosomal genomic alterations in cancer and the three-dimensional genomic architecture. Furthermore, the strength of this link is bolstered when we account for purifying selection. Our result provides evidence for a mutational mechanism of chromosomal alterations, and emphasizes the importance of spatial chromatin organization in addition to forces of natural selection for genome function. Finally, our work displays the potential of novel genomic technologies by connecting chromosomal alterations in cancer with three-dimensional genomic architecture at previously impossible megabase resolution.

References

1. R. Beroukhim et al. The landscape of somatic copy-number alteration across human cancers, Nature 463, 899-905 (2010). 2. E. Lieberman-Aiden, et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome, Science 326, 289-293 (2009).

High-Throughput DNA Structural Analysis by Hydroxyl Radical Cleavage and Capillary 153 Electrophoresis Nicholas B. Hammond1 Clare C. Rittschof2 DNA structure affects protein binding and rates of transcription, and directly informs cellular processes (e.g., 1). Since DNA structure depends on local base composition 3 Thomas D. Tullius (2), our long-term goal, in concert with the ENCODE Project, is to identify regions within the human genome with evolutionarily conserved structure. We report here on our efforts to add to the OH Radical Cleavage Intensity Database (ORChID) (3), Department of Chemistry, to improve our algorithm for predicting DNA structure, by adding cleavage data on Boston University, relatively long (300-400 base pair) DNA sequences. This necessitates development Boston, MA 02215 of an accurate, high-throughput method for DNA shape determination. [email protected] Our lab has previously developed a method to map DNA structure using hydroxyl [email protected] radical cleavage patterns (4). Hydroxyl radicals create single-stranded breaks in [email protected] DNA. The frequency of damage at a particular site depends on the local structure, because of sequence-dependent variation in backbone solvent accessibility. We use this technique, coupled with the high-throughput analytical capabilities of capillary electrophoresis, to quickly evaluate the structural variation of longer DNA sequences than was previously possible using standard gel electrophoresis. Cleavage patterns at single nucleotide resolution are quantified from the fluorescence trace of the capillary electrophoresis instrument using the open-source program CAFA (capillary automated footprinting analysis) (5). This high-throughput method enables fast and accurate acquisition of DNA structural information for improvement of the ORChID algorithm. References 1115 1. S. C. J. Parker, L. Hansen, H. O. Abaan, T. D. Tullius, and E. H. Margulies. Science, 324, 389-392 (2009). 2. R. E. Dickerson and H. R. Drew. J Mol Biol 78, 2179-2183 (1981). 3. J. A. Greenbaum, B. Pang, and T. D. Tullius. Genome Res 17, 947-953 (2007). 4. W. J. Dixon, J. J. Hayes, J. R. Levin, M. F. Weidner, B. A. Dombroski, and T. D. Tullius. Methods Enzymo 208, 380-413 (1991). 5. S. Mitra, I. V. Shcherbakova, R. B. Altman, M. Brenowitz, and A. Laederach. Nucleic Acids Res 36, e63 (2008).

When Replication Meets Transcription

The replication fork and RNA polymerase share the same DNA template making occasional collisions between the two machineries inevitable. In the case of head-on 154 collisions, the front edge of RNA polymerase collides with the lagging strand syn- Sergei M. Mirkin thesis components of the replication fork. In the co-directional case, in contrast, the rear edge of RNA polymerase meets with the leading strand synthesis components of the replication fork. The first data suggesting that head-on collisions can Department of Biology, occur both in vitro and in vivo resulting in replication fork stalling were obtained Tufts University, more than two decades ago. The mechanisms responsible for these events remained Meford, MA 02143 obscure. My lab got involved in these studies as a result of two purely serendipitous developments. First, while attempting to prove that H-DNA formed by d(G)n•d(C)n [email protected] runs can stall DNA replication in vivo, we found that it is, in fact, transcription in a specific direction through those runs that causes fork stalling (1). Recently, we showed that unusually stable rG/dC hybrids created during transcription of those runs trigger the formation of R-loops, which, in turn, stall replication forks moving co-directionally (2). Second, while attempting to understand the nature of some obscure replication stall sites in a bacterial plasmid, we found that direct physical collisions between transcription and replication apparata led to profound fork stalling (3). Furthermore, even when RNA polymerase has not cleared the promoter, its front edge represented a formidable obstacle for the replication forks moving toward it (4). Finally, we found that RNA polymerase trapped or backtracked at the terminator sequences stalls co-directional replication forks (4). It would be fair to say that these data revitalized the field resulting in many spectacular structural and functional discoveries to be discussed at this session.

References

1. M. M. Krasilnikova, G. M. Samadashwily, A. S. Krasilnikov, and S. M. Mirkin Transcrip- tion through a simple DNA repeat blocks replication elongation. EMBO J 17, 5095-5102 (1998). 2. B. P. Belotserkovskii, et al. Mechanisms and implications of transcription blockage by guanine-rich DNA sequences. Proc Natl Acad Sci USA 107, 12816-12821 (2010). 3. E. V. Mirkin and S. M. Mirkin. Mechanisms of transcription-replication collisions in bacteria. Mol Cell Biol 25, 888-895 (2005). 4. E. V. Mirkin, D. Castro Roa, E. Nudler, and S. M. Mirkin. Transcription regulatory elements are punctuation marks for DNA replication. Proc Natl Acad Sci USA 103: 7276-7781 (2006). 1116 Essential Role of the Rep, UvrD and DinG Helicases and of the RecBC Recombination Complex upon Replication – Transcription 155 Collisions in the E. coli Chromosome Anne Langlois de Septenville We are interested in the consequences of replication fork arrest in bacteria. We characterized several different reactions occurring at blocked forks prior to replica- Stéphane Duigou tion restart, which depend on the cause of arrest (reviewed in 1). Recently, we used Hasna Boubakri Escherichia coli strains carrying inverted ribosomal operons (rrn) in order to ana- Enrique Viguera lyze replication arrest caused by replication-transcription collisions. We identified three helicases required for replication upon head-on collision with transcription Bénédicte Michel* complexes: the Rep, UvrD and DinG helicases (2). Replication arrest sites could be directly visualized in ribosomal operons facing replication (provided that Rep or DinG was inactivated), and the inactivation of any combination of two of these CNRS, Centre de Génétique three accessory replicative helicases was highly detrimental for viability. All three Moléculaire, UPR3404, Gif-sur-Yvette, helicases are involved in RNA Pol removal, and DinG has an additional function F-91198, France of R-loop removal. *[email protected] In E. coli, rep uvrD and rep uvrD dinG recF mutants grow poorly particularly on rich medium (multiple replication forks conditions). We isolated mutations that suppress these growth defects; all map in RNA polymerase genes and presumably facilitate RNA Pol dislodging from DNA, which supports the idea that the Rep, UvrD and DinG helicases also facilitate replication across transcribed regions in wild-type E. coli cells (3).

In addition, increasing replication-transcription collisions by inverting an rrn operon creates a requirement for the recombination complex RecBC, specific for the degradation and the recombinational repair of DNA double-strand ends. How- ever, the key homologous recombination protein RecA is not required, suggesting a specific role for RecBC. The role of recombination proteins upon replication- transcription collisions will be discussed.

References

1. B. Michel, H. Boubakri, M. Le Masson, Z. Baharoglu, and R. Lestini. DNA Repair 6, 967-980 (2007). 2. H. Boubakri, A. Langlois de Septenville, E. Viguera, and B. Michel. EMBO J 29, 145-157 (2010). 3. Z. Baharoglu, R. Lestini, S. Duigou, and B. Michel. Mol Microbiol 77, 324-336 (2010). A Topological View of Transcription-Replication 1117 Collisions

Positive supercoiling builds-up ahead of progressing forks and DNA topoisomerases counteract this effect allowing a smooth advance (1). But the processivity of helicases outcomes topoisomerases and as completion of replication approaches the 156 DNA ends up with an excess of positive supercoiling. This supercoiling in the Virginia López unreplicated portion can migrate to the replicated portion by swivelling the forks. Here it gives rise to right-handed catenanes once replication is over (2). In bacteria, Estefanía Monturus de topoisomerase IV (Topo IV) is responsible for the decatenation of sister duplexes Carandini (3), but Topo IV is more efficient in the elimination of left-handed crosses (4). This María-Luisa Martínez-Robles apparent contradiction is known as the Topo IV paradox (5) and recent data suggests that supercoiling of the newly made sister duplexes plays an essential role to Pablo Hernández solve it (6, 7). The scenario changes significantly if transcription and replication Dora B. Krimer forks progress in opposite directions. The accumulation of positive supercoiling in Jorge B. Schvartzman* between would slow down the advancing forks and this might allow topoisomerases to cope helicases in order to maintain the replicon negatively supercoiled up to the end. If negative supercoiling ahead of the forks migrates to the replicated portion, Department of Cell Proliferation & it would give rise to left-handed catenanes once replication is over. Among other Development, Centro de Investigaciones consequences, it is known this enhances the formation of intermolecular knots during replication (8). Here we combined classical genetics with high-resolution two- Biológicas (CSIC), Ramiro de Maeztu 9, dimensional agarose gel electrophoresis and atomic force microscopy to investigate Madrid 28040, Spain this problem in bacterial plasmids and yeast minichromosomes as prokaryotic and *[email protected] eukaryotic model systems, respectively.

This research was supported by grants BFU2008-00408/BMC to JBS and BFU2007- 62670 to PH from the Spanish Ministerio de Ciencia e Innovación.

References

1. J. B. Schvartzman and A. Stasiak. EMBO Rep 5, 256-261, (2004). 2. J. J. Champoux. Annu Rev Biochem 70, 369-413, (2001). 1118 3. E. L. Zechiedrich and N. R. Cozzarelli. Gene Dev 9, 2859-2869, (1995). 4. N. J. Crisona, T. R. Strick, D. Bensimon, V. Croquette, and N. R. Cozzarelli. Genes Dev 14, 2881-2892, (2000). 5. K. C. Neuman, G. Charvin, D. Bensimon, and V. Croquette. Proc Natl Acad Sci USA 106, 6986-6991, (2009). 6. J. Baxter, N. Sen, V. Lopez-Martinez, M. E. Monturus de Carandini, J. B. Schvartzman, J. F. Diffley, and L. Aragon. Science in press, (2011). 7. M. L. Martinez-Robles, G. Witz, P. Hernandez, J. B. Schvartzman, A. Stasiak, and D. B. Krimer. Nucleic Acids Res 37, 5126-5137, (2009). 8. J. M. Sogo, A. Stasiak, M. L. Martinez-Robles, D. B. Krimer, P. Hernandez, and J. B. Schvartzman. J Mol Biol 286, 637-643, (1999).

Transcription Blockage by Guanine-Rich DNA Sequences and Possible Effects on Transcription 157 of Nascent RNA Binding to DNA Boris P. Belotserkovskii1* Richard Liu1 Various DNA sequences that interfere with transcription due to their unusual structural properties have been implicated in the regulation of gene expression and 1 Shayon Saleh with genomic instability. An important example is sequences containing G-rich Silvia Tornaletti2 homopurine-homopyrimidine stretches, for which unusual transcriptional behavior Maria M. Krasilnikova3 is implicated in regulation of immunogenesis and in other processes, such as genomic translocations and telomere function. To elucidate the mechanism of the effect of 4 Sergei M. Mirkin these sequences on transcription we have studied T7 RNA polymerase transcription Philip C. Hanawalt1 of G-rich sequences in vitro. We have shown that these sequences produce significant transcription blockage in an orientation-, length- and supercoiling-dependent manner. Based upon the effects of various sequence modifications, solution condi- 1Department of Biology, Stanford tions and substitution of inosine or deazaguanosine for guanosine, we conclude that University, Stanford, CA 94305 transcription blockage is due to formation of unusually stable RNA/DNA hybrids, which could be further exacerbated by triplex formation (1). These structures 2Department of Anatomy and Cell are likely responsible for transcription-dependent replication blockage by G-rich Biology, University of Florida, FL 32610 sequences in vivo (2). We have also analyzed the general effect of stable nascent 3Department of Biochemistry and RNA binding to DNA theoretically, and we conclude that such anchoring could interfere with transcription downstream from the anchoring point (3). Molecular Biology, Penn State University, University Park, PA 16802 Supported by a grant CA77712 from the National Cancer Institute, NIH 4Department of Biology, Tufts References University, Medford, MA 02155 1. A. Gabrielian. J Biomol Struct Dyn 26, 837-838 (2009). *[email protected] 2. B. P. Belotserkovskii, R. Liu, S. Tornaletti, M. M. Krasilnikova, S. M. Mirkin, and P. C. Hanawalt. Proc Natl Acad Sci 107(29), 12816-12821 (2010). 3. B. P. Belotserkovskii and P. C. Hanawalt. Biophysical Journal (in press). Transcription-associated Mutagenesis and Top1 1119 Activity in Budding Yeast

High levels of transcription stimulate both homologous recombination and mutagenesis in yeast, and do so by multiple mechanisms (1). Using the CAN1 gene fused to the GAL1 promoter, a novel, 2-5 bp deletion signature associated with high levels 158 of transcription has been identified. These deletions accumulate at discrete hotspots Nayun Kim that coincide with short tandem repeats of the same size, and are completely depen- Jang-Eun Cho dent on the activity of Top1 (2, 3). Top1 is a type 1B topoisomerase that nicks DNA, forming a reversible intermediate with the enzyme covalently attached at the Sue Jinks-Robertson* nick via a 3’ phosphotyrosyl linkage (4). We propose that the deletions reflect the processing of a Top1 cleavage product generated during the removal of transcrip- Department of Molecular Genetics tion-associated supercoils. To study the genetic control of deletion formation, individual 2-bp deletions hotspots have been transplanted into a frameshift reversion and Microbiology, assay where they can be studied in isolation. In this much more sensitive system, Duke University Medical Center, Top1-dependent deletions are observed even under low-transcription conditions. Durham, NC 27710 Because of the similarity of the Top1-dependent deletion signature to that recently associated with a failure to remove ribonucleotides (rNMPs) from genomic DNA *[email protected] (an RNase H2-deficient background; ref. 5), we have explored whether there is any relationship between the two. We find that rNMP-initiated deletions that accumulate in the absence of RNase H2 require the activity of Top1. This suggests a model in which presence of an rNMP at the scissile phosphate leads to an irreversible Top1 cleavage product (6) that is subsequently processed into a deletion intermediate. While all rNMP-initiated deletions appear to require Top1, there are subclasses of Top1-dependent deletions that are not elevated in the absence of RNase H2. We propose that rNMP-independent deletions reflect processing of a reversible Top1 cleavage intermediate that becomes trapped on DNA. Finally, we demonstrate that there is a synergistic relationship between rNMP-associated deletions and high levels of transcription, suggesting that rNMP levels within genomic DNA can be influenced by transcriptional status.

This work has been supported by NIH grants GM38464 and GM93197 awarded to SJR.

References

1. A. Aguilera and B. Gomez-Gonzalez. Nat Rev Genet 9, 204-217 (2008). 2. M. J. Lippert, et al. Proc Natl Acad Sci USA 108, 698-703 (2011). 3. T. Takahashi, G. Burguiere-Slezak, P. A. Van der Kemp, and S. Boiteux. Proc Natl Acad Sci USA 108, 692-697 (2011). 4. J. C. Wang. Nat Rev Mol Cell Biol 3, 430-440 (2002). 5. S. A. McElhinny, et al. Nat Chem Biol 6, 774-781 (2010). 6. J. Sekiguchi and S. Shuman. Mol Cell 1, 89-97 (1997). 1120 The E. coli Replisome and Use of Clamps to Bypass Replication Barriers

Chromosomal replicases utilize circular sliding clamps for high-processivity during 159 replication. Sliding clamps not only bind the chromosomal replicase, but they also Roxana E. Georgescu function with all 5 DNA polymerases in E. coli as well as other proteins for repair and lesion bypass. This presentation focuses on the use of the sliding clamp by the Nina Y. Yao replicase in crossing barriers that are encountered during replication. Studies have Mike O’Donnell shown that the replicase, Pol III, can rapidly hop from one sliding clamp to another clamp in order to transfer to the multiple RNA primers synthesized during lagging strand replication. However, recent studies indicate that polymerase hopping among Rockefeller University and Howard sliding clamps is of more general use, and can allow the polymerase to bypass Hughes Medical Institute, 1230 York lesions and even to circumvent a tightly bound RNA polymerase that it encounters Avenue, New York, NY 10021, USA during replication. We will present studies that demonstrate the how the replisome deals with collisions with RNA polymerase transcribing either the leading or lagging strand template. We will also present our findings on how different polymerases control the use of the clamp to form alternative replisomes for DNA damage avoid- ance and, when needed, to deal with lesions that are encountered directly. Finally, we present single-molecule studies that examine replisome action during lagging strand synthesis and the consequences for the leading strand polymerase.

An Active Clamping Role for PCNA During 160 Assembly and Function on DNA Circular clamp proteins enable processive DNA replication by tethering poly- Yayan Zhou merases to the primer-template during DNA replication. Clamps also bind to and Manju M. Hingorani coordinate the functions of several other proteins on DNA, and are therefore essential for many DNA metabolic reactions (1). Clamps are loaded onto DNA in an ATP-fueled reaction by multi-subunit AAA+ protein complexes known as Clamp Molecular Biology and Loaders. The mechanism of action of these proteins is under active investigation, Biochemistry Department, given their important role in genomic DNA replication, repair and recombina- Wesleyan University, tion (2). According to our current kinetic model of the S. cerevisiae RFC clamp loader, ATP binding activates RFC, allowing it to bind and open the PCNA clamp Middletown, CT 06459 for entry of primer-template DNA; DNA binding to RFC triggers ATP hydrolysis, [email protected] PCNA closure around DNA and release of the PCNA•DNA complex from RFC [email protected] (3). The question we are addressing is whether PCNA plays an active role in clamp assembly. The specific hypothesis is that interactions between positively charged residues on the inside of the clamp and DNA help trigger clamp closure around DNA and catalytic turnover of RFC. In order to test this hypothesis, we have generated several PCNA mutants in which individual conserved amino acids have been substituted with Alanine. Data from transient kinetic analysis of key events during clamp assembly, including ATP-bound RFC• PCNA•DNA complex association and dissociation, PCNA opening and closing, as well as phosphate release, indicates that alteration of a single PCNA-DNA contact can alter rate-limiting steps in the reaction. Thus, the kinetic data support the hypothesis that PCNA is more than just a passive ring around DNA—it is an active contributor to the clamp assembly reaction mechanism and, by extension, possibly other DNA metabolic reactions as well.

This research is supported by funding from NSF and NIH.

