Keynotes

• PROTEIN DYNAMICS, FLUCTUATIONS, AND THE FREE-ENERGY LANDSCAPE
  Hans Frauenfelder
  Los Alamos National Laboratory
  
  Motions are crucial for the function of proteins, for instance for the opening and closing of channels [1]. Three types of fluctuations have been found so far that are important, α-fluctuations that originate in the bulk solvent, β-fluctuations that come mainly from the hydration shell that surrounds the protein, and thermal vibrations that come from the entire system. Studies of these fluctuations with the Mössbauer effect, neutron scattering, and dielectric relaxation spectroscopy provide new insights [2]. They challenge the accepted wisdom, provide a new picture of how these techniques work in complex systems, and give a view of the low tiers of the energy landscape.
  
  [1] H. Frauenfelder et al., Proc. Natl. Acad. Sci. USA 106, 5129 (2009).
  [2] R. D. Young et al., Phys. Rev. Lett. 107, 158102 (2011).
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• Experimental-Computational Discoveries Achieved Through the Blue Waters and Titan Petascale Computers
  Klaus Schulten
  University of Illinois Urbana-Champaign
  
  Summary: Earlier this year, 2013, two remarkable computers have become available to computational scientists in the US, National Science Foundation-funded Blue Waters and Department of Energy-funded Titan. The machines, offering nearly 300,000 cores and GPU acceleration, permit molecular dynamics and molecular analysis calculations with NAMD and VMD of unprecedented size and time scales. BlueWaters and Titan provide, in particular, new opportunities to link experiment and theory in molecular cell biology and, in fact, already during the test period remarkable computational biology discoveries resulted from the two machines. This lecture reports on three of these discoveries.
  1. Permitting extensive experimentation and sampling, molecular dynamics simulations demonstrated that synaptotagmin, a key agent in Ca++-triggered neurotransmitter release, should act in membrane sculpting in a Ca++ activated conformation, which differs from the crystallographically observed structure.
  2. Permitting extensive transition path sampling molecular dynamics simulations investigated the mechanism of a homo-hexameric molecular motor, hexameric helicase, that translates along single-stranded RNA. The simulations revealed that the sequence of ATP binding, ATP hydrolysis, ADP + Pi release reactions affect only insignificantly protein conformations, but mainly induce a rearrangement of the six subunits relative to each other through alteration in surface-surface interactions. The resulting motion of the hexamer of subunits alters the interaction and positioning of lysine groups interacting with the central RNA such that the RNA is moved by one base per ATP hydrolysis relative to the hexamer.
  3. Molecular dynamics flexible fitting (MDFF) combining NMR, crystallography, and EM data with molecular modeling solved the atomic level structure of an entire HIV virus capsid. The protein data base (pdb) entry of the capsid with 3 million atoms is the largest pdb entry yet and documents the malleability of the single type capsid protein that assembles into a rather heterogeneous system of about 1300 proteins realizing a non-symmetric distribution of local curvature.
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• The iPlant Collaborative: A Life Sciences Cyberinfrastructure for the 21st Century
  Dan Stanzione
  University Texas at Austin
  
  The iPlant Collaborative (iPlant) is a large scale United States National Science Foundation (NSF) funded project that aims to create an innovative, comprehensive, and foundational cyberinfrastructure in support of plant biology research (and now, animal science as well). iPlant supports the diverse fields and workflows that comprise plant biology by building an open, flexible, extensible platform. iPlant provides services ranging from large scale storage and computation, to application programmer interfaces, to web-based environments for end users. In this talk, an overview of iPlant's CI capabilities will be presented, along with a discussion of how these resources take advantage of the newly-deployed cutting edge supercomputing systems at the Texas Advanced Computing Center, followed by a brief discussion of changes in computing architecture and the implications for bioinformatics applications.
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Invited Talks

• Lipids and hydrogen bonding in membrane protein function
  Ana Nicoleta Bondar
  Freie Universität Berlin
  
  Hydrogen-bond dynamics and changes in the protonation state couple to the conformational dynamics of membrane proteins. Based on extensive investigations of the dynamics of several membrane proteins, we propose that inter-helical hydrogen bonding and hydrogen bonding to lipids ensures efficient means of long-distance conformational coupling. In the absence of perturbations (mutation, protonation change), inter-helical hydrogen bonds help stabilize protein conformation. Once the protein is perturbed, however, dynamical hydrogen bonds can rearrange rapidly and help stabilize a new protein conformation. Hydrogen bonding to lipids not only affects the local structure and dynamics of the protein, but can influence the dynamics of inter-helical hydrogen bonds, effectively coupling membrane protein function to the membrane environment.
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• Macromolecular solution properties by analytical ultracentrifugation
  Borries Demeler
  University of Texas Health Science Center at San Antonio
  
  Analytical ultracentrifugation (AUC) is a powerful technique used to characterize the solution behavior of macromolecules. Being able to work in the solution state offers many advantages, because many conditions of a reaction can be easily modulated, such as pH, ionic strength, temperature, oxidation state, the presence of small molecules, and other binding partners. This allows the researcher to investigate DYNAMIC processes, study the effects of mutations, investigate oligomerization states, and follow complex multi-domain protein assembly. Unlike other techniques, AUC has virtually no size restrictions, and is suitable for a wide variety of samples and sample conditions. Advanced optical detectors and new computational methods allow us to obtain high resolution information describing sample composition (size, anisotropy, and partial concentration) as well as to measure binding strength of interactions between one or more binding partners. AUC is based on first principles and thus does not require any standards. In this talk I will present an overview of the technique, discuss some of the analysis methodology utilizing high performance computing, and review applications in AUC using illustrative examples.
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• A Novel Optimization Framework for the Design of Proteins with Post-Translational Modifications and Unnatural Amino Acids
  George A. Khoury, James Smadbeck, and Christodoulos A. Floudas
  Department of Chemical and Biological Engineering, Princeton University
  
  Most proteins being used for drug applications contain post-translational modifications (PTMs) [1]. It can become prohibitively expensive and time-consuming to experimentally modify and test the effects of large numbers of modifications. To enable academic and industrial researchers to minimize time and monetary cost searching for new functional designs, in this work we present ModDesigner, a method to in silico design proteins and peptides with post-translational modifications and unnatural amino acids.
  