References

1. G. L. Moldovan, B. Pfander, and F. Jentsch. Cell 129, 665-679 (2007). 2. M. O’Donnell and J. Kuriyan. Current Opinion in Structural Biology 16, 35-41 (2006). 3. S. Chen, M. K. Levin, M. Sakato, Y. Zhou, and M. M. Hingorani. Journal of Molecular Biology 388, 431-442 (2009). Electrostatic Properties of Bacteriophage T7 Early 1121 Promoters Recognized by E. coli RNA Polymerase

During T7 bacteriophage infection, its transcription is closely regulated by two different RNA polymerases. RNA polymerase of E. coli transcribes the early (class I) genes of T7, and newly made T7 RNA polymerase transcribes the late (class II 161 and class III) genes. The both enzymes are very specific for their own promoters. S. G. Kamzolova1 RNA polymerase of E. coli recognized three strong (A1, A2, and A3) and five week 1,2, (B, C, D, E, F) promoters in T7 DNA. All these promoters have been earlier charac- P. M. Beskaravainy * terized by their biochemical properties. Their functional characteristics were shown A. A. Sorokin1,** to differ from “consensus sequence rule” behavior thus stimulating a search of new promoter determinants. 1 Institute of Biophysics, RAS Pushchino Here electrostatic properties of the promoters were studied, and this has been Moscow Region 142290, Russia recently reported to play an important role in protein-nucleic acid recognition (1, 2). 2 Institute of Theoretical and Electrostatic potential distribution around 300 bp- fragments containing the individual promoters was calculated by Coulomb method (3) using the computer pro- Experimental Biophysics, RAS gram of Sorokin A. A. Electrostatic profiles of three strong and one weak promoters Pushchino, Moscow Region 142290, are shown in Figure 1(A-D). Electrostatic properties in the far upstream region Russia corresponding to –75 - –100 bp position are of most interest for our task since this region is known to be involved in electrostatic interaction with E. coli RNA poly- *[email protected] merase α-subunit (4). **[email protected]

Electrostatic profile of PA1 (Figure 1a) can be characterized by the presence of the most negatively charged element at –70 - –100 bp and more positive flanking sites.

Figure 1: Distribution of electrostatic potential around PA1(A), PA2(B), PA3(C) and PB(D). 1122 The promoter shares these specific electrostatic features with two E. coli ribosomal promoters (5). Although electrostatic profiles of PA2 (Figure 1b) and PA3 (Figure 1c) are not identical, they reveal some common features in the far upstream region and they share these features with T4 early promoters P114.6 and P73.0 (6). The important feature of their patterns is a continuous rise of electrostatic potential at –80 to –100 bp with an extended positive peak at –90 bp Thus, PA2 and PA3 are characterized by the presence of the positively charged element in the functionally important region of promoter DNA. At the same time, this region for PA1 is mostly negative having no pronounced positively charged area. The difference in electrostatic properties of the tandem T7 promoters can result in a difference in their functional behavior allowing a high level of gene 1 transcription to be maintained in different conditions during the phage infection.

Electrostatic properties of all weak T7 promoters (a representative example in Figure 1D) differ from that of the strong PA1, PA2 and PA3. Their electrostatic up- elements are less structurally expressed, some of them being barely distinguishable from ordinary coding regions.

Thus, there is a good correlation between electrostatic properties of early T7 promoters and their strength. All the results support that the far upstream region of T7 early promoters can be involved in modulation their activities by acting through electrostatic interaction with E. coli RNA polymerase alpha-subunit.

References

1. S. M. West, R. Rohs, R. S. Mann, and B. Honig. J Biomol Struct Dyn 27, 861-866 (2010). 2. D. Wang, N. B. Ulyanov, and V. B. Zhurkin. J Biomol Struct Dyn 27, 843-859 (2010). 3. R. V. Polozov, T. R. Dzhelyadin, A. A. Sorokin, N. N. Ivanova, V. S. Sivozhelezov, and S. G. Kamzolova. J Biomol Struct Dyn 16, 1135-43 (1999). 4. S. G. Kamzolova, V. S. Sivozhelezov, A. A. Sorokin, T. R. Dzhelyadin, N. N. Ivanova, and R. V. Polozov. J Biomol Struct Dyn 18, 325-334 (2000). 5. S. G. Kamzolova, A. A. Sorokin, P. M. Beskaravainy, and A. A. Osypov. Bioinformat- ics of genome regulation and structure II. Eds. N. Kolchanov, R. Hofestaedt, Z. Milanesi, Springer Science Business Media Inc. p. 67-74 (2006). 6. A. A. Sorokin, A. A. Osypov, T. R. Dzhelyadin, P. M. Beskaravainy, and S. G. Kamzolova. J Bioinf Comp Biol 4, 455-467 (2006).

Electrostatic Properties of T7 RNA Polymerase 162 Specific Promoters 1,2, Analysis of electrostatic properties of promoter DNA is a promising source for P. M. Beskaravainy * yielding information about promoter recognizable elements and their functioning. A. A. Osypov1 G. G. Krutinin1 Here, electrostatic properties of 17 T7 RNA-polymerase specific promoters where 1 studied, and this has been recently reported to play an important role in protein- E. A. Krutinina nucleic acid recognition (1, 2). These promoters may be classified into three groups S. G. Kamzolova1 according to their expression time and functional characteristics of the corresponding genes: class “early” (subgroup of class II), class II and class III (1). All the promoters have been erlier characterized in detail by their interaction with RNA- 1Institute of Biophysics, RAS Pushchino, polymerase and transcription initiation (3, 4). Their sequences are characterized by Moscow Region 142290, Russia comparatively extended consensus sequence (23 bp) with a high level of homology 2Institute of Theoretical and for all promoters but, despite their considerable sequence similarity, the different promoter classes differ by their strengths and by many other biochemical proper- Experimental Biophysics, RAS Pushchino, ties (3, 4). Thus, the choice of these promoters for our study was motivated by Moscow Region 142290, Russia their unusual functional characteristics differing from “consensus sequence rule” *[email protected] behavior. 1123

Figure 1: The averaged profiles of electrostatic potential distribution around T7 RNA-polymerase specific promoters belonging to different functional group.

Electrostatic potential distribution around duble-helical DNA of the promoters was calculated by the Coulombic method (5) using a new computer program (6).

Electrostatic profiles of the individual promoters were combined into three corresponding groups and the averaged electrostatic profiles of these separate groups were calculated as indicated in (6). The data obtained are shown in figure 1. The profiles of the promoters can be characterized by the presence of particular electrostatic elements in the region from –25 bp to +20 bp wich is known to be involved in contacts with T7 RNA-polymerase. These electrostatic elements are specific for the different promoter groups. The promoters belonging to class II are characterized by two valleys at ~ –18 bp to ~ –5 bp and a more positive flanking site at +5 bp. In contrast, the promoters belonging to class III has the only most expressed negative fall at ~ –18 bp and two peaks downstream at ~ +5 bp and ~ +20 bp. The promoters belonging to the “early” group are characterized a more smoothed profile. They are located within coding region transcribed by the host E. coli RNA-polymerase. This can account for a more smoothed profile of this group since the variation of average electrostatic potential is more homogeneous in nonpromoter DNA.

Thus, electrostatic patterns of T7 DNA promoters are distinguished owing to specific motifs wich may be involved as signal elements in differential recognition of T7 RNA-polymerase specific promoters by the enzyme.

References

1. S. M. West, R. Rohs, R. S. Mann, and B. Honig. J Biomol Struct Dyn 27, 861-866 (2010). 2. D. Wang, N. B. Ulyanov, and V. B. Zhurkin. J Biomol Struct Dyn 27, 843-859 (2010). 3. J. J. Dunn and F. W. Studier. Proc Natl Acad Sci USA 70, 1559-63 (1973). 4. W. T. McAllister, C. Morris, A. H. Rosenberg, and F. W. Studier. J Mo Biol 153, 527-44 (1981). 5. R. V. Polozov, T. R. Dzhelyadin, A. A. Sorokin, N. N. Ivanova, V. S. Sivozhelezov, and S. G. Kamzolova. J Biomol Struct Dyn 16, 1135-43 (1999). 6. A. A. Osypov, G. G. Krutinin, and S. G. Kamzolova. J Bioinf Comp Biol 8, 413-25 (2010). 1124 Strand Exchange Reaction Between Short Oligonucleotides Promoted by a Derivative of 1,3 – diazaadamantane 163 The DNA strand exchange reaction (SER) underlies main pathways of homologous recombination (HR) and DNA repair in different organisms. These systems of DNA 1, Anna Gabrielian * metabolism closely relate to carcinogenesis and better understanding of interac- Tatiana N. Bocharova2 tion of antitumor drugs with their components is indispensable for development of Elena A. Smirnova2 new approaches to antitumor therapy. Recently this Journal has reported antitumor intercalators and groove binders interacting with DNA double helix (1-4). Alexander A. Volodin2 Gayane Harutjunyan1 Derivatives of 1,3-diazaadamantane are promising compounds that possess essential antitumor activity on different types of experimental tumors (5). In the present study the interaction of the 1,3-diazaadamantane derivative HG122 [1′-benzil- 1Research-technological Center of 5,7-dipropil-6-oxyspiro(1,3-diazaadamantane-2,4′-piperidine)] with DNA and its Organic & Pharmacological Chemistry behavior in the system that simulate SER has been characterized. (Armenian National Academy of Earlier we described an experimental system for the study of SER between short Sciences), 26 Azatutian Avenue, oligonucleotides promoted by RecA protein from ε.coli and RecA-like human 0014 Armenia recombinases (6). RecA protein is a paradigm of the wide class of proteins of HR systems from different organisms and promotes the central stage of HR – DNA 2Institute of Molecular Genetics strand exchange. In our system fluorescent dye-tagged 21-mer oligonucleotide and (Russian Academy of Sciences), 2, unlabelled double stranded 21 bp oligonucleotides were used as single- and double Kurchatov Square 123182, Moscow, stranded substrates of SER respectively. Russia We did not detect any influence of HG122 on the RecA protein promoted DNA *[email protected] strand exchange. At the same time, it was revealed that this compound by itself exhibited the capability to facilitate SER in this system. Kinetic curves of the SER in solution with different concentrations of Hg122 were obtained for various temperatures. The final yield of SER didn’t depend on temperature in the range from 28 to 50°C that argues in favor of thermodynamics equilibrium reached by this reaction. In separate experiments we found no effect of HG122 on the melting temperature of double-stranded DNA; therefore, the observed acceleration of SER in the presence of HG122 can not be explained by trivial effect of destabilization of the DNA double helix.

Earlier, the DNA strand exchange activity was documented for Cationic Comb- type Copolymers (7) and positively charged liposome surfaces (8). We found that other polycations such as linker histones and cobalt hexamine also exhibit the capability to promote SER (manuscript in preparation). The present results provide us with the first example of the quite different class of SER facilitating compounds that contain large lipophilic core.

Figure 1: Structure of HG-122. Common feature of the strand exchange reactions promoted by polycationic agents 1125 is their low tolerance to heterology between the SER substrates. In contrast to the reactions promoted by RecA-like proteins that exhibit increased tolerance to heterology between the substrates (6) the tolerance of polycations promoted strand exchange is comparable to that of the spontaneous SER that proceeds at elevated temperatures. Hg122 promoted reaction exhibits the tolerance to heterology intermediate between the cases of the SER promoted by polycations and RecA-like proteins.

Therefore, the present results demonstrate that Hg122 exhibits interesting behavior and can serve as a model object for further studies of different aspects of DNA strand exchange reaction. Biomedical implications of this and other similar compounds activities remain to be estimated.

References

1. N. P. Bazhulina, A. M. Nikitin, S. A. Rodin, A. N. Surovaya, Yu. V. Kravatsky, V. F. Pismensky, V. S. Archipova, R. Martin, and G. V. Gursky. J Biomol Struct Dyn 26, 701-718 (2009). 2. G. Singhal and M. R. Rajeswari. J Biomol Struct Dyn 26, 625-636 (2009). 3. B. Jin, H. M. Lee, and S. K. Kim. J Biomol Struct Dyn 27, 457-464 (2010). 4. H. M. Lee, B. Jin, S. W. Han, and S. K. Kim. J Biomol Struct Dyn 28, 421-430 (2010). 5. G. L. Harutjunyan, A. A. Chachoyan, C. E. Ahajanyan, and B. T. Haribjanyan. Chem pharm J (Russia), 12, 20-24 (1996). 6. A. A. Volodin, T. N. Bocharova, E. A. Smirnova, and R. D. Camerini-Otero. J Biol Chem 284, 1495-1504 (2009). 7. W. J. Kim, Y. Sato, T. Akaike, and A. Maruyama. Nat Mater 2, 815-820 (2003). 8. K. Frykholm, B. Nordén, and F. Westerlund. Langmuir 25,1606-1611 (2009).

Transcription Factor - DNA interactions: cis Regulatory Codes in the Genome 164 The interactions between sequence-specific transcription factors (TFs) and their DNA binding sites are an integral part of the gene regulatory networks within cells. Martha L. Bulyk My group developed highly parallel in vitro microarray technology, termed protein binding microarrays (PBMs), for the characterization of the sequence specificities 1Division of Genetics, of DNA-protein interactions at high resolution. Using universal PBMs, we have Department of Medicine determined the DNA binding specificities of >500 TFs from a wide range of species. These data have permitted us to identify novel TFs and their DNA binding 2Dept. Pathology, Brigham and Women’s site motifs, predict the target genes and condition-specific regulatory roles of TFs, Hospital and Harvard Medical School, predict tissue-specific transcriptional enhancers, investigate functional divergence Boston, MA 02115, USA of paralogous TFs within a TF family, investigate the molecular determinants of TF-DNA ‘recognition’ specificity, and distinguish direct versus indirect TF-DNA 3Harvard-MIT Division of Health interactions in vivo. Further analyses of TFs and cis regulatory elements are likely Sciences and Technology (HST), to reveal features of cis regulatory codes important for driving appropriate gene Harvard Medical School, Boston, expression patterns. MA 02115, USA This research has been supported by Grants from NIH / NHGRI to M.L.B. [email protected] 1126 Cis-Regulatory Modules: Identification in silico and Understanding of Gene Regulatory Networks

Cis-regulatory modules (CRM) are segments of DNA responsible for tissue- and time- specific regulation of gene expression (1). The length of CRMs is difficult to 165 estimate directly but it is believed to vary from several hundreds to several thou- Ivan V. Kulakovskiy1,3 sands of base pairs. In multicellular eukaryotes CRMs may be located not only in Yulia A. Medvedeva2,3 the upstream vicinity of transcription start sites of the dependent genes but also at tens of thousands nucleotides upstream or downstream from the transcription 2,3 Vsevolod J. Makeev * start sites. CRMs contain multiple binding sites for protein factors regulating the transcription. Identification of CRMs in silico and prediction of their regulatory function allows one to suggest new regulatory inputs controlling expression of par- 1Engelhardt Institute of Molecular ticular genes, which makes a useful introductory step before modeling of cell sig- Biology, Russian Academy of Sciences, naling processes. CRMs make an important component of meaningful non-coding Moscow, Russia DNA. Genetic variations overlapping with CRMs contribute to functional disorders associated with non-coding DNA. 2Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Detail investigation of CRM sequences exhibit that transcription factor binding Russia sites (TFBS) form complex arrangements, probably corresponding to yet unknown regulatory code of gene expression. The simplest feature found in CRMs is clusters 3FSUE Research Institute of Genetics and of binding sites. Eukaryotic gene expression usually is controlled by many inputs Selection of Industrial Microorganims, from different regulatory circuits, so a typical CRM contains many binding sites for Moscow, Russia different transcription factors (TF), both activators and repressors, thus integrating different regulatory contributions. Thus, a typical CRM contains many sites for dif- *[email protected] ferent TFs which are rather densely packed and form a so-called heterotypic cluster (2). Sites for different TFs in such clusters are often found at specific distances from each other (3), probably facilitating correct positioning of proteins at DNA necessary for protein-protein interaction, either direct or via adapter proteins (4).

In addition, many CRMs contain many occurrences of the same binding sites, forming the so-called homotypic clusters of TFs (5, 6). The function of homotypic clustering is yet unclear; probably it is served for providing for a specific TF-concentration dependence for TF binding (7). The alternative explanation is that this type of arrangement is needed to facilitate lateral diffusion of binding factor along DNA to its functional binding position (8). The exceptional form of a homotypic cluster is a tandem repeat made from sequences, specifically bound by TFs. This type of arrangement is characterstic for some CRMs in Drosophila (9), and might serve as an origin of some CRMs in evolution.

Studying DNA sequences of CRMs often help to decipher yet unknown regulatory circuits. Two examples are touched upon: Drosophila development and human hypoxia response cascade. I’m also going to discuss how CRMs are predicted in silico, and consequences of site turnover for prediction of CRM and TFBS.

This research has been supported by Presidium of Russian Academy of Sciences Program in Molecular and Cell Biology.

References

1. E. H. Davidson. Genomic Regulatory Systems. Academic Press, San Diego, USA (2001). 2. B. P. Berman, Y. Nibu, B. D. Pfeiffer, P. Tomancak, S. E. Celniker, M. Levine, G. M. Rubin, and M. B. Eisen. Proc Natl Acad Sci 99: 757-762, (2002). 3. V. J. Makeev, A. P. Lifanov, A. G. Nazina, and D. A. Papatsenko. 31(20): p. 6016-26 (2003). 4. I. V. Kulakovskiy, A. A. Belostotskiy, and V. J. Makeev. 17th Albany conversation, Albany NY, USA (2011). 5. A. P. Lifanov, V. J. Makeev, A. G. Nazina, D. A. Papatsenko. Genome Res 13: 579-588, (2003). 6. V. Gotea, A. Visel, J. M. Westlund, M. A. Nobrega, L. A. Pennacchio, and I. Ovcharenko. 1127 Genome Res 20, 565-77, (2010). 7. D. E. Clyde, M. S. Corado, X. Wu, A. Pare, D. Papatsenko, and S. Small. Nature 426, 849-853, (2003). 8. A. Tafvizi, F. Huang, A. R. Fersht, L. A. Mirny, and A. M. van Oijen. Proc Natl Acad Sci USA 108, 563-568, (2011). 9. V. A. Boeva, M. Regnier, D. Papatsenko, and V. Makeev. Bioinformatics 22, 676-684, (2006).

Preferred Pair Distance Templates for Identification of Functional Binding Sites for Interacting Transcription Factors 166 Ivan V. Kulakovskiy1,2* We study a set of transcription factors (TFs) including the hypoxia-inducible factor Alexander A. Belostotsky3 1 (HIF-1) involved in regulation of hypoxia response in human cells. We dem- 2,3 onstrate that binding sites for a pair of interacting TFs can be found at distances Vsevolod J. Makeev that are dramatically nonrandom and depend both on selected TFs and a relative orientation of their binding sites. The set of characteristic intersite distances forms 1Laboratory of Bioinformatics and a preferred pair distance template (PPDT). System Biology, Engelhardt Institute of We started identification of high-quality binding motifs with the help of our original Molecular Biology, Russian Academy computational tool, ChIPMunk. ChiPMunk performs motif discovery integrating of Sciences, Vavilov str. 32, Moscow 119991, Russia 2Laboratory of Bioinformatics, Vavilov Institute of General Genetics, Russian Academy of Sciences, Gubkina str. 3, Moscow 119991, Russia 3Laboratory of Bioinformatics, Research Institute for Genetics and Selection of Industrial Microorganisms, 1st Dorozhny proezd 1, Moscow 117545, Russia *[email protected]

Figure 1: Distance distribution between HRE elements (corresponding to the HIF‑1α:ARNT dimer binding) and Sp1 binding sites. Y axis shows the number of genes for which at least one pair of binding sites is found at the selected distance (shown at X-axis) within the 10kb upstream promoter region. Panels: orientation of HRE in the reference to Sp1; (top) direct; (bottom) reverse. 1128 data from different experimental sources (1) including ChIP-chip and ChIP-Seq experiments (2). Both our own (3) and independent (4) benchmarks have shown that ChIPMunk can produce high-quality motifs in a reasonable time.

The motifs discovered were used to detect transcription factor binding sites in the 5’ regulatory regions of all human genes (according to the UCSC hg18 assembly).

For TFs involved in the known protein-protein interaction with the HIF1α we have observed a surprisingly large fraction of binding sites found at specific distances from HIF1α binding sites (see Figure 1). For some TF pairs the corresponding PPDTs contained quite large distances, sometimes longer than 200 bp.

Thus, with a help of PPDTs we can correctly identify composite elements (5) in regulatory segments. Moreover, PPDT can be used as a filter to remove false positive binding sites in human cis-regulatory modules. PPDT also can be used to assess functional interaction between a pair of DNA binding proteins solely by means of sequence analysis. Currently we explore how PPDT information can be used together with gene expression data to identify correct target responding to hypoxia conditions.

This research is supported by Russian Fund of Basic Research grant 10-04- 92663-Ind.

References

1. I. V. Kulakovskiy, V. J. Makeev. Biophysics 54, 667-674 (2009). 2. I. V. Kulakovskiy, V. J. Makeev, V. A. Boeva, V. J. Makeev. Bioinformatics 26, 2622-2623 (2010). 3. http://line.imb.ac.ru/ChIPMunk/Supplementary_text.pdf 4. http://cmotifs.tchlab.org/help.html 5. V. Matys, O. V. Kel-Margoulis, E. Fricke, et al., Nucleic Acids Res 34, D108-10 (2006).

Promoter Islands: The Novel Elements 167 In Bacterial Genomes Seventy-eight “promoter islands” (PIs) were delineated in E. coli genome based Konstantin S. Shavkunov on high density of potential transcription start points, high ability of RNA poly- Maria N. Tutukina merase (RNAP) binding and paradoxically low transcription efficiency (1). Among Irina S. Masulis them there are 23 PIs lying inside coding sequences (exemplified in Figure 1A) or between convergent genes, where promoter activity is not expected. The specific Olga N. Ozoline* location and high length of genomic regions (300 bp), containing overlapping transcription signals, distinguish them from clustered promoter-like sites previ- Institute of Cell Biophysics, RAS, ously found nearby highly expressed genes. In this study we compared chromatin structure for PIs and 181 “single” promoters using published ChIP-on-chip data Pushchino Moscow Region, 142290, (2-4) and validated the ability of 16 intragenic PIs + 3 PIs located between conver- Russian Federation gent genes to form “open” complexes with RNAP in vitro and in vivo. *[email protected] It turned out that PIs more frequently than “single” promoters associate with nucle- oide proteins H-NS and Fis. Interaction with RNAP was registered for all PIs (100%) if antibodies against σ subunit were used to collect complexes (Figure 1B gray bars) and only for 43% of “islands” if precipitation was carried out by β-specific reaction (Figure 1B black symbols), while complexes with “single” promoters were detected with approximately equal efficiency by these two approaches (~87% and 77%, respectively). These differences assume certain singularity in the chromatin structure within PIs. Functional analysis testified ability of all analyzed PIs to undergo local DNA melting upon interaction with RNAP in vitro and 12 “islands” form transcription bubbles in vivo, thus assuming their ability to start RNA synthesis. 1129

Figure1: (A) PI associated with genes yigF and yigG. Bars represent transcription start points predicted by PlatProm. Triangles and asterisks indicate positions of transcription bubbles registered in vitro and in vivo, respectively. (B): Sites of interaction with RNAP as registered by ChIP-on-chip technique (4) with σ- or β-specific antibodies.

However in most cases (75% for analyzed PIs and 75.6% for the total set of 78 PIs) expected products were not found among the sequenced cDNAs (5). For “single” promoters this failure was 2-fold lower (35%). Since other bacterial genomes show approximately the same distribution of PIs, we suppose that they perform evolutionary conserved biological role, not necessarily associated with RNA synthesis.

The research is supported by Grants from Russian Foundation for Basic Research (10-04-01218).

References

1. K. S. Shavkunov, I. S. Masulis, M. N. Tutukina, A. A. Deev, and O. N. Ozoline. Nucleic Acids Res 37, 4919-4931 (2009). 2. D. C. Grainger, D. Hurd, M. D. Goldberg, and S. J. W. Busby. Nucleic Acids Res 34: 4642-4652 (2006). 3. D. C. Grainger, H. Aiba, D. Hurd, D. F. Browning, and S. J. W. Busby. Nucleic Acids Res., 35: 269-278 (2007). 4. N. B. Reppas, J. T. Wade, G. M. Church, and K. Struhl. Mol Cell, 24, 747-757 (2006). 5. J. E. Dornenburg, A. M. DeVita, M. J. Palumbo, and J. T. Wade. mBio 1: e00024-10 (2010). 1130 The Influence of Aliphatic Alcohols on Oligonucleotide Hybridization

The biological activity of nucleic acids is correlates strongly with their physicochemical properties, in particular their thermodynamic properties. The thermal 168 stability of nucleic acids depends strongly on the properties of intracellular envi- Alexander A. Lomzov1,2,* ronment such as ionic strength, polarity of the medium and presence of various + 1,2 osmolites, compartmentalization and etc (1-3). The influence of monovalent (Na Irina V. Khalo and K+) and divalent (Mg2+) cations has been widely studied (4, 5). By contrast the Dmitrii V. Pyshnyi1 thermodynamics properties of nucleic acids in the presence of various co-solvents have not been studied in details previously, excepting the molecular crowding

1 effect (6). Here we begin the systematic study of the thermodynamics of DNA Institute of Chemical Biology and duplex formation in the presence of a large variety of co-solvents. In this work Fundamental Medicine SB RAS, the efficiency of oligodeoxyribolnucleotides hybridization and DNA duplex sec- Lavrent’v ave., 8, 630090, ondary structure in the presence of various aliphatic alcohols (ethanol, ethylene glycol, diethylene glycol, 2,2,2-trifluoroethanol, 2-cyanoethanol) in water solution Novosibirsk, Russia (10 mM sodium cacodylate, pH 7.2) has been characterized. It has been shown that 2Novosibirsk State University, Pirogova the increase of concentration of aliphatic alcohol in solution from 0 to 50% (v/v) st., 2, 630090, Novosibirsk, Russia does not change the conformation of DNA helix. We have demonstrated that for most alcohols (ethanol, cyanoethanol, ethylene glycol and diethylene glycol) rising *[email protected] of their concentration up to 50% in a aqueous solution lead to monotonous decrease in melting temperature for DNA duplexes of various length (12, 15 or 20 b.p.) and GC-content (27–60%). In the case of trifluoroethanol the stability of double stranded DNA decreases with the increase of the alcohol concentration up to 20 %. The higher concentrations of trifluoroethanol (up to 75%) resulted in slight stabilization of DNA duplexes. The analysis of the data obtained has shown the possibility of using a simple model for the description of thermal stability of duplexes in mixed water/alcohol solutions except 2,2,2-trifluoroethanol. The model assume that the number of solvents molecules (water and co-solvent) which are bound to DNA changes at helix-to-coil transition. The proposed model describes the dependence of melting temperatures of oligonucleotide complexes on concentration of ethanol, cyanoethanol, ethylene glycol and diethylene glycol with accuracy 1.1, 1.7, 1.0, 2.2°C, respectively.

The results obtained can be applied in hybridization analysis in various buffer conditions, for example, with the use of exciplexes (7); in studying nucleic acid – protein interactions and in the development of molecular-imprinted polymers (8).

This research has been supported by Russian Government (P1073), SB RAS, RFBR and by MCB and FBNN programs of RAS.

References

1. Y. Dalyan, I. Vardanyan, A. Chavushyan, and G. Balayan. J Biomol Struct Dyn 28, 123-131 (2010). 2. I. A. Il’icheva, P. K. Vlasov, N. G. Esipova, and V. G. Tumanyan. J Biomol Struct Dyn 27,677-693 (2010). 3. T. C. Mou, M. C. Shen, T. C. Terwilliger, and D. M. Gray. Biopolymers 70, 637-648 (2003). 4. E. N. Galyuk, R. M. Wartell, Y. M. Dosin, and D. Y. Lando. J Biomol Struct Dyn 26, 517-523 (2009). 5. R. Owczarzy, B. G. Moreira, Y. You, M. A. Behlke, and J. A. Walder. Biochemistry 47, 5336-5353 (2008). 6. D. Miyoshi and N. Sugimoto. Biochimie 90, 1040-1051 (2008). 7. E. V. Bichenkova, A. Gbaj, L. Walsh, H. E. Savage, C. Rogert, A. R. Sardarian, L. L. Etch- ells, and K. T. Douglas. Org Biomol Chem 5, 1039-1051 (2007). 8. E. V. Dmitrienko, I. A. Pyshnaya, A. V. Rogoza, and D. V. Pyshnyi. Patent of Russian Federation # 2385889, (2010). Minor Groove Ligands Based on Dimeric 1131 Bisbenzimidazoles as Inhibitors of DNA-dependent Enzymes

Recently in this Journal there have been reports of both intercalators and groove binders interacting with DNA double helix (1-4). We have reported elsewhere that 169 dimeric bisbenzimidazoles (DB(n)) differing in the length of methylene linkers can Alexander A. Ivanov1* interact with dsDNA in the minor groove (5). Sergey A. Streltsov2 Compounds DB(n) were studied as inhibitors of two DNA-dependent enzymes, Victor I. Salyanov2 namely, calf thymus DNA topoisomerase I (topo-I) (6) and the catalytic domain Olga Yu. Susova1 Dnmt3a of murine DNA methyltransferase (MTase) (7). Ligands DB[3, 4, 5, 7, 11] 3 inhibited activities of topo-I in vitro at 0.5-2.5 μM concentrations. Inhibitory activity Elizaveta S. Gromova of DB[7] was 50-fold higher than that of camptothecin, a known topo-I inhibitor. Alexei L. Zhuze2 DB[1, 2, 3, 11] reduced in vitro the MTase Dnmt3a activity by 50% at 5-12 μM concentrations. Bisbenzimidazoles DB[4, 5, 7] were shown to be less effective: 50% 1 inhibition of MTase activity was observed only at a concentration of 20 μM and Institute of Carcinogenesis Blokhin higher. Increased time of incubation of the tested DB[n] with DNA allowed for a Cancer Research Center Russian Academy substantial growth of inhibitory activity of the ligands toward both topo-I and MTase of Medical Sciences, Kashirskoye Shosse (6, 7). It should be noted that monomeric MB, a shortened DB[n] analogue, only neg- ligibly inhibited both enzymes at 200 μM concentration. We assume that the found 24, Moscow 115478, Russia inhibitory activity of DB[n] is explained by their competition with DNA-dependent 2Engelhardt Institute of Molecular enzymes for binding sites on DNA. It was previously shown that the Hoechst 33258 Biology Russian Academy of dye containing the same bisbenzimidazole motive as DB[n] bound specifically to two consecutive AT-pairs [8, 9]. Therefore, we suppose that compounds DB[n] would Sciences, Vavilova st. 32, Moscow bind with the highest specificity to the -(A/T)n-(N)m-(A/T)n- DNA site, where N, a 119991, Russia base pair, n = 2, m = 1-4, which correlates with computer modeling data. Indeed, both 3Chemistry Department, Moscow State duplex I (a highly effective topo-I cleavage site) and duplex II sensitive to MTase Dnmt3a contain sequences optimal for DB[n] binding (Figure 2). University, Moscow 119991, Russia *[email protected]

Figure 1: Structure of dimeric bisbenzimidazoles DB[n], where n = 1, 2, 3, 4, 5, 7, 11.

Figure 2: Oligonucleotide fragments used for studies of DB[n] inhibitory activities. Consecutive AT-pairs are in bold; potential binding sites of the corresponding ligands are underlined. For duplex I , the arrow shows the site cleaved by topo-I and for duplex II, cytosine residues to be methylated.

To summarize, DB[n] were proved to be inhibitors of at least two different DNA- dependent enzymes at low micromolar concentrations. Higher biological activity of DB[n] if compared with monomeric MB is due to the formation of dimeric molecules capable of binding to dsDNA in the form of bidentant ligands. 1132 The work was supported by RFBR (Grants 09-04-01126, 10-04-00809 and 11-04- 00589) and by the Program of Presidium of RAS on Molecular and Cell Biology.

References

1. N. P. Bazhulina, A. M. Nikitin, S. A. Rodin, A. N. Surovaya, Yu. V. Kravatsky, V. F. Pismensky, V. S. Archipova, R. Martin, and G. V. Gursky. J Biomol Struct Dyn 26, 701-718 (2009). 2. G. Singhal and M. R. Rajeswari. J Biomol Struct Dyn 26, 625-636 (2009). 3. B. Jin, H. M. Lee, and S. K. Kim. J Biomol Struct Dyn 27, 457-464 (2010). 4. H. M. Lee, B. Jin, S. W. Han, and S. K. Kim. J Biomol Struct Dyn 28, 421-430 (2010). 5. A. A. Ivanov, V. I. Salyanov, S. A. Streltsov, N. A. Cherepanova, E. S. Gromova, and A. L. Zhuze. Russ J. Bioorgan Chemistry 37 (2011) (in press). 6. O. Y. Susova, A. A. Ivanov, S. S. Morales Ruiz, E. A. Lesovaya, A. V. Gromyko, S. A. Streltsov, and A. L. Zhuze. Biochemistry (Moscow) 75, 695-701 (2010). 7. N. A. Cherepanova, A. A. Ivanov, D. V. Maltseva, A. S. Minero, A. V.Gromyko, S. A. Streltsov, A. L. Zhuze, and E. S. Gromova. J Enzyme Inhib Med Chem (2011) [Epub ahead of print]. 8. P. E. Pjura, K. Grzeskowiak, and R. E. Dickerson. J Mol Biol 197, 257-271 (1987). 9. M. K. Teng, N. Usman, C. A. Frederick, and A. H. Wang. Nucleic Acids Res. 16, 2671-2690 (1988).

Effect of Millimeter Electromagnetic Waves on the Water-Saline Solutions of DNA and 170 DNA-Ligand Complexes Poghos O. Vardevanyan1 Ara P. Antonyan1 There have been recently studies of various factors that affect the stability of DNA, DNA-ligand and RNA-ligand complexes (1-6). In this paper we examine the effect 1 Mariam A. Shahinyan of radiation and ionic strength on the thermostability of DNA and DNA-ligand Margarita A. Torosyan2 complexes. The effect of non-thermal (∼50 µW/cm2) (50,3 and 64,5 GHz are reso- Armen T. Karapetian2 nant of water hexagonal structure frequencies) millimeter electromagnetic waves (MM EMW) on water-saline solutions of DNA and DNA-ligand complexes with EtBr and Hoechst 33258 is studied. Experimental data showed that radiation 1Department of Biophysics of Yerevan increased the thermostability of the investigated samples. The differences between r nr the melting temperatures of radiated (Tm ) and non-radiated (Tm ) DNA and its State University, A.Manoogian str. 1, r nr complexes with ligands (δTm = Tm -Tm ) depended on the ionic strength; the greater Yerevan 0025, Armenia the ionic strength, the smaller is the δTm. 2Physics Department of Yerevan State It is known, that the hydration of DNA decreases with increase of the Na+ concen- University of Architecture and + tration. Therefore, decrease of the value of δTm at high concentration of Na points Construction, Teryan str. 105, bldg.2, to the non significant effect of the radiation of the structure of hydration shell of Yerevan 0005, Armenia DNA and its complexes with the ligands, at these conditions. This conclusion is supported by our experimental results according to which the radiation of DNA and [email protected] DNA-ligand complexes by non-resonant of water hexagonal structure frequency MMEMW (48.3 GHz) caused no changes in the thermostability of the samples at all investigated concentrations of Na+.

Experimental results obtained made it possible to suggest that radiation, like ionic strength of solutions, increases the thermostability of DNA and DNA-ligand complexes. References 1133 1. A. Borkar, I. Ghosh, and D. Bhattacharyya. J Biomol Struct Dyn 27, 695-712 (2010). 2. N. P. Bazhulina, A. M. Nikitin, S. A. Rodin, A. N. Surovaya, Yu. V. Kravatsky, V. F. Pismensky, V. S. Archipova, R. Martin, and G. V. Gursky. J Biomol Struct Dyn 26, 701-718 (2009). 3. G. Singhal and M. R. Rajeswari. J Biomol Struct Dyn 26, 625-636 (2009) 4. B. Jin, H. M. Lee, S. K. Kim. J Biomol Struct Dyn 27, 457-464 (2010). 5. H. M. Lee, B. Jin, S. W. Han, and S. K. Kim. J Biomol Struct Dyn 28, 421-430 (2010). 6. Y. Dalyan, I. Vardanyan, A. Chavushyan, and G. Balayan. J Biomol Struct Dyn 28, 123-131 (2010).

Dynamics of Two Combined Reversible Binding Ligands to DNA

Kinetic investigations on ligand binding to DNA describe the binding process as a function of time and allow getting information on rates of formation and dissociation of DNA-ligand complexes. Usually one works on binding kinetics of a single 171 ligand binding on DNA. For examples of current study of ligand binding to nucleic 1 acids, see references 1-5. Valery B. Arakelyan Gohar G. Hovhannisyan2 In this paper binding of two different ligands with DNA is considered under Poghos O. Vardevanyan2 condtions in which both ligands occupy the same adsorption centers with different binding constants, and each ligand occupies different number of absoption centers. The ligand binding and dissociation of the complex can be represented as a discrete 1Department of Molecular Physics Markov process, and the derived differential equations describe the kinetics of com- of Yerevan State University, bined binding of various ligands to DNA. Analysis of the data show that depending on A.Manooqian str. 1, Yerevan 0025, adsorption parameters various modes of ligands binding to DNA may be realized. Armenia Comparison of experimental curves of ligands interacting with DNA as a function 2Department of Biophysics of Yerevan of time with theoretical curves allows to determine the rate constants of formation State University, A.Manooqian str. 1, and dissociation of DNA-ligand complex It is shown that the kinetics of DNA occupancy with a ligand of given type depends on the ratios between binding Yerevan 0025, Armenia constant rates and concentration of ligands as well. Depending on their ratios, it [email protected] may be possible that nonmonotonic change of number of ligands adsorbed on DNA over time may lead to inversion of ligand occupancy.

References

The complex formation of podophyllotoxin and its semi synthetic derivative – etoposide with DNA has been investigated by absorption and thermal denaturation methods. The data show that these ligands have a destabilizing effect on DNA. The

values of binding constant (Kb) obtained and the number of binding sites (n) of 172 these ligands with DNA indicate that both ligands bind with DNA by two ways – Ara P. Antonyan1 specifically with limited sites of binding corresponding to intercalative (or semi- Anna S. Ryazanova2 intercalative) mechanism and non specifically (external with one chains of DNA) with unlimited binding sites. For the various ways of binding to nucleic acids see Hrachik R. Vardapetyan2* references 1-5. It has been shown that the value of Kb of etoposide to DNA is higher than that of podophyllotoxin. The data obtained by these methods reveal that both ligands interact with DNA and possess a higher affinity with AT sequences. 1Department of Biophysics, Yerevan State University, Yerevan, 0025, Armenia The data obtained also show that these ligands bind with DNA immediately and the 2Biomedical faculty, Russian-Armenian citotoxicity may be conditioned by inhibition of topoisomerses I and II of mammalian DNA, as well as a destabilization and damage to DNA itself. (Slavonic) University, Yerevan, 0051,

Armenia References [email protected] 1. N. P. Bazhulina, A. M. Nikitin, S. A. Rodin, A. N. Surovaya, Yu. V. Kravatsky, V. F. Pismensky, *[email protected] V. S. Archipova, R. Martin, and G. V. Gursky. J Biomol Struct Dyn 26, 701-718 (2009). 2. G. Singhal and M. R. Rajeswari. J Biomol Struct Dyn 26, 625-636 (2009). 3. B. Jin, H. M. Lee, and S. K. Kim. J Biomol Struct Dyn 27, 457-464 (2010). 4. H. M. Lee, B. Jin, S. W. Han, and S. K. Kim. J Biomol Struct Dyn 28, 421-430 (2010). 5. Y. Dalyan, I. Vardanyan, A. Chavushyan, and G. Balayan. J Biomol Struct Dyn 28, 123-131 (2010).