  We develop a new integer linear optimization design formulation to introduce the modified amino acids, drawing on our recently developed forcefields [2]. Given a ligand or an ensemble of ligand conformations of a peptide bound in a receptor protein, the formulation maximizes the favorable (Van der Waals and hydrogen bond) contacts and minimizes steric clashes while maintaining or enhancing the binding free energy. To reduce the combinatorial complexity of the problem, logical restraints to preserve the residue-wise charge and hydrophobicity are applied to reduce the number of allowed modifications in each position. For each configuration, the initial contacts, clashes, and hydrogen bonds are populated. Next, for each configuration, each design position is modified, a local energy minimization is performed, and the new contacts, clashes, hydrogen bonds and interaction energy are populated. Finally, the ILP is solved to global optimality, generating a rank-ordered list of designs using integer cuts. The importance of using a flexible template in using these design metrics is demonstrated using an ensemble of conformers generated from a molecular dynamics simulation.
  
  The algorithm is designed to serve as Stage 3: Modification Selection of our generalized de novo design framework [3,4], with Stage 1 being an optimization driven Sequence Selection, and Stage 2 being Fold Specificity and statistical mechanics-based Approximate Binding Affinity calculations. We first benchmark the forcefield which is based on only physics ability to predict IC50 values. Next, we describe our efforts to use the method to design new variants of Compstatin to inhibit Complement C3c beginning with the extremely potent Compstatin analog [5], Variant E1. In all efforts we begin with template sequences and structures containing only the 20 amino acids discovered to experimentally bind and inhibit their target receptors [6-8]. In this way, the modified amino acids serve to fine-tune specificity and potentially enhance the affinity of a given inhibitor.
  
  References:
  1. Walsh C. Posttranslational modification of proteins: expanding nature's inventory. Englewood, Colo.: Roberts and Co. Publishers; 2006. xxi, 490 p. p.
  2. Khoury GA, Thompson J, Smadbeck J, Floudas CA. ForcefieldPTM: Development and Testing of a First Generation AMBER Forcefield for Post-Translational Modifications In Preparation.
  3. Bellows ML, Fung HK, Floudas CA. Recent Advances in De Novo Protein Design. Process Systems Engineering: Wiley-VCH Verlag GmbH & Co. KGaA; 2011. p 207- 232.
  4. Smadbeck J, Bellows-Peterson ML, Khoury GA, Taylor MS, Floudas CA. Protein WISDOM: A Workbench for In Silico De novo design of bioMolecules. Journal of Visualized Experiments In Press.
  5. Mallik B, Katragadda M, Spruce LA, Carafides C, Tsokos CG, Morikis D, Lambris JD. Design and NMR Characterization of Active Analogues of Compstatin Containing Non- Natural Amino Acids. Journal of Medicinal Chemistry 2004;48(1):274-286.
  6. Bellows ML, Taylor MS, Cole PA, Shen L, Siliciano RF, Fung HK, Floudas CA. Discovery of Entry Inhibitors for HIV-1 via a New De Novo Protein Design Framework. Biophysical Journal 2010;99(10):3445-3453.
  7. Bellows ML, Fung HK, Taylor MS, Floudas CA, López de Victoria A, Morikis D. New Compstatin Variants through Two De Novo Protein Design Frameworks. Biophysical Journal 2010;98(10):2337-2346.
  8. López de Victoria A, Gorham RD, Bellows-Peterson ML, Ling J, Lo DD, Floudas CA, Morikis D. A New Generation of Potent Complement Inhibitors of the Compstatin Family. Chem Biol Drug Des 2011;77(6):431-440.
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• Probing functional dynamics in enzymes using simulations
  Donald Hamelberg
  Georgia State University
  
  Molecular dynamics is now commonly used as a complementary tool to experiments in understanding the dynamical behavior of biomolecules. Application of advanced sampling methods, such as accelerated molecular dynamics, to probe long time scale dynamics is fast becoming indispensible in the field, since conformational dynamics is generally accepted to be important in enzyme catalysis. However, the precise role of conformational dynamics in enzymes remains unresolved. We present a rationale of how enzyme dynamics is coupled to the reaction step and affects the catalytic rate. Our atomistic simulations provide insights into the general interplay between enzyme conformational dynamics and catalysis from an atomistic perspective, and the results are in notable agreement with experiments. We further discuss our efforts in finding a fast, yet accurate, advance sampling method to probe long time scale dynamics in biomolecules.
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• Correlation networks of rhinovirus capsid dynamics
  Carol Post
  Purdue University
  
  Human rhinovirus (HRV) and other members of the enterovirus genus bind small-molecule antiviral compounds in a cavity buried within the viral capsid protein VP1. These compounds block the release of the viral protein VP4 and RNA from inside the capsid during the uncoating process. In addition, the antiviral compounds prevent “breathing” motions, the transient externalization of the N-terminal regions of VP1 and VP4 from the inside of intact viral capsid. The site for externalization of VP1/VP4 or release of RNA is likely between protomers, distant to the binding cavity for antiviral compounds. Molecular dynamics (MD) simulations were conducted to explore how the antiviral compound, WIN 52084, alters properties of the HRV 14 capsid through long-distance effect. The Pearson correlation coefficient of atomic positions, typically used to search MD trajectories for correlated motions, failed to detect any long-range correlations. Nonetheless, an alternative metric of the radial distance uncovered a number of long-range correlations of capsid dynamics, which have not been previously recognized. In the absence of WIN, correlated radial motion is observed between residues separated by as much as 85 Angstroem, a remarkably long distance. We developed a framework to analyze these long-distance, concerted motions using a network based on the radial correlation coefficients. The most frequently populated path segments of the network were localized near the fivefold symmetry axis and included those connecting the N termini of VP1 and VP4 with other regions, in particular near twofold symmetry axes, of the capsid. Moreover, the presence of WIN destroys this radial correlation network, suggesting that the underlying motions contribute to a mechanistic basis for the initial steps of VP1 and VP4 externalization and uncoating.
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• Biological Applications of the UNRES Force Field
  Harold Scheraga
  Cornell University
  