Yarrowia Lipolytica Yeast Possesses an Atypical 173 Catabolite Repression Catabolite repression was thoroughly studied in the bacterium Escherihia coli Igor G. Morgunov and, though not so well, in the yeast Saccharomyces cerevisiae (1, 2). Glucose and Svetlana V. Kamzolova related sugars repress the transcription of genes encoding enzymes required for the utilization of alternative carbon sources. The different sugars produce signals which modify the conformation of certain proteins that, in turn, directly or through G. K. Skryabin Institute of Biochemistry a regulatory cascade affect the expression of the genes subject to catabolite repres- and Physiology of Microorganisms, sion. These genes are not all controlled by a single set of regulatory proteins (3, 4),

Russian Academy of Sciences, pr-t but there are different circuits of repression for different groups of genes (2). Catab- olite repression allows the respective microorganisms effectively use carbohydrate Nauki 5, Pushchino, Moscow Region substrates, which first assimilate one of the two available substrates (commonly, a 142290, Russia carbohydrate), whereas the assimilation of the other substrate starts only after the [email protected] first substrate is fully consumed from the medium. The degree of catabolite repression varies very significantly in microorganisms. For example, glucose suppresses the expression of invertase in Saccharomyces cerevisiae by 800 times, whereas the expression of aconitate hydratase, cytochrome oxidase, and isocitrate dehydrogenase, are suppressed not more than by 10 times (1). glycerol + oleate GK PC IL MS 0,2 1135 12

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glucose + hexadecane PDH PC IL MS 12 0,2

8 0,1 4

0 0 0612 18 24 30 36 61218243036 Time (h) Time (h) Figure: The growth of Yarrowia lipolytica in the two-substrate medium (left) and key enzymes (write) in these conditions. Variation of • biomass,  glycerol,  oleic acid,  glucose and  hexadecane (expressed in g/l); enzyme activity (expressed in U/mg of protein) of GK (glycerolkinase), PC (pyruvate carboxylase), PDH (pyruvate dehydrogenase), IL (isocitrate lyase) and MS (malate synthase).

The non-conventional yeast Yarrowia lipolytica is an organism of great biotech- nological interest due to its ability to excrete organic acids and proteins to the medium. The phenomenon of catabolite repression in Yarrowia lipolytica yeast is poorly studied.

The aim of this work was to study the metabolism of Yarrowia lipolytica yeast in media containing two different carbon sources: glycerol/oleic acid; glucose/oleic acid; glucose/hexadecane and its regulation.

It is evident from the Figure that when Yarrowia lipolytica was cultivated on the mixture of glycerol and oleic acid, the concentration of these substrates started to decrease just from the first hours of cultivation. Moreover, the utilization of these two substrates went concurrently, although glycerol was utilized at a higher rate than oleic acid. Glycerol kinase (the key enzyme of glycerol metabolism) and two key enzymes of the glyoxylate cycle responsible for the metabolism of fatty acids (isocitrate lyase and malate synthase) were induced from the fist hours of cultivation and remained active to the end of the cultivation period. These results suggest that glycerol is more easily utilizable substrate than oleic acid and probably other fatty acids; however, glycerol does not suppress the metabolism of fatty acids. In contrast, upon the cultivation of Yarrowia lipolytica on the mixture of glucose 1136 and oleic acid, the latter substrate began to be utilized only when the concentration of glucose decreased from 10 to less than 2.5 g/L (on the 12th hour of cultivation). The glycolytic enzymes pyruvate dehydrogenase and pyruvate carboxylase were induced from the first hours of cultivation and remained at high levels until the exhaustion of glucose in the medium. At the same time, the activities of isocitrate lyase and malate synthase were very low during the metabolism of glucose, but were rapidly induced after the exhaustion of glucose in the medium. These data can be interpreted in such a manner that glucose at rather high concentrations suppresses enzymes involved in the metabolism of fatty acids.

When Yarrowia lipolytica was grown on the mixture of glucose and hexadecane, the dynamics of growth and substrate consumption was typical of the diauxie phenomenon. Indeed, the utilization of hexadecane began only in several hours after the time when glucose was completely exhausted in the cultivation medium. In this case, the exhaustion of glucose arrested growth and the culture resumed growth only after a lag period. The assay of enzymes showed that the glycolytic enzymes pyruvate dehydrogenase and pyruvate carboxylase were active during the phase of growth on glucose, whereas the enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were active during the phase of growth on hexadecane.

Thus, experiments on the cultivation of Yarrowia lipolytica on two substrate pairs: glycerol/oleic acid and glucose/oleic acid and glucose/hexadecane indicated that glycerol does not suppress the assimilation of oleic acid, whereas glucose suppresses it in such a manner that oleic acid begins to be consumed only after the concentration of glucose in the medium falls to about zero.

References

1. K. D. Entian, H. J. Shuller. Glucose Repression (Catabolite Repression) in Yeast. In: Yeast Sugar Metabolism. Biochemistry, Genetics, Biotechnology and Applications, F. K. Zimmerman, K. D. Entian (Eds.), Technomoc Publishing, Basel, Switzerland, pp. 409-434 (1997). 2. J. M. Gancedo. Microbiol Mol Biol Rev 62, 334-361 (1998). 3. F. B. Guo and Y. Lin. J Biomol Struct Dyn 26, 413-420 (2009). 4. I. H. Cho, Z. R. Lu, J. R. Yu, Y. D. Park, J. M. Yang, M. J. Hahn, and F. Zou. J Biomol Struct Dyn 27, 331-345 (2009). New Insights into Protein-DNA Electrostatic 1137 Interactions: Beyond Promoters to Transcription Factors Binding Sites

Electrostatic properties of genome DNA are well recognized to influence its interactions with different proteins such as histones (1, 2), and in particular the primary 174 recognition and regulation of transcription by RNA-polymerase. This enzyme may Eugenia A. Krutinina identify promoters and evaluate their strength due to the peculiarity of their elec- Gleb G. Krutinin trostatic profiles (3-7). The same problem of recognition of a limited number of specific sequences in the long DNA molecule faces also transcription factors. To Svetlana G. Kamzolova reveal the role of electrostatic properties here we studied binding sites of different Alexander A. Osypov* families of these proteins.

The analysis of the profiles using DEPPDB – DNA Electrostatic Potential Properties Institute of Cell Biophysics of RAS, Database – showed a number of common features, which can be illustrated with the Pushchino Moscow Region, cAMP-CRP complex binding sites in the genome DNA in E.coli K12 (Figure 1). 142290, Russia The averaged profiles of the DNA electrostatic potential aligned around the CRP *[email protected] dimer binding sites centers exhibit the pronounced rise in the negative potential value with the characteristic W-like profile in the concensus area of 16 bp (TGTGA-N6-TCACA palindrome). The extensive (around 150-300 bp long), symmetrical overall potential rise can not be explained by the influence of the concensus itself and reflects the sequence organization of the flanking regions, contributing to the high potential area formation. Apparently this sequence organization was selected evolutionary to support the binding site recognition by the regulation protein molecule and its retention.

It is worth noting that this high potential area is relatively AT-enriched though doesn’t possess any textual consensus properties. Such enrichment is commonly accepted as facilitating the DNA melting in the promoter regions. However, it is clearly not the case in the present system, as the promoter core (and the initially

Figure1: Averaged electrostatic profile of the cAMP-CRP complex binding sites in the genome DNA in E. coli K12 (above) and the GC content (below). The binding site consensus region is highlighted with gray. 1138 DNA melting point) lies downstream of the CRP binding sites, sometimes more than 100 bp apart. So the function of this enrichment is rather the formation of the high electrostatic potential trap for the CRP complex, especially given the fact of the symmetry of this electrostatic valley, which wouldn’t be the case if due to the promoter core influence.

The same overall properties, though vary in particular details, are typical to binding sites of other families of transcription factors in a diverged range of bacterial taxa.

The data obtained reveal the role of electrostatic properties of DNA in the recognition of the transcription regulation proteins binding sites, further confirming their universal importance in the protein-DNA interactions beyond the classical promoter- RNA polymerase recognition and regulation. They demonstrate the necessity of the studies of the electrostatic properties of genome DNA in addition to the traditional textual analysis of its sequence.

References

1. S. M. West, R. Rohs, R. S. Mann, and B. Honig. J Biomol Struct Dyn 27, 861-866 (2010). 2. D. Wang, N. B. Ulyanov, and V. B. Zhurkin. J Biomol Struct Dyn 27, 843-859 (2010). 3. R. V. Polozov, T. R. Dzhelyadin, A. A. Sorokin, N. N. Ivanova, V. S. Sivozhelezov, S. G. Kamzolova. J Biomol Struct Dyn 16(6), 1135-43 (1999). 4. S. G. Kamzolova, A. A. Sorokin, T. D. Dzhelyadin, P. M. Beskaravainy, and A.A. Osypov. J Biomol Struct Dyn 23(3), 341-346 (2005). 5. S. G. Kamzolova, V. S. Sivozhelezov, A. A. Sorokin, T. R Dzhelyadin, N. N. Ivanova, and R. V. Polozov. J Biomol Struct Dyn 18(3), 325-334 (2000). 6. A. A. Sorokin, A. A. Osypov, T. R. Dzhelyadin, P. M. Beskaravainy, and S. G. Kamzolova. J Bioinform Comput Biol 4(2), 455-467 (2006). 7. A. A. Osypov, G. G. Krutinin, and S. G. Kamzolova. J Bioinform Comput Biol 8(3), 413-425 (2010). The Role of Electrostatics in Protein-DNA 1139 Interactions in Phage Lambda

Physical properties of DNA are known to be essential for RNA polymerase-promoter recognition. Especially electrostatic interactions between promoter DNA and RNA polymerase is of considerable importance in regulating promoter function (1-5), 175 just like the electrostatic interactions between histones and DNA determine the positioning of the nucleosomes and the expression of the genome (6, 7). Gleb G. Krutinin Eugenia A. Krutinina One of the elements that may play a crucial role in the promoter strength regulation Svetlana G. Kamzolova is the so-called “up-element”, which interacts with the alpha-subunit of the RNA polymerase and thus facilitates its binding to the promoter. It was shown earlier that Alexander A. Osypov* the T4 phage strong promoters with pronounced “up-element” have high levels of the electrostatic potential within it (3). Institute of Cell Biophysics of RAS, Pushchino Moscow Region, Using DEPPDB – DNA Electrostatic Potential Properties Database (5) – we studied electrostatic properties of the lambda phage genome DNA. We observed that the 142290, Russia strong lambda phage promoters have pronounced “up-element” compared to the *[email protected] absence of it in weak promoters. Promoters with intermediate strength possess weak “up-element” (Figure 1).

Strong promoters also have the characteristic heterogeneity of the electrostatic profile, known to differentiate promoters and coding regions. Pseudopromoters are located in the region of high potential value with a prominent electrostatic trap. It is reported that RNA polymerase binds them frequently and rests there for a long time (8).

Not only promoters are marked with peculiarities in the electrostatic properties. Regulator proteins binding sites within the operators also have electrostatic features that correlate with binding ability of the corresponding regulatory proteins. Apparently the sequence text itself shows weaker correlations. Another region that shows a considerable increase in the electrostatic potential value is the attachment site where the initial integration to the host genome DNA occurs.

Figure1: Electrostatic profile of a strong (3) weak (2) and intermediate-strength (1) promoters of the phage lambda. The “up-element” area is highlighted with gray. 1140 The role of electrostatics in the DNA-protein interaction is directly confirmed by the rigorous experiment evidence described in ref. 8. Using the visualization of the individual molecules of RNA polymerase and phage DNA observed is the spatial non-homogeneity of their interactions. The frequency of binding correlates with the absolute value of the electrostatic potential along the DNA molecule.

These data highlight the universal role of electrostatics in the protein interactions with the genome DNA.

References

1. R. V. Polozov, T. R. Dzhelyadin, A. A. Sorokin, N. N. Ivanova, V. S. Sivozhelezov, and S. G. Kamzolova. J Biomol Struct Dyn 16(6), 1135-43 (1999). 2. S. G. Kamzolova, A. A. Sorokin, T. D. Dzhelyadin P. M., Beskaravainy, and A. A. Osypov, J Biomol Struct Dyn 23(3), 341-346 (2005). 3. S. G. Kamzolova, V. S. Sivozhelezov, A. A. Sorokin, T. R Dzhelyadin, N. N. Ivanova, and R. V. Polozov. J Biomol Struct Dyn 18(3), 325-334 (2000). 4. A. A. Sorokin, A. A. Osypov, T. R. Dzhelyadin, P. M. Beskaravainy, and S. G. Kamzolova. J Bioinform Comput Biol 4(2), 455-467 (2006). 5. A. A. Osypov, G. G. Krutinin, and S. G. Kamzolova. J Bioinform Comput Biol 8(3), 413-425 (2010). 6. S. M. West, R. Rohs, R. S. Mann, and B. Honig. J Biomol Struct Dyn 27, 861-866 (2010). 7. D. Wang, N. B. Ulyanov, and V. B. Zhurkin. J Biomol Struct Dyn 27, 843-859 (2010). 8. Y. Harada, T. Funatsu, K. Murakami, Y. Nonoyama, A. Ishihama, and T. Yanagida. Biophys J 76, 709-715 (1999).

The Study of the Nucleic Acid Base-Stacking by 176 Monte Carlo Method: Extended Cluster Approach In the last two years we have seen publications in this Journal on nucleic acids based Victor I. Danilov* on density functional theory and correlated ab initio calculations which examined Vladimir V. Dailidonis sugar-phosphate backbone, base stacking, hydrogen bonding, even ring expansion Tanja van Mourik and metal interactions. (1-3). Here a Metropolis Monte Carlo method based on the extended cluster approach is used to investigate adenine-thymine (A/T), adenine- Herbert A. Früchtl uracil (A/U) and guanine-cytosine (G/C) base associates in a cluster containing 400 and 800 water molecules. It is shown that during the simulation each Watson- Institute of Molecular Biology and Crick base pair is transformed into a more favorable stacked configuration. Genetics, National Academy of Sciences The results obtained allow to observe the whole process of convergence for the of Ukraine, 150 Zabolotnoho St., Kyiv, first time (for more information, visit the Web site http://vd.bitp.kiev.ua). Ukraine 03680 *[email protected]

Table: Energetic characteristics of the transformation of hydrogen-bonded base pairs to the stacked associates and of the base stacking reaction in water cluster (in kcal mol).

Transformation ΔUtot ΔUww ΔUwb Ubb

AT base pair > A/T stacked dimer –15.9 –3.4 –18.8 6.2 AU base pair > A/U stacked dimer –1.4 12.6 –21.8 7.8 GC base pair > G/C stacked dimer –32.3 20.3 –50.4 19.7 A 1 T > A/T stacked dimer –15.8 –13.0 –1.5 –4.3 A 1 U > A/U stacked dimer –8.8 –3.2 –1.9 –3.6 G 1 C > G/C stacked dimer –12.4 –6.7 –3.5 –2.3 It follows from this Table that all stacked associates in the water cluster are ener- 1141 getically more preferable to the corresponding Watson-Crick base pairs. The changes in the interaction energies show that the water–base interaction (ΔUwb) is the determining factor in favoring the stacked species over the base pair in an aqueous cluster. This may be due to the smaller hydrophobic surface of the stacks. The study showed that stacked dimers hydrate better than the hydrogen-bonded associates. The formation of the A-T, A-U and G-C base pairs in the water clusters was found to be energetically unfavorable, primarily due to the destabilizing contribution of the base-water interactions. The data allow us to calculate the transformation energy (ΔUtot) and its various contributions for all the three stacked associates investigated in the water cluster. These results are given in the Table.

As can be seen, the formation of all stacked dimers was found to be favorable, with transformation energies ranging from −8.8 to −15.8 kcal/mol. The preference for the formation of these stacks results mainly from the favourable change in the water-water interaction (Uww) and partly from the base-base interaction (Ubb) during the base association reaction.

In contrast to the Watson-Crick base pairs, the formation of all stacked associates is highly favorable. The water energy change, associated with the structural rearrangement of the water molecules around the bases during their association, contributes most to the stabilization of the stacks. The stacked associates are significantly less stabilized by the base-base interaction in comparison with the H-bonded base pairs. Yet to a lesser extent this applies to the water-base interactions. Thus, the water– water interaction is one of the main factors promoting stacked dimer formation, and the data are a direct confirmation of the crucial role of the water-water interaction in base stacking reported earlier in Ref. (4-6).

References

1. D. Vasilescu, M. Adrian-Scotto, A. Fadiel, and A. Hamza. J Biomol Struct Dyn 27, 465-476 (2010). 2. P. Sharma, S. Sharma, A. Mitra, and H. Singh. 19. J Biomol Struct Dyn 27, 65-81 (2009). 3. G. V. Palamarchuk, O. V. Shishkin, L. Gorb, and J. Leszczynski. J Biomol Struct Dyn 26, 653-661 (2009). 4. V. I. Danilov, I. S. Tolokh, Nature of the stacking of nucleic acid bases in water: a Monte Carlo simulation. J Biomol Struct Dyn, 2, 119-130 (1984). 5. V. I. Danilov and T. van Mourik. Molecular Physics 106, 1487-1494 (2008). 6. V. I. Danilov, V. V. Dailidonis, T. van Mourik, and H. Fruchtl. (in preparation). 1142 Coarse-Grained Model of Nucleic Acids

Atomistic simulations of nucleic acids are prohibitively expensive and, consequently, reduced models of these compounds are of great interest in the field. Most coarse-grained models developed so far implemented harmonic (1, 2) or Go-like 177 potentials (3). Here we present physics-based coarse-grained model of nucleic acids based on our recently developed method of coarse-graining (4). The model is Maciej Maciejczyk1* built of six types of rigid bodies connected by virtual bonds. Bases are represented Aleksandar Spasic2 by several (three to five) centers of van der Waals and electrostatic interactions Adam Liwo3 (dipolar beads), deoyribose is approximated by single van der Waals interaction center (neutral bead) and phosphate group is represented by charged van der Waals Harold A. Scheraga4 sphere (charged bead). Recently developed method of coarse-graining was used for parametrization of two-body long-range interactions between coarse-grained objects (4). Bonded part of interaction energy was determined by fitting analytical 1Department of Physics and Biophysics expression to potentials of mean force computed for the model system. Umbrella University of Warmia and Mazury sampling and WHAM methods were applied to the model three-nucleotide water- Olsztyn 10718, Poland solvated system. Rigid-body equations of motion were integrated with simplectic rotation matrix constraint method (5). Good numerical stability of microcanonical 2Department of Biochemistry and simulation was achieved for timesteps up to 25 fs for the model DNA duplex. Biophysics, University of Rochester Medical Center, Rochester, References NY 14642, USA 1. W. R. Rudnicki, G. Bakalarski, and B. Lesyng. J Biomol Struct Dyn 17, 1097-1108 (2000). 2. M. Maciejczyk, W. R. Rudnicki, and B. Lesyng. J Biomol Struct Dyn 17, 1109-1116 (2000). 3 Faculty of Chemistry University of 3. E. J. Sambirski, V. Oritz, and J. J. de Pablo. J Phys: Condens Matter, 21, 034105-034118 Gdan´sk, Gdansk 80216, Poland (2009). 4. M. Maciejczyk, A. Spasic, A. Liwo, and H. A. Scheraga. J Comp Chem 31, 1644-1655 (2010). 4Baker Laboratory of Chemistry and 5. A. Kol, B. B. Laird, and B. J. Leimkuhler. J Chem Phys 107, 2580-2588 (1997). Chemical Biology, Cornell University, Ithaca, NY 14853, USA *[email protected]

Statistical Thermodynamics of DNA Sequences Based 178 on a Two State Model for the Base Pair Steps 1,2 A statistical thermodynamic framework is constructed for investigating the stability Garima Khandelwal of DNA sequences and their potential for interaction with ligands and proteins, on Rebecca A. Lee2,4 the lines of structure based thermodynamics of Hilser and coworkers (1). Each base B. Jayaram1,2,3 pair step (the dinucletotide) is considered to be either closed (as in the intact double 4 helical DNA where in the Watson-Crick pairing and inter-strand and intra-strand David L. Beveridge stacking is intact; or open ( as in the locally molten state in which the Watson- Crick pairing and inter-strand stacking is completely lost and intra-strand stacking 1Department of Chemistry is partially disrupted). A sequence of n bases is characterized by n-1 base pair steps, leading to a total of 2(n-1) microstates which can be enumerated explicitly. The bp 2Supercomputing Facility for step free energies are adapted from experiment (2). This facilitates construction of Bioinformatics and Computational Biology the partition function and development of thermodynamic parameters as statistical 3School of Biological Sciences, mechanical ensemble averages over all the microstates. As an internal consistency check, the computed average free energies are plotted against experimental melting Indian Institute of Technology Delhi, temperatures of 44 oligonucleotide sequences all at ~1M salt concentration, which Hauz Khas, New Delhi-110016, India gives a correlation coefficient of 0.89. The methodology has the potential to address 4Department of Chemistry and Molecular questions concerning, sequence and stability, cooperativity in DNA-ligand/protein binding etc. Biophysics Program, Wesleyan University, Middletown, CT-06459, USA References [email protected] 1. V. J. Hilser, B. Garca-Moreno E., T. G. Oas, G. Kapp, and S. T. Whitten. Chemical Reviews [email protected] 106(5), 1545-1558 (2006). 2. J. SanataLucia Jr. PNA, 95, 1460-1465 (1998). [email protected] 3. G. Khandelwal, R. A. Lee, B. Jayaram, and D. L. Beveridge. (Manuscript in Preparation). [email protected] In Silico Analysis of Structural Properties of Fungal 1143 DNA Sequences and Promoter Prediction

Promoters play a central role in transcription initiation and gene regulation, so it is necessary for these DNA regions to be differentiated from non-promoter sequences. Sequence motif based computational methods have not been able to identify 179 these regions with high degree of sensitivity and precision. On the other hand Manju Bansal* several experimental and computational studies have shown that promoter sequences possess some special properties, which are common across all organisms. In Venkata Rajesh Yella the current in silico study, we examined genomic regions upstream of genes of S. cerevisiae with respect to their transcription start sites as well as the upstream Molecular Biophysics Unit, regions in all 7 yeast species with respect to their translation start sites, to see the differences in their predicted structural features (stability, bendability and Indian Institute of Science, nucleosome positioning preference, curvature, 2, 3). The core promoter regions Bangalore 560012, India show different structural properties like lower stability, lpwer bendability and *[email protected] slightly higher curvature compared to other neighbouring regions (1). Moreover we also observed that TATA-containing promoters are less stable, more flexible and more curved compared to TATA-less promoters, but both have similar nucleosome positioning preferences. The variability of structural properties of TATA-less and TATA-containing promoters may be due to differences in their role in regulation of gene expression. Detailed analysis of these promoters, comparison with results for prokaryotic promoters (4, 5) as well as quantitative prediction ability of our in-house software ‘PromPredict’ will be presented.

References

1. A. Kanhere and M. Bansal. Nucleic Acids Res 33, 3165-3175 (2005). 2. F. Cui and V. B. Zhurkin. J Biomol Struct Dyn 27, 821-841 (2010). 3. D. Wang, N. B. Ulyanov, and V. B. Zhurkin. J Biomol Struct Dyn 27, 843-859 (2010). 4. V. Rangannan and M. Bansal. Mol BioSyst 5, 1758-1769 (2009). 5. V. Rangannan and M. Bansal. Bioinformatics 26, 3043-3050 (2010).

Temperature-Induced Unfolding Thermodynamics of DNA Hairpins Containing Internal Loops and Their Targeting with Complementary Strands 180 Luis A. Marky The main focus of our research is to further our understanding of the physico- Iztok Prislan chemical properties of nucleic acid structures. In particular, to investigate the melting behavior of unusual DNA structures, by determining their complete unfolding Cynthia Lee thermodynamic profiles. In this work, we use a set of DNA hairpins as a model Hui-Ting Lee to mimic a common motif present in the secondary structures of mRNA, i.e., a stem-loop motif with an internal loop in the stem. Specifically, we used a combination of UV spectroscopy and differential scanning calorimetry (DSC) melting Department of Pharmaceutical Sciences, techniques to determine the unfolding thermodynamics of a set of hairpins with University of Nebraska Medical sequence: d(GCGCT GTAACT GTTACT GCGC, where T corresponds to inter- n 5 n n Center, 986025 Nebraska Medical nal loops with n = 1, 3 or 5 and “T5” is an end loop of 5 thymines, as shown in the figure. The UV melting curves of each hairpin show monophasic transition with Center, Omaha, NE 68198-6025

TMs that are independent of strand concentration, confirming their intramolecular formation. Analysis of the DSC profiles indicates that the unfolding of each hairpin results from the typical compensation of a unfavorable enthalpy (breaking of base-pair stacks) and favorable entropy contributions (release of ion and water molecules). The increase in the size of the internal loop from 2 to 10 thymines yielded: a) lower TMs and similar enthalpy contributions; b) lower heat capacity values that correlated with the lower releases of structural water molecules; and c) higher ion releases. Furthermore, we used isothermal titration calorimetry and DSC to 1144 investigate directly and indirectly, respectively, the thermodynamics of the reaction of each hairpin with their partially complementary strands. The results showed that all three targeting reactions yielded favorable free energy contributions that were enthalpy driven. Overall, this approach works because of the favorable heat contributions resulting from the formation of base-pair stacks involving the unpaired bases of the loops. Supported by Grant MCB-0315746 from the NSF.

DNA Recognition in RNA Polymerase III Transcription

Eukaryotic transcription requires the coordinated activities of enhancers that rec- 181 ognize specific DNA target sites, co-activators and general transcription factors Nicholas Taylor to recruit RNA polymerases to their transcription start sites (1). Among the three Gary Male eukaryotic RNA polymerases, recruitment of RNA polymerase II is by far the most complex process requiring a large number of different factors. In comparison, Christoph W. Müller* recruitment of RNA polymerase I and III is simpler and therefore more amenable for detailed structure-function analyses. RNA polymerase I and III are responsible for Structural and Computational Biology producing non-coding RNAs such as pre-RNA and tRNA. Together, they contribute up to 80% to the total transcriptional activity in growing cells and need to be tightly Unit, European Molecular Biology regulated. In the last years there has been increasing awareness that mis-regulation Laboratory (EMBL), 69117 Heidelberg, of RNA polymerase I and III is associated with different types of cancer (2). Germany Recruitment of RNA polymerase III to the transcription start site requires binding of *[email protected] the general transcription factors TFIIIB and TFIIIC. TFIIIC is a large DNA-binding complex with a total molecular weight of 0.6 MDa composed of six polypeptides. TFIIIC can be subdivided into two subcomplexes, that initiate RNA polymerase III transcription by binding to two internal promoter sites in tRNA genes named A-and B-box. We are using a combination of X-ray crystallography, electron microscopy and biochemistry to gain insights into the structural organization of the RNA polymerase III enzyme and its general transcription factors (3-5). During the last years our group has determined crystal structures corresponding to ~2/3 of the entire TFIIIC complex and obtained a low-resolution EM reconstruction of TFIIIC that serves as a starting point to assemble the entire complex. We will present insights into DNA recognition by the RNA polymerase III-specific general transcription factor complex TFIIIC resulting from this studies.

References

1. R. D. Kornberg. Proc Natl Acad Sci 104, 12955-61 (2007). 2. R. J. White. Trends Genet 27, 622-629 (2008). 3. A. Mylona, C. Fernandez-Tornero, P. Legrand, M. Haupt, et al. Mol Cell 24, 221-232 (2006). 4. C. Fernandez-Tornero, B. Bottcher, M. Riva, C. Carles, et al. Mol Cell 25, 813-23 (2007). 5. C. Fernandez-Tornero, B. Bottcher, U. J. Rashid, et al. EMBO J. 29, 3762-72 (2010).

Protein Sliding and Jumping Along DNA

Interactions between proteins and nucleic acids are ubiquitous and central to the 182 life of cells. The remarkable efficiency and specificity of protein-DNA recogni- Yaakov (Koby) Levy tion presents a major theoretical puzzle given the size of the genome, the largea number of molecular species in vivo at a given time, and the crowded environment they inhabit. Our research is motivated at quantitatively advancing our understand- Department of Structural Biology, ing of the kinetics and mechanisms of protein-DNA recognition, the molecular Weizmann Institute of Science, Israel and physical principles of fast association, and protein recruitment by DNA. For the first time, we have visualized protein sliding along DNA where the protein binds DNA nonspecifically and performs a helical motion when it is placed in the major groove. Using coarse-grained models we found that the spiral motion along the sugar-phosphate rail is typical to various DNA-binding motifs. This stochastic 1145 dynamics that is governed by electrostatic forces has similar structural features to the specific binding mode of the protein with the DNA. In our study, we address the question of the linkage between the molecular architecture of DNA-binding proteins and the search mechanism. We have explored the interplay between the molecular characteristics of the proteins (e.g., DNA recognition motifs, degree of flexibility, and oligomeric states) and the nature of sliding, intersegment transfer events and the overall efficiency of the DNA search.

Indirect Readout by the P22 Repressor Protein: How Charge and Solvent Organization Aid Binding Site Discrimination 183 Lydia-Ann Harris To form a lysogen, the repressor of bacteriophage P22 (P22R) must bind and dis- Gerald Koudelka criminate between its six DNA binding sites on the P22 chromosome. Comparison of the six naturally occurring binding site sequences shows that the sequences of the symmetrically-arrayed outermost base pairs in these sites are highly conserved, but Department of Biological Sciences, the sequence of the innermost bases are not (Figure 1). Crystallographic studies show University at Buffalo (SUNY), the conserved bases are directly contacted by bound protein, but the non-conserved central bases are not (1). Nonetheless through a sequence recognition strategy called 109 Cooke Hall, Buffalo, New York “indirect readout”, the affinity of P22R for DNA varies with the non-contacted base 14260 sequence (2). We wish to understand the mechanism of indirect readout. [email protected] P22R DNA binding causes the non-contacted region to assume a B’-DNA configura- [email protected] tion (1). The minor groove of B’-DNA is very narrow and contains a spine of highly ordered solvent molecules (1). We hypothesize that indirect readout depends on P22R’s ability to either sense and/or impose B’ structure on the bases in the non-contacted of its binding site. Thus the ease with which the non-contacted bases can assume B’ configuration by P22R determines the protein’s affinity for the binding site.

We showed that P22R binding sites lacking a minor groove N2-NH2 group on the base pairs at the non-contacted positions bind P22R ~12-fold better than those that do not contain this group at these positions. This finding identifies the N2-NH2 group as the mediator of indirect readout by P22R. We also found that changing either E44 or E48, which are directly juxtaposed to the DNA phosphate backbone near the non-contacted bases (1) (Figure 2), to uncharged residues eliminates the ability of P22R to recognize non-contacted base sequence. This finding suggests that these residues function to force the P22R-bound DNA into the B’ configuration. An N46A mutation completely blocks P22R’s ability to bind to sites bearing sequences in the non-contacted region, which cannot normally assume the B’ configuration. This finding suggests that N46 is responsible for inducing and/or stabilizing B’ conformation in the non-contacted bases in P22R-DNA complexes.

Figure 1: DNA sequences of naturally occuring operators in the P22 chromosome. The contacted and conserved bases are boxed and in bold. The central non-contacted bases are positions 8-11. 1146

Figure 2: Positions of E44 and E48 relative to the DNA backbone. The P22R protein is above and the DNA is below. E44 and E48 are spacefilled (yellow) the nearest phosphates are spacefilled (cyan) (2). We have found P22R sensitivity to non-contacted base sequence, directly affects repressor’s ability to sense sequence changes in the contacted bases. We provide evidence that this effect may be due to the spine of hydration which may run along the minor groove from one end of the binding site to the next. Together these results provide a fascinating picture of how P22R uses indirect readout in binding site discrimination.

References

1. D. Watkins. Biochemistry 47, 2325-2338 (2008). 2. L. Wu, A. Vertino, and G. B. Koudelka. J Biol Chem 267, 9134-9135 (1992).

A Kinetic Code for SRY-Dependent Transcriptional 184 Activation Based on DNA Intercalation The HMG (High-Mobility-Group) box defines a superfamily of DNA-bending Joseph Racca* proteins remarkable for partial DNA intercalation by “cantilever” side chains. Here, Yen-Shan Chen1 we describe the role of the cantilever residue of SRY, the human male-determining Nelson Phillips1 factor encoded by the Y chromosome. Mutations at this and related sites abutting 1 the sharp DNA bend cause gonadal dysgenesis leading to chromosome sex rever- Agnes Jansco-Radek sal. A yeast-one-hybrid model was designed to enable rapid and efficient screen- Michael Weiss1 ing of mutational libraries in relation to the database of clinical substitutions and Elisha Haas2* evolutionary relationships among HMG boxes. Our results demonstrate (a) that the only allowed cantilevers are those observed among mammalian SRY and related SOX sequences (Ile, Met, Leu, and Phe); (b) that mutations associated with de novo 1Department of Biochemistry, sex reversal in vivo markedly impair DNA binding; and (c) that variant cantilevers Case Western Reserve University, are associated with a range of kinetic on- and off-rates. To correlate the biophysical properties of the bent protein-DNA complex with gene-regulatory activity, Cleveland, OH 44106 a transfection assay was developed in an immortalized rodent embryogenic cell 2Faculty of Life Sciences, Bar Ilan line derived from the differentiating gonadal ridge at the time of onset of SRY University, Ramat Gan, Israel 52900 expression. A biological read-out was provided by real-time quantitative PCR analysis of the endogenous Sox9 gene, a key downstream target of SRY in the *[email protected] male transcriptional program. Remarkably, transcriptional potency was observed to correlate with the kinetic life time of the bent DNA complex and not its thermodynamic stability. Whereas a variant Val cantilever (too short to fully insert within the DNA double helix) suffices in vitro to direct formation of a well- organized but short-lived SRY-DNA complex of near-native DNA bend angle, no gene activation was observed in the rodent cell culture model. We propose that the active cantilever side chains act as a kinetic anchor to provide a long-lived DNA bend, in turn enabling formation of a functional male-specific transcriptional pre-initiation complex. The Mode of DNA Recognition and Dimerization 1147 Specificity of MITF Revealed by Three Crystal Structures

The Microphthalmia-associated Transcription Factor (MITF) regulates the expression of pigment-cell specific genes in melanocytes, the mature pigment producing 185 cells of the skin and hair follicles. In addition, MITF function is of major interest in Vivian Pogenberg1* the investigation of the mechanisms leading to melanoma. Viktor Deineko1 1 MITF belongs to the superfamily of basic Helix-Loop-Helix leucine zipper tran- Morlin Milewski scription factors (b-HLH-Zip). Like other b-HLH-Zip factors, MITF can bind a Alexander Schepsky2 subset of the canonical palindromic E-box sequence (CANNTG) as well as related Eirikur Steingrimsson2 asymmetric motives like M-box (TCATNTG); nevertheless the exact mechanism in 1 which MITF recognizes the correct promoters of target genes is not yet fully under- Matthias Wilmanns stood. Within the b-HLH-Zip family, MITF associates with the Tfe factors, but no heterodimeric complex containing MITF and the related Myc, MAX or USF-1 have 1EMBL-Hamburg, c/o DESY, been observed, raising the question how this discrimination is achieved. Notkestrasse 85, 22603 Hamburg, We determined the crystal structure of MITF in its apo form and of two different Germany complexes of MITF with DNA containing two different target motives (E-box 2Department of Biochemistry and and M-box). We pursued the study measuring interactions between these DNA motives and several MITF mutants with known phenotype in mice, using different Molecular Biology, Faculty of Medecine, technique such as using Isothermal Titration Calorimetry, Fluorescence Anisotropy University of Iceland, 101 Reykjavik, or EMSA. Comparing structural, biophysical and biological data, this study reveals Iceland a particular mechanism of DNA recognition by MITF and how MITF discriminates between the E and M boxes. In addition, our data demonstrate an unusual mode of *[email protected] dimerization that might explain how MITF select its heterodimerization partners.

A Convenient Fluorescence Assay of Polyamide-DNA Interactions 186 Polyamides (PA) are distamycin-type ligands of DNA that bind the minor groove and are capable of sequence selective recognition (1). This capability provides Cynthia M. Dupureur* a viable route to their development as therapeutics. PAs have limited intrinsic James K. Bashkin fluorescence behavior, making this signal poorly suited for binding assays, and Karl Aston the introduction of a dye to this structure requires additional synthetic steps and can change uptake for cell-based assays (2). The most commonly performed Kevin J. Koeller assays of DNA binding by PAs involve complex techniques like calorimetry (3), Kimberly R. Gaston surface plasmon resonance (3), and footprinting (4). Presented here is a simple and Gaofei He convenient fluorescence assay for polyamide-DNA binding. PAs are titrated into a sample of a hairpin DNA featuring a TAMRA dye attached to an internal dU a few nt away from the PA binding site. In a study of 12 polyamides, PA binding Department of Chemistry and the Center leads to a steady, reproducible decrease in fluorescence intensity that can be used for Nanoscience, University of Missouri to generate binding isotherms. The assays works equally well with both short St. Louis, St. Louis, MO 63121, USA (6-8 ring) and long (14 ring) polyamides, and Kd values ranging from 0.5 to at least 300 nM (depending on PA composition and target sequence), were readily *[email protected] obtained using a simple monochrometer configuration. Stronger Kd values were confirmed with a PCR fragment and quantitative footprinting analysis. Competi- tion assays indicate that the effects of the dye are typically negligible when the dye-labeled dU residue is outside the binding site. When the dye is placed within the predicted binding site, binding experiments show disturbances in the classical binding trend. This effect provides a means to confirm the binding site in DNAs with more than one potential binding site. 1148 This research is supported by NIH R01 AI083803-02.