  Our UNited RESidue force field (UNRES), developed with Adam Liwo and coworkers, has been made available on a website ((http://UNRES.PL). It has undergone blind tests in the recent CASP10 exercise, with interesting success so that it is now being applied in studies of biological complexes. In addition, the coarse-grained UNRES philosophy has been extended as NARES-2P to treat DNA and RNA (Y. He, M. Maciejczyk, S. Oldziej, H.A. Scheraga, and A. Liwo, Phys. Rev. Letters, in press). These recent results will be discussed in detail in this CBSB13 workshop talk.
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• Generality of parallel replica dynamics and applicability to biological systems
  Arthur Voter
  Los Alamos National Laboratory
  
  Many important processes in chemistry, physics, materials science, and biology take place on time scales that exceed what can be accessed directly with molecular dynamics simulation. Often the dynamics on these longer time scales consists of infrequent transition events that take the system from one state to another. Our research program has been aimed at developing methods for extending the accessible molecular dynamics simulation time for this type of system, methods that make as few approximations as possible. Our focus is on an accelerated molecular dynamics (AMD) approach, in which we let the trajectory itself find an appropriate way to escape from each state, but we coax it into doing so more quickly. One of these AMD methods, parallel replica dynamics (ParRep), achieves this by running many replicas of the system in parallel. Implemented carefully, ParRep can give arbitrarily accurate dynamics for infrequent event systems, and can give substantial computational speedup, up to the number of processors, when the events are very infrequent. A requirement for ParRep is that the distribution of first-passage times is exponential, as it is naturally for deep states that lose their memory long before the next escape event. A recent mathematical analysis in the context of overdamped Langevin dynamics [ C. Le Bris, T. Lelievre, M. Luskin, and D. Perez, Monte Carlo Methods and Applications 18, 119 (2012)] has shown that the ParRep method is even more general than we previously realized. In essence, a system that does not have exponentially distributed first-passage times can be converted to one that does, simply by applying the ParRep dephasing procedure for a sufficiently long time, yielding the so-called quasi-stationary distribution (QSD). This opens the possibility of using ParRep on a much broader range of complex systems than we previously thought possible, such as systems where each \\}"state\\\" actually consists of many (perhaps a huge number of) substates, and for which there may not be a good separation of time scales between the equilibration time within the state and the time for escape from the state. I will describe the ParRep method, explain how it is applied in this new QSD-based context, and discuss the implications for treating biological systems, which are the subject of this conference.
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• Wanted: The Best Models in Systems Biology
  Eberhard Voit
  Georgia Institute of Technology
  
  One of the grand challenges of computational systems biology is the translation of an actual biomedical phenomenon into a computational format that can be used for interrogation, analysis, manipulation, and optimization. This translation not only requires an understanding of the functional structure and regulation of the actual system underlying the phenomenon, but also the choice of effective mathematical formulations. Alas, nature has not provided us with a list of optimal functions or a handbook of best practices, with the consequence that we simply do not know how to select optimal mathematical or computational representations. In the first part of this presentation, I will identify the challenges faced during model selection and present power-law models as reasonable defaults, at least to get started with model design and analysis. In the second part I will demonstrate to what degree we may obtain a glimpse of the actual shapes of processes within the context of biochemical and metabolic pathway systems.
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• Microsecond simulations at electronic structure quality: heading toward a biological force field from accurate water models
  Feng Wang
  University of Arkansas
  
  Developing accurate force fields that are efficient for biological free energy simulations is a grand challenge. High force field accuracy requires sophisticated energy expressions to model many-body effects that are consequence of quantum mechanics. Without relying on complicated many-body energy expressions, the adaptive force matching (AFM) method developed in the Wang group robustly models the quantum mechanical potential energy surface by systematically optimizing the energy expressions for each specific system. Acting as a bridge that connects quantum-mechanical potential energy surface to simulations at extended time and length scales, AFM force fields can make reliable predictions of key experimental properties. In this talk, we present studies of free energy landscape of the ubiquitous biological solvent, water. Microsecond simulations are reported investigating ice-liquid and liquid-liquid phase transitions in water.
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• Continued Sampling: Using data aware feedback and control for peptide conformational search
  Tom Woolf
  John Hopkins University
  
  Sampling till completion is a new framework for conformational search that uses a coupled feedback between running computational dynamics and a large database. By using this feedback we can get improved convergence as well as more efficient sampling. We have applied this concept to sampling of peptide degrees of freedom and the first part of the presentation will highlight the challenges in making this idea work by addressing both hardware and software issues. This includes efforts to create rapid query evaluations and our use of coupled CPU and GPU systems. The second part of the presentation will focus on the nature of the efficiency gains and how the concept may be helpful for sampling within the Onsager-Machlup framework for understanding conformational transitions. This enables us to view certain degrees of freedom as more important for the transitions than others and ties into dynamic importance sampling and effective transfer entropy approaches.
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Outstanding Young Researcher Award Talks

• Multiscale Modeling of G Protein-Coupled Receptors (GPCRs)
  Jianing Li
  Department of Chemistry, University of Chicago
  
  G-protein-coupled receptors (GPCRs) are crucial membrane proteins for cellular signal transduction, representing therapeutic targets of 30-50% marketed drugs. Through conformational changes induced by delicate protein-ligand interactions, GPCR can be activated or deactivated by different ligands, many of which are candidates for new drugs. Despite the exceptional pharmaceutical importance, structure-based GPCR drug development is still challenging due to scarce known structures and unclear activation/deactivation mechanism. Using robust multiscale modeling techniques, we have developed multiscale models for GPCR drug discovery. Starting from atomistic resolution, we simulated the ligand-induced GPCR activation and deactivation to elucidate how a ligand induces structural and energetic changes to activate/deactivate GPCR. Our large-scale simulations suggest that the ligand induces rotation of a tryptophan and initiates GPCR activation/deactivation. Next, we developed mixed-resolution models to tackle tasks that require high-resolution GPCR structures such as homology model refinement and docking. Solvent and membrane are represented by lower-resolution models to improve computation efficiency, while the accuracy is maintained comparable to high-resolution models. Finally, we also designed “aggressive” coarse-grained models for high-throughput discovery of GPCR ligands. We have utilized our latest coarse-graining methodology to optimize our coarse-grained models for good approximation of GPCR-ligand interactions. In summary, our study provides a full spectrum of GPCR-ligand models from low to high resolutions with a variety of potential applications, shining a light on the path to practical structure-based GPCR drug development.
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• In silico investigation of structure Isu1 and its complex with Jac1 using the coarse-grained UNRES force field
  Magdalena Mozolewska
  Chemistry Department, University of Gdansk (Poland)
  
  Yeast proteins Isu1 (Iron Sulfur protein) and Jac1 (J-protein) are a part of a huge ATP system and both interact with the Ssq1 molecular chaperone. In bacteria, the equivalent proteins are IscU (for Isu1) and HscB (for Jac1), respectively. Isu1 has influence on iron homeostasis in the mitochondrion where it is involved in assembling of iron-sulfur proteins. It can also be involved in the repair of iron sulfur clusters. Jac1 is involved with Hsp70 and Isu1 in Fe-S cluster biogenesis in mitochondria. The iron-sulfur clusters are the most ancient co-factors of proteins involved in many essential processes such as catalysis and electron transfer. The release of the Fe/S cluster from Isu1, and its transfer and incorporation into recipient apoproteins (Apo) is facilitated by components of the ISC assembly machinery including the ATP-dependent Hsp70 chaperone Ssq1, the DnaJ-like cochaperone Jac1, the nucleotide exchange factor Mge1, and the monothiol glutaredoxin Grx5. The disturbances of the balance of these processes can have very serious and dangerous consequences. In Homo sapiens, such disturbances can cause serious diseases such as cerebellar ataxia, myopathy, Friedreich’s ataxia, microcytic anemia, tumor suppressor. The aim of this work was to model the tertiary structure of Isu1 and predict the structure of the complex of Isu1 and Jac1, and also to investigate the interactions between Isu1 and Jac1 that may be crucial for understanding the machinery of yeast mitochondrial chaperone system. We also wanted to verify the results of earlier experimental studies, which suggest that Jac1 interacts with Isu1 mainly by Leu105, Leu109 and Tyr163. We used the I-TASSER server and YASARA as tools to model, by means of homology modeling, the tertiary structure of Isu1. Docking of Isu1 to Jac1 was carried out by using the ZDOK server. Because the complexes are large, we used coarse-grained molecular dynamics with the UNRES force field to optimize the geometry of the complexes; such an approach enabled us to run simulations at effectively millisecond timescale, which is inaccessible to all-atom simulations. We also modeled and, by means of coarse-grained molecular dynamics with UNRES, assessed the stability of complexes, in which we mutated the putative Isu1-binding residues of Jac1 to alanine residues. The simulated differences in the stability of complexes with mutated variants of Jac1 are in qualitative agreement with experiment.
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• Efficient arbitrary order implementation of anisotropic electrostatic interactions in the CHARMM simulation package
  Frank Pickard
  Laboratory of Computational Biology, National Institutes of Health
  
  Most molecular dynamics simulations are carried out using isotropic atom-atom potentials to model non-bonded interactions. Such potentials can be insufficient to accurately model a variety of physical properties present in biologically relevant molecules. A proper description of the anisotropy of the electrostatic interactions is of particular importance, as it directly affects a variety of structural and transport properties such as hydrogen bonding and diffusion. We have recently developed a novel, algorithm to efficiently calculate coulombic forces in the CHARMM simulation package using an arbitrary order multipole expansion. Our algorithm formally scales more efficiently than all current multipole based electrostatic implementations, and allow the inclusion of interactions through hexadecapole-hexadecapole for an additional cost of about an order of magnitude versus charge-charge potentials. We present details of the algorithm, implementation and initial calculations enabled by this work.
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• Elucidating the mechanistic dependence of Clp ATPase assisted protein remodeling on substrate protein topology
  Andrea Kravats
  Department of Chemistry, University of Cincinnati
  
  Clp ATPases actively participate in bacterial protein quality control, specializing in the selective destruction of damaged proteins. They assemble as hexameric ring structures encompassing a narrow central pore. Repetitive cycles of ATP binding and hydrolysis induce large scale conformational changes in highly flexible central pore loops to drive substrate protein(SP) unfolding and translocation. Remarkably, Clp ATPases are exceptionally versatile macromolecular machines, degrading proteins with diverse geometry and topology. While experiments have indicated that various SP\'s are processed by these assemblies at different rates, the molecular details of these processes remain unclear. We developed a novel coarse grained model coupled with Langevin dynamics simulations to elucidate the molecular details of Clp assisted unfolding and translocation of an all-alpha SP on biologically relevant timescales (1). To further investigate the effects of protein topology on Clp assisted unfolding and translocation, we have performed additional simulations with an alpha/beta folded SP (2). The results indicate a well preserved initial unfolding event by unraveling at the C-terminus for both SP’s, though the overall unfolding and translocation pathways diverge. The effective forces required for unfolding and translocation and the rate limiting step in the degradation pathway are determined by the mechanical stability of the SP. Due to the higher mechanical resistance of the alpha/beta SP, larger forces are required for SP processing. Translocation occurs in discrete steps for both SP’s, indicating a conserved power stroke mechanism. These results are in agreement with recent single molecule experiments. Assistance from accessory domains located on the exterior of the ATPase depends on unfolded intermediate structures and the respective timescales. Other minimalist approaches to simulating such large biomolecular systems employ mechanical pulling. Our results indicate that this approach captures few molecular details of Clp assisted mechanisms. AFM type simulations of mechanical unfolding reveal multiple unfolding pathways. Additionally, mechanical translocation by pulling through a rigid pore with constant velocity requires larger effective forces, while pulling with constant force results in simultaneous unfolding and translocation.
  1. A. Kravats, M. Jayasinghe, G. Stan. Proc. Natl. Acad. Sci. USA 108,2234-2239 (2011).
  2. A. Kravats, S. Tonddast-Navaei, G. Stan. Manuscript in preparation.
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• Molecular dynamics simulations of the folding of a five-helix bundle protein
  Yanxin Liu
  Department of Physics, University of Illinois at Urbana-Champaign
  