References

1. P. B. Dervan and B. S. Edelson. Curr Opin Struct Biol 13, 284-299 (2003). 2. K. S. Crowley, D. P. Phillion, S. S. Woodard, B. A. Schweitzer, M. Singh, H. Shabany, B. Burnette, P. Hippenmeyer, M. Heitmeier, and J. K. Bashkin. Biorg Med Chem Lett 13, 1565-1570 (2003). 3. H. Mackay, T. Brown, P. B. Uthe, L. Westrate, A. Sielaff, J. Jones, J. P. Lajiness, J. Kluza, C. O’Hare, B. Nguyen, Z. Davis, C. Bruce, W. D. Wilson, J. A. Hartley, and M. Lee. Bioorg Med Chem 16, 9145-9153 (2008). 4. J. W Trauger and P. B. Dervan. Methods Enzymol 340, 450-466 (2001).

A Mechanism to Understand the Dynamics 187 of Tryptophan Repressor Trp repressor was an early paradigm for understanding DNA binding, but presented Balasubramanian Harish many still-unsolved mysteries, including the non-cooperative binding of L-trypto- Jannette Carey phan, the presence of a buried water layer at the DNA interface, and extensive local dynamics in the DNA-binding domains. The crystal structure of a temperature-sensitive mutant of E. coli TrpR now suggests a mechanism for the dynamics Chemistry Department, Princeton of the wildtype protein. One subunit of the mutant crystal structure shows an exten- University, Princeton, sive rearrangement in the DNA-binding region, whereas the other subunit is in the NJ 08544-1009, USA wildtype conformation. Comparison with isomorphous wildtype crystals reveals that the distorted conformation is not an artifact of crystallization. Reinvestigation [email protected] of NMR data for the mutant shows that this conformation can explain NOEs that [email protected] are not accounted for by a wildtype-like structure. A survey of the extensive NMR data on the wildtype protein reveals that many puzzling features in the dynamics of the protein could also be explained by the presence of this alternative conformation. This hypothesis is presently being tested by NMR and molecular dynamics simulations.

Physicochemical Features of Protein-DNA Interactions and the Identification of Interface 188 Region in DNA-Binding Proteins Pinak Chakrabarti1,2* Sucharita Dey1 The use of physicochemical features of protein-protein and protein-DNA interfaces can act as useful constraints in docking studies (1-3). Amino acid residues important 2 Arumay Pal for structure and function are evolutionary conserved, and tend to cluster together Mainak Guharoy2 at the binding site (4, 5). Even though recently, there is substantial progress on our understanding of the interactions between DNA and proteins (6-8), still we lack quantitative or semi quantitative description of the surfaces involved. Following 1Bioinformatics Centre the protocol used in protein-protein interfaces (5), here we analyze the clustering 2Department of Biochemistry, Bose of conserved residues in protein-DNA interfaces and show how this and other features, such as the propensity of residues to occur in the interfaces, can be used for Institute, P-1/12 CIT Scheme VIIM, the discrimination of the real interface from other random surface patches. Kolkata 700054, India *[email protected] In 129 non-redundant interfaces from 126 protein-DNA complexes, 81% have the conserved positions clustered within the overall interface region – indicated by rho *[email protected] (ratio of parameters representing the degree of clustering of conserved residues relative to the overall interface) being greater than 1. The use of rho can identify the interface (with rank 1) from other randomly generated surface patches in ~46% of the cases. The incorporation of the Euclidean distance of the composition of a surface patch from the average value in all protein-DNA interfaces improves the efficiency by another 6%. While the effectiveness of the clustering in the dis- 1149 crimination of the real interface is rather mediocre, the use of Rp (9) (the number of a residue type in a patch multiplied by its propensity to occur in the interface, summed over all the residues in the patch) can identify 81% of the interfaces with rank 1. Another parameter Dp, the number of potential hydrogen bond donors in the patch gives an accuracy of ~65%.

References

1. S. Ahmad, M. M. Gromiha, and A. Sarai. Bioinformatics 20, 477-486 (2004). 2. Y. Mandel-Gutfrend and H. Margalit. Nucleic Acids Res 26, 2306-2312 (1998). 3. S. Biswas, M. Guharoy, and P. Chakrabarti. Proteins 74, 643-654 (2009). 4. S. Ahmad, O. Keskin, A. Sarai, and R. Nussinov. Nucleic Acids Res 36, 5922-5932 (2008). 5. M. Guharoy and P. Chakrabarti. BMC Bioinformatics 11, 286 (2010). 6. S. M. West, R. Rohs, R. S. Mann, and B. Honig. J Biomol Struct Dyn 27, 861-866 (2010). 7. D. Wang, N. B. Ulyanov, and V. B. Zhurkin. J Biomol Struct Dyn 27, 843-859 (2010). 8. C. Carra and F. A. Cucinotta. J Biomol Struct Dyn 27, 407-427 (2010). 9. R. P. Bahadur, P. Chakrabarti, F. Rodier, and J. Janin. J Mol Biol 336, 943-955 (2004).

Getting a Firm Grip on DNA: Understanding DNA Recognition at the Finger-finger Interface of Zinc Finger Proteins 189 Ankit Gupta1,2 Zinc finger nucleases hold tremendous potential for site-specifically Amy Rayla1 editing genomes in a variety of organisms (1). However, their util- Ryan G. Christiansen3 ity is predicated on the ability to efficiently create sequence-specific C2H2 Zinc Finger Proteins (ZFPs) for a wide variety of target sequences. Each zinc finger Nathan D. Lawson1 module typically binds to a 3 bp core DNA element (DNA triplet). Zinc finger Gary D. Stormo3 modules have been characterized that can specify most of the sixty-four possible 1,2, DNA triplets. These modules can be assembled into multi-finger zinc finger pro- Scot A. Wolfe * teins (modular assembly) that bind extended target sites (9-12 bp), where typically three to four zinc fingers are employed for efficient DNA recognition. However, 1Program in Gene Function and modularly-assembled ZFPs often show poor specificity presumably due to incom- patible specificity determinants at the finger-finger interface (2). To understand the Expression influence of these interactions on DNA recognition, we employed bacterial one- 2Department of Biochemistry and hybrid selections (3, 4) to identify groups of amino acid residues at the interface of Molecular Pharmacology, University two-finger modules that specify all sixteen 2 bp interfaces between the two DNA triplets. In total, we could identify two-finger modules that specify >90% of these of Massachusetts Medical School, targets. Further analysis of the selected modules suggests the presence of com- Worcester, MA 01605, USA plex interactions at the finger-finger interface that contribute to context-dependent 3Department of Genetics, Washington effects on specificity. These selected finger pairs are functional in vivo, as zinc finger nucleases employing these modules generate targeted lesions in zebrafish. University School of Medicine, Ultimately, understanding finger-finger interactions will allow the rational design St. Louis, MO 63108, USA of multi-finger ZFPs for their use as artificial proteins and aid the assignment of *[email protected] specificities for naturally-occurring zinc finger proteins.

References

1. F. D. Urnov, E. J. Rebar, M. C. Holmes, H. S. Zhang, and P. D. Gregory. Nat Rev Genet 11, 636-646 (2010). 2. C. L. Ramirez, J. E. Foley, D. A. Wright, F. Muller-Lerch, S. H. Rahman, T. I. Cornu, R. J. Winfrey, J. D. Sander, F. Fu, J. A. Townsend, T. Cathomen, D. F. Voytas, and J. K. Joung. Nature methods 5, 374-375 (2008). 3. X. Meng, M. B. Noyes, L. J. Zhu, N. D. Lawson, and S. A. Wolfe. Nat Biotechnol 26, 695-701 (2008). 4. M. B. Noyes, X. Meng, A. Wakabayashi, S. Sinha, M. H. Brodsky, and S. A. Wolfe. Nucleic Acids Res 36, 2547-2560 (2008). 1150 Intrinsic DNA Shape of Fis-DNA Binding Sites Determines Binding Affinity

The bacterial nucleoid-associated protein Fis (factor for inversion stimulation) binds to DNA of various DNA nucleotide sequences at binding affinities varying 190 in three orders of magnitude. Johnson and coworkers recently reported 11 crystal Tianyin Zhou structures of the Fis protein bound to high- and low-affinity sites (1). A common Ana Carolina Dantas Machado feature of these Fis-DNA complexes is the narrow minor groove in the central region of the DNA targets. Without the input of any structural data derived from Remo Rohs* experimental studies, we predicted the shape of the naked Fis-DNA binding sites based on our Monte Carlo algorithm (2). For this purpose, we generated an ideal Molecular and Computational Biology, B-DNA double helix of a given sequence without any structural identity of dinucleotide steps and sampled the three-dimensional structure of high- and low-affinity Department of Biological Sciences, Fis-DNA binding sites. In agreement with the hypothesis presented by Johnson University of Southern California, 1050 and coworkers (1), we find that the shape of the naked binding site with the highest Childs Way, RRI 404C, Los Angeles, Fis-DNA binding affinity already assumes a shape with a minor groove narrow- ing in its central region similar to the one observed in the crystal structure of the CA 90089, USA complex. In contrast, the Fis-DNA site with the lowest binding affinity exhibits in *[email protected] its unbound state a shape that is essentially the one of average B-DNA. Although the Fis protein binds DNA largely non-specifically, the finding that DNA shape determines binding affinity indicates a similar mechanism as the one that we previously described for the sequence-specific DNA recognition by the papillomavirus E2 protein. The E2 protein binds to DNA that is intrinsically bent as observed in the protein-DNA complex with high affinity and to DNA that is essentially straight with low affinity (2). The data presented for the larger set of Fis-DNA binding sites further validates our structure prediction method and emphasizes the important role of DNA shape in protein-DNA recognition (3, 4). Moreover, protein-DNA binding affinity was shown to be correlated with gene regulatory control (5), thussuggesting that structural data that explains binding affinity leads to a better understanding of protein-DNA recognition, both for largely non-specific DNA interactions with architectural proteins and highly specific DNA readout by transcription factors.

References

1. S. Stella, D. Cascio, and R. C. Johnson. Genes Dev 24, 814-26 2. R. Rohs, H. Sklenar, and Z. Shakked. Structure 13, 1499-509 (2005). 3. R. Rohs, S. M. West, A. Sosinsky, P. Liu, R. S. Mann, and B. Honig. Nature 461, 1248-53 (2009). 4. R. Rohs, X. Jin, S. M. West, R. Joshi, B. Honig, and R. S. Mann. Annu Rev Biochem 79, 233-69 (2010). 5. S. V. Nuzhdin, A. Rychkova, and M. W. Hahn. Trends Genet 26, 51-3 (2010). Structural Studies of Nucleic Acid-Binding Proteins 1151 Involved in Regulating Cell Differentiation and Pluripotency

Transcription factors play a central role in regulating cell differentiation and de-differentiation. The use of “cocktails” of transcription factors to promote the 191 reprogramming of adult fibroblasts into induced pluripotent stem cells (iPS) has Udo Heinemann* generated tremendous interest in biology and medicine. The originally reported sets Florian Mayr of iPS generating factors contained Oct4, Sox2, Klf4 and c-Myc (1) or Oct4, Sox2, Nanog and Lin28 (2). Here we report on structural and biochemical studies of two Anja Schuetz of these proteins, Klf4 and Lin28. Macromolecular Structure and Klf4 (Krueppel-like factor 4) is a zinc-finger transcription factor required for the maturation of epithelial tissues. Crystal structure analyses of two different zinc- Interaction Group, Max-Delbrück Center finger fragments of Klf4 reveal that the two C-terminal C2H2 zinc-finger motifs for Molecular Medicine, of Klf4 are required for DNA site specificity and the induction of macrophage Robert-Rössle-Str. 10, 13125 Berlin, differentiation (3). The N-terminal zinc finger, conversely, inhibits the otherwise cryptic self-renewal capacity of Klf4. A Klf4 zinc-finger domain mutant induces Germany self-renewal and block of cell maturation. *[email protected]

Lin28 is a highly conserved RNA-binding protein and was described to modulate the processing of let-7 microRNA precursors (4). The small protein contains a cold- shock domain (CSD) and a tandem array of retroviral-type CCHC zinc fingers. Both protein motifs are presumably involved in RNA binding. Crystal structure analysis reveals that the Lin28 CSD resembles the bacterial cold shock proteins (5-8). The presence of conserved nucleotide-binding subsites of the surface of Lin28 CSD suggests a common mode of DNA or RNA single-strand binding of Lin28 and bacterial cold shock proteins (9, 10).

References

1. K. Takahashi, K. Tanabe, M. Ohnuki, M. Narita, T. Ichikasa, K. Tomoda, and S. Yamanaka. Cell 131, 861-872 (2007). 2. J. Yu, M. A. Vodyanik, K. Smuga-Otto, J. Antosiewicz-Bourget, J. L. Frane, S. Tian, J. Nie, G. A. Jonsdottir, V. Ruotti, R. Stewart, I. I. Slukvin, and J. A. Thomson. Science 318, 1917-1920 (2007). 3. A. Schuetz, D. Nana, C. Rose, G. Zocher, M. Milanovic, J. Koenigsmann, R. Blasig, U. Heinemann, and D. Carstanjen. Cell Mol Life Sci DOI 10.1007/s00018-010-0618-x (2011). 4. S. R. Viswanathan and G. O. Daley Cell 140, 445-449 (2010). 5. H. Schindelin, M. Herrler, G. Willimsky, M. A. Marahiel, and U. Heinemann. Proteins: Struct Funct Genet 14, 120-124 (1992). 6. H. Schindelin, M. A. Marahiel, and U. Heinemann. Nature 364, 164-168 (1993). 7. H. Schindelin, W. Jiang, M. Inouye, and U. Heinemann. Proc Natl Acad Sci USA 91, 5119-5123 (1994). 8. U. Mueller, D. Perl, F. X. Schmid, and U. Heinemann. J Mol Biol 297, 975-988 (2000). 9. K. E. A. Max, M. Zeeb, R. Bienert, J. Balbach, and Heinemann. J Mol Biol 360, 702-714 (2006). 10. K. E. A. Max, M. Zeeb, R. Bienert, J. Balbach, and U. Heinemann. FEBS J 274, 1265-1279 (2007). 1152 The Role of DNA Shape on Protein-DNA Binding Affinity and Specificity

Gene regulation requires highly specific interactions between proteins and their DNA binding sites. This high level of binding specificity in protein-DNA readout is 192 achieved through the recognition of both linear sequence (base readout) and three- Remo Rohs dimensional structure (shape readout) (1). DNA shape is specifically recognized by a variety of protein families, and we have identified two different ways of modulating DNA shape. One widely observed phenomenon is the variation of the shape Molecular and Computational Biology, of the double helix as a function of nucleotide sequence. This mode of sequence- dependent DNA shape was first found to affect the DNA binding specificity of the Department of Biological Sciences, Hox protein Scr (Sex combs reduced) (2). A general study showed the critical role University of Southern California, 1050 of DNA shape on the binding of other transcription factors to their target sites and Childs Way, RRI 404C, Los Angeles, on the genome packaging through nucleosome formation (3). CA 90089, USA Whereas the findings on the readout of sequence-dependent DNA shape are based [email protected] on the analysis of crystal structures, we now expanded the description of DNA shape as binding affinity and specificity determinant including systems for which no structural data is available. We used our Monte Carlo algorithm (4) for predicting the shape of various DNA binding sites. In one study we relate binding affinity of the architectural protein Fis (factor for inversion stimulation) to the shape of high- and low-affinity Fis-DNA binding sites (see poster abstract by Zhou et al.). In another study the DNA structure predictions of multiple binding sites of the eight Drosophila Hox proteins and their cofactors Extradenticle and Homothorax, as identified in SELEX-seq experiments, reveal intrinsic DNA shape as a selection criterion contributing to binding specificity (see poster abstract by Liu et al.).

In addition to the sequence dependence of DNA shape, we identified a different mode for varying the shape of DNA binding sites. In the case of DNA recognition by the tumor suppressor p53, the base pairing geometry at specific positions of the p53 response element deviates from standard-Watson-Crick geometry (5). Certain base pairs assume Hoogsteen geometry, thus affecting local DNA shape and in turn the interaction with arginine residues that were found to be frequently mutated in human tumors. If applicable to other systems, non-Watson-Crick base pairs would effectively extend the four letter genomic alphabet, demonstrating the importance of considering three-dimensional structure (6).