  The five-helix bundle lambda-repressor fragment is a fast-folding protein. A length of 80 amino acid residues puts it on the large end among all known microsecond folders. We studied the folding of a novel lambda-repressor fast-folding mutant in explicit solvent using all-atom molecular dynamics (MD) simulation. By means of a recently developed tempering method, we observed reversible folding and unfolding of lambda-repressor in a 10-microsecond trajectory. The folding kinetics was also investigated through a set of MD simulations run at different temperatures that together covered more than 125 microseconds. The protein was seen to fold into a native-like topology at intermediate temperature, and a slow-folding pathway was identified. The simulations suggest new experimental observables for better monitoring of the folding process, and a novel mutation is expected to accelerate folding. For a different lambda-repressor mutant, a short refolding time of 2 microseconds was reported in an earlier pressure-jump experiment. To investigate this pressure-jump induced fast folding behavior, MD simulations of more than 35 microseconds were carried out on the same lambda-repressor construct. High-pressure denatured states, generated through a high-temperature unfolding and high-pressure equilibration simulation procedure, were found to contain a significant amount of helical structure. Upon pressure drop, the protein refolded into the native state in 20 microseconds. The folding in the simulation is slower than the one measured in pressure-jump experiment, but faster than the folding time of 80 microseconds measured in temperature-jump experiment. A complete unfolding and refolding process was observed in the trajectory, which permitted the characterization of high-pressure denatured states and refolding pathway. The pressure jump simulations carried out for this study can be employed in the future to investigate slow-folding proteins through 10 to 100 microseconds MD simulations by inducing a fast folding phase.
  Reference:
  Structural characterization of lambda-repressor folding from all-atom molecular dynamics simulations. Y. Liu, J. Strumpfer, P. L. Freddolino, M. Gruebele, and K. Schulten. Journal of Physical Chemistry Letters, 3:1117-1123, 2012.
  Misplaced helix slows down ultrafast pressure-jump protein folding. M. B. Prigozhin, Y. Liu, A. J. Wirth, S Kapoor, R. Winter, K. Schulten and M. Gruebele. Proceedings of the National Academy of Sciences, in press, 2013.
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Contributed Talks

• Water and Hydrogen Bond Dynamics in the Channelrhodopsin-2 Light-Gated Proton Channel
  Christopher Mielack
  Department of Physics, Freie Universität Berlin (Germany)
  
  Channelrhodopsin-2 is a light gated cation channel used to control the trans- membrane potential in neurobiology applications. Understanding how channel- rhodopsin-2 works is important, because detailed knowledge of the active mechanism could help us understand and predict mutant proteins with specific characteristics of the reaction cycle. As a first step toward this aim, we investigate the coupling between water and protein dynamics for different protonation states of carboxylates thought to participate in proton-transfer reactions. We perform all- atom classical molecular dynamics simulations of the channelrhodopsin chimaera [1] in a hydrated POPC lipid membrane patch. We find that the protonation state largely affects interhelical hydrogen bonds within the pore region, and thus the opening of the ion-conducting channel. More- over, the protonation state impacts on a hydrogen-bonding network connecting the retinal binding pocket to the cytoplasmic side of the protein. This network allows passage of water only when the internal Asp195 is unprotonated. Our results highlight a strong coupling between the protonation state, water and protein hydrogen-bond dynamics in channelrhodopsin-2.
  This work has been supported by the Marie Curie International Reintegration Grant (FP7-PEOPLE-2010-RG 276920) and by the SFB 1078 Protonation Dynamics in Protein Function.
  References: [1] H. E. Kato, F. Zhang, O. Yizhar, C. Ramakrishnan, T. Nishizawa, K. Hirata, J. Ito, Y. Aita, T. Tsukazaki, S. Hayashi, and et al., Nature, vol. 482, pp. 369 - 74, Feb 2012.
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• Molecular Insights into the Reversible Fibril Formation of Tau Protein
  Yin Luo
  Department of Physics, Fudan University (P.R. China)
  
  The aggregation of the microtubule-associated tau protein into neurofibrillary tangles is considered as one of the important factors leading to Alzheimer disease. The thermal stability of such amyloid fibrils and the structures of the monomers are very important to understand the mechanisms of protein aggregation and fibril formation. Using a combined computational and experimental approach, we first studied the monomeric and oligomeric structures and three- and four-repeat of tau proteins -- K18 (130 aa) and K19 (99 aa), and then examined the stability of fibril-like oligomers. We found that the formation of tau protein fibril displays temperature-dependent feature and the fibril formed at elevated temperature can dissociated into monomer upon cooling. We performed replica-exchange molecular dynamics (REMD) simulations on both monomers and oligomers of K18 and K19. The simulations revealed that both tau protein monomers and oligomers have structural transitions around 330-340 K. We found that although tau is an intrinsically disordered protein, some regions have a high preference to adopt alpha-helix and beta-sheet structures. K18 and K19 monomers show distinct differences in contact probability map and number of salt-bridge, implying that repeat 2 (residues 275-305) significantly influences the global structure of tau protein. Our calculated C-alpha secondary chemical shifts of K18 and K19 monomers shows comparable correlations with the NMR-measured chemical shift [1]. REMD simulations on K18 and K19 oligomers showed that tau protein fibril would be stabilized at high temperature and dissociated at low temperature. Subsequent experiments confirmed that in the absence of heparin, the tau fibril can be formed and stabilized at 343 K, and dissociates back to monomers when cooling to 275 K. Addition of heparin promotes tau fibril formation and prevents cold dissociation process. Since K18 and K19 are highly charged, with K18 having 32 charged residues (+10 net charges) and K19 having 25 charged residues (+7 net charges), negatively charged heparin may locked the tau proteins and stabilize the fibril structures. Potential energy calculation indicated that these temperature-sensitive properties are largely correlated with the electrostatic interactions, especially the protein-solvent electrostatic interactions. [2]
  References:
  1. Marco D. Mukrasch, Jacek Biernat, Martin von Bergen, Christian Griesinger, Eckhard Mandelkow, and Markus Zweckstetter. \"Sites of Tau Important for Aggregation Populate beta-Structure and Bind to Microtubules and Polyanions.\" The Journal of Biological Chemistry (2005), 280: 24978-24986.
  2. Yin Luo, Paul Dinkel, Xiang Yu, Martin Margittai, Jie Zheng, Ruth Nussinov, Guanghong Wei, and Buyong Ma. \"Molecular Insights into the Reversible Formation of Tau Protein Fibrils.\" Chemical Communications (under review).
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• Strucuture prediction of single-span transmembrane dimers
  Sabareesh Subramaniam
  Departemnt of Biochemistry, University of Wisconsin(Madison)
  