References

1. R. Rohs, X. Jin, S. M. West, R. Joshi, B. Honig, and R. S. Mann. Annu Rev Biochem 79, 233-69 (2010). 2. R. Joshi, J. M. Passner, R. Rohs, R. Jain, A. Sosinsky, M. A. Crickmore, V. Jacob, A. K. Aggarwal, B. Honig, and R. S. Mann. Cell 131, 530-43 (2007). 3. R. Rohs, S. M. West, A. Sosinsky, P. Liu, R. S. Mann, and B. Honig. Nature 461, 1248-53 (2009). 4. R. Rohs, H. Sklenar, and Z. Shakked. Structure 13, 1499-509 (2005). 5. M. Kitayner, H. Rozenberg, R. Rohs, O. Suad, D. Rabinovich, B. Honig, and Z. Shakked. Nat Struct Mol Biol 17, 423-9 (2010). 6. B. Honig and R. Rohs. Nature 470, 472-3. Exploring the DNA Binding Specificities of 1153 Homeodomain Complexes using SELEX-sequencing

Hox genes encode an evolutionarily conserved set of homeodomain-containing transcriptional regulators that play critical roles in the development of all meta- zoans. Although first discovered in Drosophila because of their role in anterior 193 (A)-posterior (P) axial patterning, these genes are now known to assign morpho- Matthew Slattery1,4,* logical identities along the AP axes in both vertebrates and invertebrates (1). Hox 2,3 proteins typically bind to degenerate AT-rich DNA sequences. This low degree of Todd R. Riley sequence specificityin vitro contrasts with the highly gene-specific regulation Hox Namiko Abe1 proteins carry out in vivo. Complicating the Hox specificity problem is that this Harmen J. Bussemaker2,3 family of proteins, which are encoded by eight Hox paralogs in Drosophila and 1 39 Hox genes in vertebrates, all bind to very similar DNA sequences via identical Richard S. Mann DNA-contacting residues in their homeodomains (2). One way in which Hox proteins achieve a higher degree of DNA binding specificity is to bind cooperatively 1Department of Biochemistry and with cofactors. One such cofactor is a heterodimer composed of Extradenticle (Exd; Pbx in vertebrates) and its binding partner Homothorax (Hth; Meis in vertebrates), Molecular Biophysics, both homeodomain proteins (2). Together, Exd-Hth bind cooperatively with Hox Columbia University, 701 West 168th proteins, allowing them to recognize features of the DNA that cannot be read in the Street, HHSC 1104, New York, absence of these cofactors. NY 10032, USA To explore the DNA binding specificities of the Hox transcription factors and their 2Columbia University, Department cofactor Exd, we have combined the traditional method of SELEX (Systematic of Biological Sciences, 1212 Amsterdam Evolution of Ligands by Exponential Enrichment), which can be used to select DNA binding sites, with next generation sequencing technology (e.g. Illumina Avenue, New York, NY 10027 sequencing). This combination, termed SELEX-seq, allows the identification of 3Columbia University, Center for the complete repertoire of binding sites for any Hox-Exd combination after only a Computational Biology and small number of SELEX rounds. Subtle differences in DNA binding preferences among the Hox-Exd complexes can be clearly observed using this technology. In Bioinformatics, 1130 St Nicholas general Hox-Exd complexes seem to use a modular binding site architecture, in Avenue, New York, NY 10032 which an octomer core sequence determines the DNA signature motif for various 4 Current address: Institute for Genomics Hox-Exd dimers while flanking nucleotides adjust the absolute affinity of a signature motif. Importantly, these motifs can explain certain aspects of in vivo Hox and Systems Biology, University function, and our findings suggest that signature motif selectivity is achieved in part of Chicago, 900 East 57th Street KCBD by Hox-Exd recognition of structural features of the DNA. 10115, Chicago, IL 60637, USA

References *[email protected]

1. T. Iimura and O. Pourquie. Dev Growth Differ 49, 265-275 (2007). 2. R. S. Mann, K. M. Lelli, and R. Joshi. Curr Top Dev Biol 88, 63-101 (2009). 1154 Dimerization with Extradenticle Unlocks Latent DNA Binding Specificities that Discriminate Hox Proteins in Drosophila

Hox proteins constitute a subclass of the large homeodomain family of transcription 194 factors. They play a crucial role in body plan and tissue specification throughout Todd R. Riley1,2,* the animal kingdom (1). In the transcriptional network of the cell, each Hox protein Matthew Slattery3,4 targets a distinct set of genes. Little is currently understood about the differences 3 in DNA binding specificity that underlie these functional differences. In particular, Namiko Abe Hox monomers have been shown to bind DNA with similar sequence preferences Harmen J. Bussemaker1,2 (2, 3). We developed a method (“SELEX-seq”) that couples in vitro selection of Richard S. Mann3 pools of random DNA with massively parallel sequencing. In combination with a novel computational method for analyzing the data, based on a biophysical model, it allows us to quantify relative binding affinities at unprecedented resolution for all 1Columbia University, Department of 12-mer sequences. Application of SELEX-seq to dimers of each of the eight Droso- Biological Sciences, 1212 Amsterdam phila Hox proteins with the co-factor Extradenticle (Exd) revealed the DNA binding specificities for this family of homeodomain proteins. Only in the context of Avenue, New York, NY 10027 the Exd-Hox complex is the identity of the Hox protein manifested, through major 2Columbia University, Center for differences in how base identities in the core of the binding site are recognized. Our Computational Biology and analysis reveals that most Hox-Exd dimers have a unique DNA recognition signature. These differences in binding specificity in vitro may go a long way towards Bioinformatics, 1130 St Nicholas explaining the functional differences between the Hox proteins observed in vivo. Avenue, New York, NY 10032 3Department of Biochemistry and We present a novel method, based on a biophysical model of the SELEX procedure, which is capable of estimating binding affinities between a protein or protein Molecular Biophysics, Columbia complex of interest and all oligonucleotide sequences of a given length with great University, 701 West 168th Street HHSC accuracy. Relative affinities are obtained by comparing the sequence composition 1104, New York, NY 10032, USA of later rounds to that of the initial round. In this comparison our method takes into 4 account the significant sequence biases in the initial pool of dsDNA molecules. Current address: Institute for Genomics While the higher sequence counts associated with the later rounds of SELEX may and Systems Biology, University of be preferable in terms of statistical accuracy, the accumulation of PCR-amplifi- Chicago, 900 East 57th Street KCBD cation noise and binding-site saturation leads to systematic error in the estimated affinities. We developed a procedure that uses the earlier and later rounds together, 10115, Chicago, IL 60637, USA and thereby obtains affinities that are both accurate and precise. *[email protected] The unprecedented depth of the sequencing data allowed us to systematically determine the effective length of the Exd-Hox dimer. While a 12nt degenerate consensus binding motif emerged for all Hox identities, we found that there exist striking differences in how different Exd-Hox dimers interact with the central hexanucleotide of the binding site. By quantifying the differences in affinity for each class of binding motif, we were able to identify a unique DNA recognition signature for each of the Exd-Hox dimers.

References

1. S. Banerjee-Basu and A. D. Baxevanis. Nucleic Acids Res 29, 3258-3269 (2001). 2. M. F. Berger, G. Badis, A. R. Gehrke, S. Talukder, A. A. Philippakis, L. Pena-Castillo, T. M. Alleyne, S. Mnaimneh, and O. B. Botvinnik, et al. Cell 133, 1266-1276 (2008). 3. M. B. Noyes, R. G. Christensen, A. Wakabayashi, G. D. Stormo, M. H. Brodsky, and S. A. Wolfe. Cell 133, 1277-1289 (2008). Structural Determinants of DNA-binding Specificity 1155 for Drosophila Hox Proteins

Hox proteins are homeodomain transcription factors that help to define cellular and tissue identities, and thus diversify body patterning on the anterior-posterior 195 axis (1). Understanding the DNA-binding specificities of Hox proteins will there- 1, fore provide insight into the regulation of Hox target genes and animal morpho- Peng Liu * genesis. Advanced by the SELEX-seq method, the complete repertoire of DNA Remo Rohs2 sequences that bind to Drosophila Hox proteins with their cofactors Extradenticle Richard Mann3 (Exd) and Homothorax (Hth) has been obtained. A range of unique DNA-binding 1, specificities has been observed among these Hox proteins, raising the question of Barry Honig ** how such diverse DNA-binding specificities are generated. 1Howard Hughes Medical Using multiple sequence alignments of Hox proteins, we identified partially conserved residues that correlate with DNA-binding specificity. Possible functions Institute, Center for Computational of these residues were inferred by mapping them onto available 3D structures. In Biology and Bioinformatics, Department parallel, using Monte Carlo simulations (2), we predicted the width of DNA minor of Biochemistry and Molecular groove for different Hox binding sites identified by SELEX-seq. We find that DNA sequences preferred by Hox proteins that control posterior patterning have Biophysics, Columbia University, 1130 similar minor groove shape, and this shape is different than that of DNA sequences St. Nicholas Avenue, New York, preferred by Hox proteins that define anterior morphology. In particular, within NY 10032, USA the Exd-Hox binding site, high-affinity DNA sequences for Hox protein Scr have 2 two narrow regions while sequences preferred by Hox protein Ubx have only one. Molecular and Computational Biology As revealed by previous studies (3, 4), the additional narrow minor groove of Scr Program, Department of Biological induces enhanced negative electrostatic potential, which attracts Arg3 of Scr. Our Sciences, University of Southern work leads to a new understanding of the structural basis of specific DNA-binding for Hox proteins in Drosophila, linking DNA binding site preferences to common California, Los Angeles, California DNA shapes. 90089, USA 3Department of Biochemistry and References Molecular Biophysics, Columbia 1. R. S. Mann, K. M. Lelli, and R. Joshi. Curr Top Dev Biol 88, 63-101 (2009). 2. R. Rohs, H. Sklenar, and Z. Shakked. Structure 13, 1499-1509 (2005). University, 701 West 168th Street, HHSC 3. R. Joshi, J. M. Passner, R. Rohs, R. Jain, A. Sosinsky, M. A. Crickmore, V. Jacob, A. K. 1104, New York, NY 10032, USA Aggarwal, B. Honig, and R. S. Mann. Cell 131, 530-543 (2007). 4. R. Rohs, S. M. West, A. Sosinsky, P. Liu, R. S. Mann, and B. Honig. Nature 461, 1248-1253 * [email protected] (2009). ** [email protected] 1156 DNA Shape Patterns and Binding Specificities for Drosophila Hox Proteins

The Hox family of transcription factors is essential for the patterning of the anteri- or-posterior axis, stem cell maintenance, and motor neuron specification in Droso- phila (1-3). This family of transcription factors carries out highly specific gene regulation; however, the source of specificity for binding of Hox proteins to DNA is poorly understood. Hox proteins, such as Ultrabithorax (Ubx) and Sex combs reduced (Scr), bind as monomers to degenerate DNA sequences with little specificity. In the presence of the homeodomain-containing cofactors Extradenticle (Exd; Pbx in vertebrates) and Homothorax (Hth; Meis in vertebrates), Hox proteins bind 196 DNA sequences with a high degree of specificity, in part by recognizing sequence- Dana R. Holcomb* dependent DNA structural features, such as minor groove shape and electrostatic potential (4). Thomas D. Tullius In order to determine DNA structural features that are associated with specific Department of Chemistry, binding to Hox proteins, we are employing hydroxyl radical cleavage chemistry to probe the structures of DNA sequences that have been identified as high-affinity Boston University, binding sites by the Mann and Bussemaker labs using the SELEX-seq technique. Boston, MA 02215 The hydroxyl radical cleavage pattern depends on the local solvent accessibility of *[email protected] each nucleotide in a duplex DNA molecule, thereby yielding a representation of the sequence-dependent shape of a DNA molecule. We compare our results with Monte Carlo-based computational predictions of DNA structure made by the Rohs and Honig laboratories (5). We also use the ORChID (OH Radical Cleavage Inten- sity Database) and ORChID2 algorithms developed by our laboratory (6) to compute the sequence-dependent structure of SELEX-seq-identified binding sites.

In previous work our laboratory used ORChID to show that similar DNA structures can arise from different DNA sequences (6). This result suggests that nucleotide sequence similarity alone may not be enough to recognize all classes of Hox binding sites. By experimentally determining the shapes of DNA sequences that bind to Hox proteins, and then comparing our experimental structural results to Monte Carlo- and ORChID-based computational predictions of structure, we can identify DNA shape-based binding preferences for Hox transcription factors.

References

1. J. S. Dasen and T. M. Jessel. Curr Topics Devel Biol 88, 169-200 (2009). 2. S. D. Hueber and I. Lohmann. Bioessays 30, 965-979 (2008). 3. S. Tumpel, L. M. Wiedemann, and R. Krumlauf. Curr Topics Devel Biol 88, 103-137 (2009). 4. R. S. Mann, K. M. Lelli, and R. Joshi. Curr Topics Devel Biol 88, 63-101 (2009). 5. R. Rohs, S. M. West, P. Liu, and B. Honig. Curr Opin Struct Biol 19, 171-177 (2009). 6. J. A. Greenbaum, B. Pang, and T. D. Tullius. Genome Res 17, 947-953 (2007). Protein-DNA Interactions in the p53/DNA System 1157

The tumor suppressor protein p53 is a transcription factor (TF) that, in response to various types of cellular stress, regulates the expression of a variety of genes involved in cell-cycle control, apoptosis, DNA repair, and cell differentiation (1), by first binding sequence-specifically to defined DNA targets (2). Abrogation of p53 sequence-dependent binding is implicated in ~50% of all known cancers (1). p53 molecules consist of four major functional domains (3). The N-terminus contains a transactivation domain; the core domain contains the sequence-specific DNA binding domain (DBD); and the C-terminal domain (CTD) includes a tetramerization domain (TD) and a regulatory domain that contain a separate sequence non-specific DNA binding activity. The core domain of p53 contains 95% of the missense mutations identified in human tumors (4). This highlights the importance of sequence- 197 specific DNA binding by p53 in maintaining genomic integrity and preventing tumor formation. The consensus DNA response element (RE) consists of two deca- Itai Beno meric half-sites with the general form RRRCWWGYYY (R=A,G;W=A,T;Y=C,T), Karin Rosenthal separated by a variable number of base pairs (2). The WW doublet in the CWWG Michael Levitine center of p53 half-sites is uncontacted with the protein in the crystal structure of p53DBD/DNA complexes (5, 6). Nonetheless, this position is highly conserved in Lihi Shaulov p53 binding sites. Tali E. Haran*

We will present our recent study (7) demonstrating that p53DBD bind consensus sequences differing in the CWWG center with different binding affinities and espe- Department of Biology, Technion, cially binding cooperativity. The binding cooperativity spans five orders of magni- Technion City, Haifa 32000, Israel tude and is encoded in the structural properties of this region and in particular in the *[email protected] torsional flexibility of the CWWG motif, as determined experimentally by us. The torsionally flexible CATG motif, connected with binding sites related to cell-cycle arrest genes, is bound with high affinity and low cooperativity by p53DBD. The torsionally rigid CAAG and CTAG motifs are bound with lower affinity and high cooperativity. These motifs are abundant in binding sites associated with low affinity apoptosis-related genes. Our results provide a molecular and structural basis to recent findings that DNA binding cooperativity of p53 modulate the decision between cell-cycle arrest and apoptosis (8). p53 sits at a hub of cellular network controlling many genes, mostly by its function as a TF that binds sequence-specifically to p53 REs. Knowledge of the full spectrum of p53 cellular connectivity is a prerequisite for a true understanding of what can go wrong when p53 function is disrupted. We have determined, using protein- binding microarrays, the binding affinity of p53DBD-TD to a large fraction of all possible combinations of p53 REs, and will present these results.

References

1. B. Vogelstein, D. Lane, and A. J. Levine. Nature 408, 307-310 (2000). 2. W. S. El-Deiry, S. E. Kern, J. A. Pietenpol, K. W. Kinzler, and B. Vogelstein. Nature Gen 1, 45-49 (1992). 3. A. C. Joerger and A. R. Fersht. Annu Rev Biochem 77, 557-582 (2008). 4. M. Olivier, R. Eeles, M. Hollstein, M. A. Khan, C. C. Harris, and P. Hainaut. Hum Mutat 19, 607-14 (2002). 5. Y. Cho, S. Gorina, P. D. Jeffrey, and N. P. Pavletich. Science 265, 346-355 (1994). 6. M. Kitayner, H. Rozenberg, N. Kessler, D. Rabinovich, L. Shaulov, T. E. Haran, and Z. Shakked. Mol Cell 22 741-753 (2006). 7. I. Beno, K. Rosenthal, M. Levitine, L. Shaulov, and T. E. Haran. Nuc Acids Res In Press (2011). 8. K. Schlereth, R. Beinoraviciute-Kellner, M. K. Zeitlinger, A. C. Bretz, M. Sauer, J. P. Charles, F. Vogiatzi, E. Leich, B. Samans, M. Eilers, C. Kisker, A. Rosenwald and T. Stiewe. Mol Cell 38, 356-368 (2010). 1158 Modulating DNA Binding Activity of P53 by Base Sequence Effects and Amino-Acid Mutations

Our structural studies of the DNA binding domain of the tumor suppressor protein p53 and its complexes with various DNA targets demonstrate that four p53 molecules bind to two decameric DNA half-sites to form a dimer of dimers stabilized by protein-DNA and protein-protein interactions. The 3-D architecture of the 198 complex and its stability are dependent on the identity of the DNA half-sites and on the DNA spacer between them. Several hot-spot mutations in the DNA bind- H. Rozenberg ing domain of p53 abolish the protein’s DNA binding activity and its function as A. Eldar a tumor suppressor. In certain cases, such activities can be restored by second-site M. Kitayner suppressor mutations. Our high-resolution crystal structures of wild-type p53, its tumor-derived p53 mutants and the restored proteins in their free and DNA-bound Y. Diskin-Posner states provide a framework for understanding the molecular and structural basis of A. Kapitkovsky p53 disfunction as a result of oncogenic mutations and its restoration by suppressor O. Suad mutations and potential drug molecules. Z. Shakked*

Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel *[email protected]

DNA Mechanics, Nucleosome Positioning and 199 p53-DNA Recognition For many years, it has been assumed that the nucleosome positioning is defined Victor B. Zhurkin* entirely by the energy of DNA deformation (bending and twisting) when it is Feng Cui wrapped around the histone core [see the retrospective review by Trifonov (1) and Difei Wang the accompanying commentaries (2, 3); many of the relevant detailed papers are in (4-17)]. However, when the high-resolution NCP crystal structure was solved (18), it became clear that bending of the nucleosomal DNA differs drastically from that Laboratory of Cell Biology, NCI, NIH in the other protein-DNA complexes. The DNA bending in nucleosome is accom- Bethesda, MD 20892 panied by strong lateral displacements of the DNA axis (Slide) that are critical for formation of the DNA superhelical path in nucleosome (19). These severe DNA *[email protected] deformations (denoted Kink-and-Slide), occur as a result of interactions with histone arginines which penetrate the DNA minor groove asymmetrically, so that their side chains are closer to one DNA strand (13).

Recent computer simulations of oligonucleotide duplexes suggest that the sequence- dependent deformability of DNA depends on the imposed constraints that mimic the presence of bound protein. In particular, when the Slide constraints observed in the nucleosome were used, the energy of the Kink-and-Slide deformation increased in the order TA  CA:TG  CG (13). Bending into the minor groove brings the highest increase in the deformation energy of DNA (19). Therefore, selection of the ‘correct’ sequences for the minor-groove bending is likely to be the most critical part in the process of finding the optimal position of nucleosomes on DNA. According to our current understanding (12, 13), the optimal minor-groove bending patterns contain the TA and CA:TG dimers in the pyrimidine-purine context YYRR. Importantly, this sequence preference explains the exceptionally high stability of nucleosomes formed by the “TG repeat” (20) and the “601 sequence” (21) containing numerous TTAG and TTAA fragments, respectively. In addition to folding in nucleosomes, the DNA Slide is implicated in the sequence- 1159 specific recognition of DNA by the tumor suppressor protein p53 (22). The shear- ing deformation of the DNA axis caused by p53 binding (23-25) is consistent with the Kink-and-Slide conformation described above. Therefore, structural organization of a p53 binding site in chromatin can regulate its affinity to p53 – for example, exposure of the DNA site on the nucleosomal surface would facilitate p53 binding to the response element (26). Our results indicate that there is a complex interplay between the structural codes encrypted in eukaryotic genomes – one code for DNA packaging in chromatin, and the other code for DNA recognition by transcription factors (TFs). The two codes appear to be generally consistent with each other. At least in some cases, such as p53 (26) and the glucocorticoid receptor (27), the DNA wrapping in nucleosomes can facilitate the binding of a TF to its cognate sequence, provided that the latter is properly exposed in chromatin.