  a Membrane proteins are an important class of proteins whose structural characterization with experimental methods is challenging. We have developed a computational method to predict the 3D structure of dimers of single-span transmembrane helices. This method, called CaTM , currently predicts symmetric homodimers and will be extended to other oligomeric configurations. CaTM is designed to predict a certain subset of oligomeric interactions - structures that are mediated by the formation of weak interhelical hydrogen bonds - along with favorable packing of the side chains. These hydrogen bonds, which occur between the Cα atoms (the donor) and a backbone carbonyl group on the opposite helix (Cα-H⋯O=C ), are frequently observed in the transmembrane regions of proteins. These backbone-to-backbone interactions need to be permitted by the side chain packing, requiring the presence of small amino acids at the interface. We have determined that only a fraction of the possible oligomeric space can accommodate Cα-H⋯O=C hydrogen bonds. CaTM takes advantage of these factors, incorporating techniques to prune the search space by exploiting the geometric and sequence requirements imposed by Cα-H⋯O=C hydrogen bonds. CaTM uses a precomputation stage where poly-glycine backbones are used to compile a “grid” of possible dimeric geometries/backbones that can accomodate Cα-H...O=C hydrogen bonds. This is followed by the compilation of “sequence rules” that specify incompatible amino acids for each position and backbone in the grid. Sequence rules are computed using a van der Waals function that detects atomic overlaps; a residue is deemed incompatible if it cannot be accommodated into a position with a favorable van der Waals score. A target sequence is threaded onto all the backbones in the grid that satisfy the sequence rules and an energy of association is computed for each backbone. The energy of association consists of a hydrogen bond term and a van der waals energy function. It is observed that the energy computed by CaTM is the lowest for structures that are closest to the NMR models. CaTM is capable of efficient large-scale screening and has been applied on the entire human genome. CaTM predicts the structure of known Cα-H⋯O=C mediated transmembrane homo-dimers with near atomic precision. For other proteins whose structures are yet undetermined, the predictions are corroborated by experimental results. The CaTM program is built using the open source C++ modeling library MSL.
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• Mechanism of Substrate Protein Binding and Release during the Allosteric Cycle of p97*
  Sam Tonddast-Navaei
  Department of Chemistry, University of Cincinnati
  
  ATPases associated with various cellular activities (AAA+) form a superfamily of ring-shaped motor proteins that utilize cyclical allosteric motions to remodel or translocate substrate proteins (SP) through a narrow central pore. The p97 ATPase is a homo-hexameric, double-ring, member of this superfamily that encloses a central channel with non-uniform width. A narrower compartment is present within the D1 ring and a larger cavity within the D2 ring, separated by a constriction formed by six His amino acids. The underlying mechanism of interaction between p97 and its SPs is currently unclear. We use molecular dynamics simulations to probe the interaction between p97 and an extended peptide substrate (SsrA-SsrA). Using mechanical force, SP was pulled into the pore from the D1 towards the D2 compartment or in the opposite direction. Our results reveal that the work needed for displacement of the SP in the D1-D2 direction is lower compared to the D2-D1 direction. These distinct energetic requirements originate in structural aspects and chemical properties of the pore lining. Whereas van der Waals interactions are dominant within the D1 pore, interactions within the D2 pore are strongly electrostatic. Arg599 and Glu554, two charged amino acids inside the D2 pore, provide the largest contribution to the interaction with SP and hinder translocation. SP threading requires smaller forces when the SP is pulled from the D1 side due to lower barrier to rotation of the His317 side chains in the direction of the D2 pore. Based on additional simulations of SP binding to two allosteric conformations of p97, we propose that SP binding and release from the pore involves a lever mechanism. In the open pore state, binding to the cavity wall of D2 ring takes place primarily through interactions with the side chain of Arg599 in which electrostatic interactions, including hydrogen bonds, with SP backbone are dominant. ATP-driven conformational changes of the D2 ring alter the chemical environment in the closed pore state. In this state, Glu554 side chains project further into the pore and dominate the interaction through van der Waals contacts with the SP backbone. Mutations at the two sites, in each of the states, indicate the specific requirement of these side chains for interaction with the substrate. * S. Tonddast-Navaei, G. Stan (manuscript submitted)
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• Simulation of epidemics with quarantine in host-pathogen interactions
  Akos Dobay
  Institute of Evolutionary Biology and Environmental Studies, University of Zürich (Switzerland)
  
  Simulation of epidemics with quarantine in host-pathogen interactions
  Abstract: The Susceptible-Infected-Recovered (SIR) represents a general framework in epidemiology based on ordinary differential equations (ODEs). It could be used to predict the size of epidemics, assuming the key parameters of the model can be estimated. Quarantine is one possible solution to limit the size of epidemics. Typically, infected individuals are removed from the population and are prevented from having physical contact with healthy individuals. A key factor for the success of a quarantine strategy is the carrying capacity of the facility. Here we develop a model where we explicitly introduce the carrying capacity of the quarantine facility into an SIR framework. We show how the model can address the propagation and control of contact and sexually-transmitted infections. We illustrate this by a case study of the city of Zurich during the 16th century. We also show how this framework can be used to address the degree of virulence in a host-pathogen interaction such as cell-virus interactions.
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• A Novel Screening Tool Indicative of Primary Sjogren‘s Syndrome versus Sicca Symptoms
  Michael Brown
  Biostatistics and Epidemiology, University of Oklahoma Health Science Center
  
  Jögren‘s syndrome (SS) is a chronic, progressive disease characterized by dry eyes and dry mouth resulting from lymphocytic infiltration of the lacrimal and salivary glands, respectively. Although SS is defined by keratoconjunctivitis sicca and xerostomia (dry eyes and mouth), the full spectrum of disease can involve a complex myriad of systemic symptoms similar to other rheumatic diseases. The American-European Consensus Group (AECG) classification criteria for SS require serologic and hematologic tests as well as the expertise of at least two clinical specialties for diagnosis. As such, proper diagnosis of SS is often challenging. In order to develop a model to better predict SS, we first assessed the predictive value of a commonly used screening tool, the AECG six-question phone interview, in 432 European American (EA) individuals with dry eyes and mouth using structural equation modeling (SEM) and logistic regression (LR). Then, using responses to 440 general health and medical history questions from 436 EA participants, we developed a novel predictive model using genetic algorithm (GA), SEM and LR. The six-question phone interview, which in our experience results in ~30% classification of SS, yielded an ill-fitting model with low prediction accuracy (~56% under violated assumptions) in silico. Our novel model generated from the general health and medical history questionnaire data satisfies multiple statistical fit criteria, obtains an accuracy of ~72%, and is clinically substantive. This is the first study to examine the value of general health and medical history questions in screening individuals for SS. Our predictive model, which includes questions indicative of dry eyes/mouth, history of autoimmunity, and fatigue and pain, is more accurate than the standard phone interview in distinguishing participants that will meet criteria for SS. Hence, this model could aid in the identification of SS patients a priori, thereby decreasing the cost of research studies and increasing the likelihood of patients receiving proper diagnoses and treatment.
  (co-authors: Gerard G. Dumancas1, Indra Adrianto1, Christopher J. Lessard1,2, Jennifer A. Kelly1, Kiely Grundahl1, Astrid Rasmussen1, Hal Scofield1, Kathy Sivils1,2, Courtney Montgomery1 1Arthritis and Clinical Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA; 2Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA)
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• Assessment of protein simulations in explicit and implicit solvents
  Duy Hua
  Medicinal Chemistry & Molecular Pharmacology, Purdue University
  
  Implicit solvent models have become increasingly popular in molecular dynamics simulations of biomolecules. Implicit solvent formalism is advantageous in comparison to explicit solvent formalism because it allows a reduction of system size, and presumingly a faster rate of phase space exploration. A long-standing question is whether implicit solvent formalism is realistic while being efficient. Much effort has been spent on determining the accuracy in estimating free energy from implicit solvent simulations. Phase space coverage is, however, a less frequently evaluated quantity. In order to compare the extent of phase space coverage between simulations in explicit and implicit solvents, we carried out multiple molecular dynamics simulations of free and bound SH2 domain in FACTS and TIP3P solvent models. Further, we assessed the effects that explicit and implicit solvents have on protein structure and dynamics. We utilized measurements such as dihedral angle and root-mean-squared fluctuation (RMSF) of backbone heavy atoms for comparison of structural and dynamical properties. Results show dissimilar distributions of phi-psi angles and RMSF values between simulations in TIP3P and in FACTS. The differences observed can be potentially attributed to interactions of protein residues with water molecules.
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• Investigating Ion Channel Activity of Human Islet Amyloid Polypeptide (amylin) Using Molecular Dynamics Simulations
  Jun Zhao
  Department of Chemical Engineering, University of Akron
  
  Formation of receptor-independent channels by hIAPP in the membrane is regarded as one of the membrane-damaging mechanisms associated with the dysfunction and death of pancreatic islet -cells in type II diabetes. Here, we investigate dynamic structure, ion conductivity, and membrane interactions of hIAPP channels in the DOPC bilayer using molecular dynamics simulations. In the simulated lipid environments, a series of annular-like hIAPP structures with different sizes and topologies are compatible with the doughnut-like images obtained by AFM, and with those of modeled channels for our peptide, and antimicrobial peptide PG-1, suggesting that loosely-associated -structure motifs can be a general feature of toxic, unregulated channels. All channels induce directional permeability of multiple ions across the bilayers. This study represents a first attempt to delineate some of the main structural features of the hIAPP channels, for a better understanding of the origin of amyloid toxicity and the development of pharmaceutical agents.
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• Molecular simulation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) bound to an open channel blocker GlyH-101.
  Shanthi Nagarajan
  Physiology and Pharmacology, Oregon Health Science University
  
  Cholera is an acute diarrhoeal disease caused by Vibrio cholera. The World Health Organization (WHO) estimates 3-5 million chorea cases worldwide, especially in underdeveloped countries, resulting in 100,000-120,000 deaths per year. Cholera toxin activates adenylate cyclase in cells of the intestinal mucosa, resulting in increased levels of intracellular cAMP, opening of apical cystic fibrosis conductance regulator (CFTR) Cl- channels and excessive salt and water secretion. CFTR channel blockers identified through high throughput screening could lead to treatments for several diarrheal diseases, but the molecular mechanism of blocker binding remains to be resolved. We have investigated CFTR structure and the mechanism of blocker binding by employing all atom molecular dynamics simulation. CFTR homology models were developed based on the crystal structure of the homologous ABC Transporter, Sav1866, captured in the outward facing, NBD dimerized state. The predicted structure of the transmembrane region of the current model has been refined according to experimental data. We studied the open channel blocker, GlyH101, initially docked in the CFTR model using an induced-fit procedure. Extensive MD simulation was applied to optimize the binding pose and identify key interactions between the blocker and pore elements. The CHARMM force field parameters for GlyH101 were optimized using the CGenFF parameterization procedure; and QM target data was generated using a Gaussian program for charge and dihedral fitting. The entire simulation was carried out using the NAMD Langevin dynamics protocol, with the CFTR channel inserted in pre-equilibrated, POPC lipid bilayer surrounded by TIP3P water molecules. Key interactions predicted in silco were compared with the results of cysteine-scanning and mutational experiments. To our knowledge this is the first report of a CFTR-open channel blocker simulation. Supported by NIH and the C.F. Foundation.
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• Understanding the biophysical nature of LSD1/CoREST dynamics using enhanced sampling simulation
  Nadeem Vellore
  Department of Medical Chemistry, University of Utah
  