References

1. E. N. Trifonov. Phys Life Rev 8, 39-50 (2011). 2. A. Travers. Phys Life Rev 8, 53-55 (2011). 3. V. B. Zhurkin. Phys Life Rev 8, 64-66 (2011). 4. A. Travers, E. Hiriart, M. Churcher, M. Caserta, and E. Di Mauro. J Biomol Struct Dyn 27, 713-724 (2010). 5. F. Xu and W. K. Olson. J Biomol Struct Dyn 27, 725-739 (2010). 6. E. N. Trifonov. J Biomol Struct Dyn 27, 741-746 (2010). 7. P. De Santis, S. Morosetti, and A. Scipioni. J Biomol Struct Dyn 27, 747-764 (2010). 8. G. A. Babbitt, M. Y. Tolstorukov, and Y. Kim. J Biomol Struct Dyn 27, 765-780 (2010). 9. D. J. Clark. J Biomol Struct Dyn 27, 781-793 (2010). 10. S. M. Johnson. J Biomol Struct Dyn 27, 795-802 (2010). 11. G. Arya, A. Maitra, and S. A. Grigoryev. J Biomol Struct Dyn 27, 803-820 (2010). 12. F. Cui and V. B. Zhurkin. J Biomol Struct Dyn 27, 821-841 (2010). 13. D. Wang, N. B. Ulyanov, and V. B. Zhurkin. J Biomol Struct Dyn 27, 843-859 (2010). 14. S. M. West, R. Rohs, R. S. Mann, and B. Honig. J Biomol Struct Dyn 27, 861-866 (2010). 15. Y. V. Sereda and T. C. Bishop. J Biomol Struct Dyn 27, 867-887 (2010). 16. I. Gabdank, D. Barash, and E. N. Trifonov. J Biomol Struct Dyn 26, 403-411 (2009) 17. I. Gabdank, D. Barash, and E. N. Trifonov. J Biomol Struct Dyn 28, 107-121 (2010). 18. C. A. Davey, D. F. Sargent, K. Luger, A. W. Mäder, and T. J. Richmond. J Mol Biol 319, 1097-1113 (2002). 19. M. Y. Tolstorukov, A. V. Colasanti, D. M. McCandlish, W. K. Olson, and V. B. Zhurkin. J Mol Biol 371, 725-738 (2007). 20. T. E. Shrader and D. M. Crothers. Proc Natl Acad Sci USA 86, 7418-7422 (1989). 21. P. T. Lowary and J. Widom. J Mol Biol 276, 19-42 (1998). 22. S. R. Durell, R. L. Jernigan, E. Appella, A. K. Nagaich, R. E. Harrington, and V. B. Zhurkin. In Sarma, R. H. and Sarma, M. H. (Eds.): Structure, Motion, Interaction and Expression of Biological Macromolecules. Proceedings of the Tenth Conversation, 1997. New York, Adenine Press, 1998, (2) pp. 277-296. 23. K. A. Malecka, W. C. Ho, and R. Marmorstein. Oncogene 28, 325-33 (2009). 24. Y. Chen, R. Dey, and L. Chen. Structure 18, 246-56 (2010). 25. M. Kitayner, H. Rozenberg, R. Rohs, O. Suad, D. Rabinovich, B. Honig, and Z. Shakked. Nat Struct Mol Biol 17, 423-9 (2010). G.26. Sahu, D. Wang, C. B. Chen, V. B. Zhurkin, R. E. Harrington, E. Appella, G. H. Hager and A. K. Nagaich. J Biol Chem 285, 1321-1332 (2010). 27. M. Becker, C. Baumann, S. John, D. A. Walker, M. Vigneron, J. G. McNally, and G. L. Hager. EMBO Rep 3, 1188-94 (2002). 1160 Yeast Nucleosomes Mapped with High Resolution by Paired-end Sequencing Exhibit Strong Positioning Patterns

Despite intense efforts, recently summarized in a series of publications (1-14), 200 understanding nucleosome positioning continues to be challenging. Recent Feng Cui1,* advances in massively parallel shotgun sequencing techniques allowed detecting Hope A. Cole2 nucleosome positions across the genomes of yeast (15, 16), nematode (17) and 2 human (18). Millions of DNA fragments derived in the course of micrococcal David J. Clark nuclease cleavage were partially sequenced at the 5’ end. As a result, the ‘properly’ Victor B. Zhurkin1 trimmed nucleosome core particles have been mixed up with the over-digested or incompletely trimmed nucleosomes. To resolve this ambiguity, we used the paired- end sequencing technique and obtained ~15 million paired reads that are uniquely 1 Laboratory of Cell Biology, NCI, NIH mapped to the yeast genome (19). The lengths of DNA fragments obtained in 2Program in Genomics of Differentiation, this way span the interval from 120 bp (over-digested core particles) to 180 bp NICHD, NIH, Bethesda, MD 20892 (poorly trimmed nucleosomes), with the highest occurrence at 150 bp. The data for 147-152 bp-long fragments (~5 million) were compared with the two published *[email protected] datasets, one with the same length interval, 147-152 bp (~72,000) (16), and the other with the estimated length 147 bp (~50,000) (20).

The high-resolution nucleosomes in our new dataset are depleted in the promoter regions and positioned regularly downstream of gene starts, consistent with the earlier studies (16, 20). This regularity was further supported by the start-to-start distance correlation function having periodicity of 160-170-bp, close to the yeast nucleosome repeat ~165 bp. A detailed analysis of this distance correlation rendered a clear 10-bp periodicity, more pronounced than in the two published datasets (16, 20). Moreover, a stronger variation of occurrence of the AT-containing fragments (21) was found in the paired-end nucleosomes (19). This new dataset is being used to analyze the occurrence of the DNA dimers, trimers and tetramers in various positions in the nucleosome core particles, and to further develop our scheme for prediction of the nucleosome positioning (9).

References

1. A. Travers, E. Hiriart, M. Churcher, M. Caserta, and E. Di Mauro. J Biomol Struct Dyn 27, 713-724 (2010). 2. F. Xu and W. K. Olson. J Biomol Struct Dyn 27, 725-739 (2010). 3. E. N. Trifonov. J Biomol Struct Dyn 27, 741-746 (2010). 4. P. De Santis, S. Morosetti, and A. Scipioni. J Biomol Struct Dyn 27, 747-764 (2010). 5. G. A. Babbitt, M. Y. Tolstorukov, and Y. Kim. J Biomol Struct Dyn 27, 765-780 (2010). 6. D. J. Clark. J Biomol Struct Dyn 27, 781-793 (2010). 7. S. M. Johnson. J Biomol Struct Dyn 27, 795-802 (2010). 8. G. Arya, A. Maitra, and S. A. Grigoryev. J Biomol Struct Dyn 27, 803-820 (2010). 9. F. Cui and V. B. Zhurkin. J Biomol Struct Dyn 27, 821-841 (2010). 10. D. Wang, N. B. Ulyanov, and V. B. Zhurkin. J Biomol Struct Dyn 27, 843-859 (2010). 11. S. M. West, R. Rohs, R. S. Mann, and B. Honig. J Biomol Struct Dyn 27, 861-866 (2010). 12. Y. V. Sereda and T. C. Bishop. J Biomol Struct Dyn 27, 867-887 (2010). 13. I. Gabdank, D. Barash, and E. N. Trifonov. J Biomol Struct Dyn 26, 403-411 (2009). 14. I. Gabdank, D. Barash, and E. N. Trifonov. J Biomol Struct Dyn 28, 107-121 (2010). 15. I. Albert, T. N. Mavrich, L. P. Tomsho, J. Qi, S. J. Zanton, S. C. Schuster, and B. F. Pugh. Nature 446, 572-576 (2007). 16. T. N. Mavrich, I. P. Ioshikhes, B. J. Venters, C. Jiang, L. P. Tomsho, J. Qi, S. C. Schuster, I. Albert, and B. F. Pugh. Genome Res 18, 1073-1083 (2008). 17. A. Valouev, J. Ichikawa, T. Tonthat, J. Stuart, S. Ranade, H. Peckham, K. Zeng, J. A. Malek, G. Costa, K. McKernan, A. Sidow, A. Fire, and S. M. Johnson. Genome Res 18, 1051-1063 (2008). 18. D. E. Schones, K. Cui, S. Cuddapah, T-Y. Roh, A. Barski, Z. Wang, G. Wei, and K. Zhao. Cell 132, 887-898 (2008). 19. H. A. Cole, B. H. Howard, and D. J. Clark. Submitted for publication (2011). 20. Y. Field, N. Kaplan, Y. Fondufe-Mittendorf, I. K. Moore, E. Sharon, Y. Lubling, J. Widom, and E. Segal. PLoS Comp Biol 4, e1000216 (2008). 21. S. C. Satchwell, H. R. Drew, and A. A. Travers. J Mol Biol 191, 659-675 (1986). DNA Shape Patterns and Evolutionary Constraint in 1161 Nucleosome Positioning Sequences

The process of wrapping DNA around a nucleosome core particle affects the functions encoded in the underlying sequence. Therefore, it is important to understand the biological signals responsible for nucleosome positioning. Nucleosome positioning 201 is one of the current ‘hot’ issues and the various approaches and hypotheses on what 1, positions nucleosomes are discussed extensively in recent publications, particularly Stephen C. J. Parker * in an issue of this journal devoted exclusively to this subject (1-14). One of the Sean M. West2,3 major factors affecting the nucleosome positioning is DNA sequence. Despite the Eric Bishop4 observation that nucleosome positioning is sequence-directed, the core particle 5 makes no base-specific contacts with DNA. These circumstances suggest that Remo Rohs nucleosome positioning may be encoded through DNA structural properties and Barry Honig2 not necessarily the primary sequence. To study this, we introduce a new algorithm Thomas D. Tullius4,6 named ORChID2 that uses hydroxyl radical cleavage patterns to predict the single- 1 nucleotide resolution shape of a DNA molecule. Analysis of ORChID2 DNA shape Elliott H. Margulies profiles in well-positioned nucleosome sequences reveals a periodic and symmetric pattern. Additionally, there is more information encoded in the DNA shape than the 1National Human Genome Research primary sequence. Information content is consistently highest at regions where the Institute, National Institutes of Health, minor groove is narrow—suggesting a fundamental property of protein-DNA shape recognition in nucleosome positioning. Finally, we present a new algorithm named Bethesda, Maryland, USA Chai2 that measures the single-nucleotide resolution evolutionary conservation of 2Howard Hughes Medical Institute, DNA shape. Overlaying Chai2 constraint results on ORChID2 shape profiles in Center for Computational Biology nucleosome positioning sequences reveals interesting evolutionary conservation patterns. We suggest that selective pressure operating on DNA shape can influ- and Bioinformatics, Department of ence nucleosome positioning dynamics, and therefore be a major force in genome Biochemistry and Molecular Biophysics, evolution. Columbia University, New York, USA References 3Center for Genomics and Systems

1. A. Travers, E. Hiriart, M. Churcher, M. Caserta, and E. Di Mauro. J Biomol Struct Dyn 27, Biology, Department of Biology, 713-724 (2010). New York University, New York, USA 2. F. Xu and W. K. Olson. J Biomol Struct Dyn 27, 725-739 (2010). 3. E. N. Trifonov. J Biomol Struct Dyn 27, 741-746 (2010). 4Program in Bioinformatics, Boston 4. P. De Santis, S. Morosetti, and A. Scipioni. J Biomol Struct Dyn 27, 747-764 (2010). 5. G. A. Babbitt, M. Y. Tolstorukov, and Y. Kim. J Biomol Struct Dyn 27, 765-780 (2010). University, Boston, Massachusetts, USA 6. D. J. Clark. J Biomol Struct Dyn 27, 781-793 (2010). 5Molecular and Computational Biology 7. S. M. Johnson. J Biomol Struct Dyn 27, 795-802 (2010). 8. G. Arya, A. Maitra, and S. A. Grigoryev. J Biomol Struct Dyn 27, 803-820 (2010). Program, Department of Biological 9. F. Cui and V. B. Zhurkin. J Biomol Struct Dyn 27, 821-841 (2010). Sciences, University of Southern 10. D. Wang, N. B. Ulyanov, and V. B. Zhurkin. J Biomol Struct Dyn 27, 843-859 (2010). 11. S. M. West, R. Rohs, R. S. Mann, and B. Honing. J Biomol Struct Dyn 27, 861-866 (2010). California, Los Angeles, California, USA 12. Y. V. Sereda and T. C. Bishop. J Biomol Struct Dyn 27, 867-887 (2010). 6 13. I. Gabdank, D. Barash and E. N. Trifonov. J Biomol Struct Dyn 26, 403-411 (2009). Department of Chemistry, Boston 14. I. Gabdank, D. Barash, and E. N. Trifonov. J Biomol Struct Dyn 28, 107-121 (2010). University, Boston, Massachusetts, USA *[email protected] 1162 Molecular Dynamics Simulations on DNA Y10K

Recent progress in the area molecular dynamics (MD) simulations on DNA over the last 10 years will be presented, with an emphasis on force field developments, convergence and stability at the nano- to microsecond level, and the state of valida- 202 tion based on comparisons with diverse experimental physical methods. Results on the sequence effects on dynamical structure based on MD on all tetranucleotide David L. Beveridge steps by the ABC consortium will be described, and current research on DNA axis bending and flexibility will be included. Dept of Chemistry and Molecular Biophysics Program Wesleyan University, Middletown, CT [email protected]

Six Sensations: Six Second Genetic Codes

Human mind can accept the idea that in addition to classical triplet code some 203 other, “second genetic code” could exist. And its discovery, of course, would be Edward N. Trifonov1,2 a great scientific sensation. But the human mind can not possibly imagine that there could be several different codes. This is why every newly discovered code (and there are, actually, many) is called by the same generic name “second genetic 1 Genome Diversity Center, Institute code”. The second codes, indeed, all different, have been solemnly announced six of Evolution, University of Haifa, times (1-6), in 1988, 2001, 2001, 2006, 2008 and 2010. Mount Carmel, Haifa 31905, Israel Another, well known feature of human mind is fading memory. This is illustrated 2 Department of Functional Genomics and by the above time series. The sensational discoveries are honored by the traditional Proteomics, Faculty of Science, title of the “second genetic code”, and only the amnesia keeps the discoverers from Masaryk University, Kotlarska 2, making grimaces.

CZ-61137 Brno, Czech Republic What is good about the amnesia, however, it encourages to make yet new sensa- [email protected] tional discoveries (as by the short memory perception none are made before).

Will the sevenths Second Genetic Code be discovered? (Chicken would give all previous codes different numbers, as it counts till seven).

References

1. G. Kolata. New York Times May 13 (1988). 2. Second genetic code could provide clues to schizophrenia, bipolar disorder. CBCNews March 12 (2008). 3. E. Young. New scientist August 9 (2001). 4. N. Wade. New York Times July 25 (2006). 5. T. Hughes. The FASEB Journal 22, 262-268. (2008). 6. J. R. Tejedor and J. Valcárcel. Nature May 6 (2010). The UvrA•UvrB DNA Damage Sensor: Structure 1163 and Mechanism

During nucleotide excision repair (NER), the UvrA•UvrB (AB) damage sensor identifies lesion-deformed DNA within a sea of undamaged DNA. We report the first 204 structure of the AB sensor and of a novel UvrA conformer. In the structure of the AB David Jeruzalmi* sensor, a central UvrA dimer is flanked by two UvrB molecules, all linearly arrayed along a DNA path predicted by biochemical studies. DNA is predicted to bind to Danaya Pakotiprapha UvrA in the complex within a narrow and deep groove that is compatible with native Martin A. Samuels duplex DNA only. In contrast, the shape of the corresponding surface in all other Koning Shen UvrA structures is wide and shallow and appears compatible with various types of lesion-deformed DNA. These differences point to conformation switching between Johnny Hu the two forms as a component of the genome-scanning phase of damage sensing. We also show that the highly conserved signature domain II of UvrA, which is adja- Department of Molecular and Cellular cent to the proximal nucleotide-binding site, mediates a critical nexus of contacts to UvrB and to DNA. Moreover, in the novel UvrA conformer, the disposition of this Biology, Harvard University, domain is altered such that association with either UvrB or DNA is precluded. Con- 52 Oxford Street, Cambridge, comitantly, nucleotide is uniquely absent from the proximal binding site. Thus, the MA 02138, USA. signature domain II is implicated in an ATP-hydrolysis-dependent conformational change that detaches UvrA from both UvrB and DNA after initial damage recogni- *[email protected] tion. Finally, the disposition and number of UvrB molecules in the AB complex, both unanticipated, suggest that once UvrA departs, UvrB localizes to the site of damage by helicase-mediated tracking along the DNA. Together these results permit a high-resolution model for the dynamics of early stages in NER (1-6).

Research in the Jeruzalmi lab is supported by the NSF (MCB-0918161) and NIH (GM084162).

References

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Registration Fee: The registration fee listed below also includes all receptions, 4 continental breakfasts, Friday, June 17th night big feast, and one evening dinner and Saturday lunch. We appreciate an early registration because it allows us to organize the affairs properly, and to encourage this, there is a discount for early registration. Fee must be paid along with this completed from.

Check Box Registration Dates Student Fees† Fee for all others Before December 15 '10 $450 $575 Dec 16 ‘10-Feb 15 '11 $500 $625 Feb 16 '11 – Mar. 31 '11 $550 $675 April 1 '11 – April 30 '11 $600 $725 After May 1, '11 $650 $775 † Student registration must be accompanied by photo copy of student id or letter from registrar.

Reservation for Accommodation at Empire Commons: We collect fees and process accommodation at the Univesity's brand new airconditioned, 24 hr internet connected (bring your own laptop and pay $15 for connection) apartment houses (Empire Commons) built for our students. Each apartment contains 4 single bed rooms with a full double bed, a living room, kitchen and two bath rooms etc. This time we have access only to a limited number of buildings at Empire Commons; So we serve on a first come basis.

Check Box Type of Occupancy Accommodation fees Single Occupancy, 4 nights $260 Saturday June 20th, the 5th night $ 65 If you want Internet in your room pay $ 15 I am making my own arrangement; possible hotels are below

If you want to make your own arrangement, the nearest hotels are: Marriott -Courtyard (518-435-1600; 1-800-321-2211), Hilton Garden Inn, SUNY Albany (518-453-1300) Extended Stay (518-446-0680;1-800-398-7829) and Fairfield Inn (518-435-1800). They are just across the Univ, and the Empire Commons.

What are you planning to do? I am an invited speaker chair guest at the 17th Conversation I wish to present a poster/posters; I wish to present a poster and a lecture based on my poster.

Last Name (CAPS or type) ______First Name ______Male/Female

EMails (CAPS): ______

Phone ______Fax ______

Your Institution: ______Country ______

Meal preference: No preference, Kosher, Vegetarian

Payment for registration and Accommodation must be together if you plan to live in Empire Commons

If paying by credit card do the following: Complete the above form, and the credit card form on page 2; then email them as attachments to Prof. Sarma: [email protected]. You can also fax the forms to 518-452-4955

If paying by check or money order do the following: They should be drawn in any bank located in the US. Make check payable to Adenine Press (Federal ID = 14-1626143) and send form and check to the following address:

Prof. R. H. Sarma, Chemistry Bldg, SUNY at Albany, Albany NY 12222 USA email:[email protected] phone:518-456-9362/fax:518-452-4955

Registration fee will be refunded fully if cancelled by email by May 1, 2011; accommodation fee will be refunded fully if cancelled by June 1 2011; late cancellation will result in the loss of the fees.

ADENINE PRESS, INC.

www.adeninepress.com

Pay 17th Conversation Fees by Credit Card or Wire Transfer Please fill the form below and email this to Prof. Sarma at [email protected] along with your registrationform. You may also fax the forms to 518-452-4955.

1. Total fees from the registration form: ______

2. Type of CC: MC VISA AMX

3. CC number, Exp. date, & security code CC #: ______Exp: date: ______Security Code ______

4. Name under which CC is issued: Name: (First) ______(Last) ______

5. Billing Address where credit card statements are received by the customer: (address) ______(address) ______City ______State _____ Zip ______Country ______

6. Email address to inform the customer of the results of processing charges on CC email: ______phone no: ______fax: no: ______

For electronic wire transfer use the information below. You must add an additional $50.00 to the invoice amount because our bank charges us this fee to receive wire transfers. Name of bank = Key bank NA; Address of Bank = Albany, NY 12203 USA Account Owner = Adenine Press; Account Number = 325450035846; ABA Code 021300077Swift code = KEYB-US-33

2066 Central Avenue, Schenectady NY 12304 USAPhone: 518-456-0784, Fax: 518-452-4955, e-mail: [email protected]