  Aberrant demethylation of the H3-histone protein, an important epigenetic marker, is responsible for activating various regulatory genes involved in cancer development. Lysine Specific Demethylase (LSD1) in complex with its co-repressor protein CoREST catalyzes the demethylation of the H3-histone N-terminal tail and is a rapidly emerging epigenetic target for developing drugs against cancer and neurodegenerative disorders. The high sequence conservation of the H3 peptide within the SNAIL/SCRATCH superfamily of transcription factors unravels the potential of LSD1-CoREST as a multiple binding site for other small molecules, peptides, proteins, and chromatin. However, little is known to date on the relationship between LSD1/CoREST molecular recognition and the mechanisms of epigenetic regulation. Using long, brute-force MD simulation, we have previously shown that LSD1/CoREST is a highly dynamic nanoscale clamp; we proposed that the H3-histone N-terminal tail acts as an allosteric modulator by reducing the rotation motion of the clamp upon binding. Here, we present new studies on LSD1/CoREST clamp dynamics and substrate recognition using focused enhanced-sampling simulations and the integration of CPU peta-scale computing and GPU-accelerated calculations. We elucidate the recognition mechanism displayed by the H3-histone pocket and the thermodynamic forces driving binding. Overall, our study reveals an unprecedented view on LSD1/CoREST molecular recognition and paves the route for a more general understanding of epigenetic regulation at the molecular level.
  REFERENCES
  1. Shi, Y., Nat. Rev. Genet. 2007, 8, 829.
  2. Arrowsmith et al. Nat. Rev. Drug Disc. 2012, 11, 384. Br> 3. Forneris et al. LSD1: oxidative chemistry for multifaceted functions in chromatin regulation. Trends in biochemical sciences 2008, 33,181.
  4. Baron, R.; Vellore, N. A., LSD1/CoREST is an allosteric nanoscale clamp regulated by H3-histone-tail molecular recognition. PNAS 2012, 109, 12509.
  5. Baron, R.; Vellore, N. A., LSD1/CoREST reversible opening-closing dynamics: discovery of a nanoscale clamp for chromatin and protein binding. Biochemistry 2012, 51, 3151.
  6. Vellore NA.; Baron, R. An Induced-Fit Mechanism is Consistent With LSD1/CoREST- H3-Histone Molecular Recognition and Non-Covalent Binding as Revealed by Molecular Dynamics Simulations, J. Mol. Recogn. In-prep 7. Robertson et al. PLoS Comput. Biol. 2013, under review.
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Posters

• Characterization of a novel class of putative Sadenosylmethionine decarboxylase-like protein from Leishmania donovani
  Pragati Agnihotri
  Central Drug Research Institute
  
  In addition to the S-adenosylmethionine decarboxylase (AD) that is present in all organisms, trypanosomatids including Leishmania spp. pos sess an additional copy, referred to as the putative S-adenosylmethionine decarboxylase-like protein (ADL). Phylogenetic analysis confirms that ADL is unique to trypanosomatids, has several unique features, such as the lack of autocatalytic cleavage and distinct evolutionary lineage, even from AD of trypanosomatids. we have cloned, expressed, purified and characterized ADL from Leishmania donovani (L. donovani) using biophysical, biochemical and computational techniques. In Trypanosoma ADL was found to be enzymatically dead but playing an essenti al regulatory function by forming a heterodimer complex with AD. Biophysical studies show that unlike Trypanosoma, L. donovani ADL binds S -adenosylmethionine (SAM) and putrescine, which are the natural substrates of AD. Computational modeling and docking studies showed that i n comparison to ADs of other organisms including human, residues involved in putrescine binding are partially conserved while SAM binding residues are different. In silico protein-protein interaction study reveals that ADL of L. donovani can interact with AD. These results in dicate that ADL protein of L. donovani is a novel protein and may play an and essential role with protein and its completely different mode of function is completely different from known proteins of S-adenosylmethionine decarboxylase super family and may be considered as a potential suitable drug target.
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• Single Disulfide Bond Disruption in the 3 Integrin Subunit
  Lihie Levin
  Tel-Aviv University
  
  The integrins are a family of membrane receptors that attach a cell to its surrounding and play a crucial function in cell signaling. The combination of internal and external stimuli alters a folded non-active state of these proteins to an extended active configuration. The 3 subunit of the platelet IIb3 integrin is made of well-structured domains rich in disulfide bonds. During the activation process some of the disulfides are re-shuffled by a mechanism requiring partial reduction of some of these bonds; any disruption in this mechanism can lead to inherent blood clotting diseases. In the present study we employed Molecular Dynamics simulations for tracing the sequence of structural fluctuations initiated by a single cysteine mutation in the 3 subunit of the receptor. These simulations showed that in-silico protein mutants exhibit major conformational deformations leading to possible disulfide exchange reactions. We suggest that any mutation that prevents Cys560 from reacting with one of the Cys567-Cys581 bonded pair, thus disrupting its ability to participate in a disulfide exchange reaction, will damage the activation mechanism of the integrin. This suggestion is in full agreement with previously published experiments. Furthermore, we suggest that rearrangement of disulfide bonds could be a part of a natural cascade of thiol/disulfide exchange reactions in the IIb3 integrin, which are essential for the native activation process.
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• The effect of the load on the shape of Kap FABP4 complex
  Ortal Amber Vitos
  Tel-Aviv University
  
  The Gene-Activating lipophilic compounds are carried into the nucleus when enclosed inside a Fatty acid Binding Proteins (FABP). This protein is recognized by the karyopherins (Kap) through its Nuclear Localization Signal (NLS). The system is capable to discriminate between activating and non activating compounds (linoleate vs. palmitate); even that it is hidden deep inside the hydrophobic binding site of the FABP. The mode of this discrimination is still unresolved. In the present study we introduced molecular dynamics as a tool for clarifying the mechanism of the selectivity. In the first phase we simulated the complex between a short NLS peptide with Kap (ARM) and on the basis of the solution structure of this complex, projected the required properties of FABP4-ARM complexes. On simulations of the FABP4-ARM complexes each of the proteins retained their individual structure, but the relative orientation of the protein, one with respect to the other, differed in accord with the nature of the load. FABP4 that bears a transportable ligand forms a complex that resembles the solution structure of the peptide-Kap complex. When the FABP4 is in its Apo state or loaded by non-transportable ligand, different contacts are establish affecting the overall shape of the complex and its electrostatic <93>fingerprint<94>. It is suggested that these variations are large enough to affect the recognition of the binary complex by the Kap and its interactions with the GF Rich proteins that are crucial for the passage through the nucleopore.
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• Computer simulation of solvation forces
  Adil Güler
  Marmara University
  
  In this work the solvation forces in colloidal solutions are investigated by means of extensive usage of computer simulations. The double-layer model is used. The results are investigated in comparison with the experiments and the theoretical works and also some previous simulation works.
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• Modeling the effect of length and crowding on the mechanics of microtubule protofilaments.
  Kelly Theisen
  University of Cincinnati
  
  Cytoskeletal filaments, such as microtubules (MTs), have a variety of cellular roles. They serve as tracks for molecular motor transport, participate in cell division, and are regulated by a diverse range of associated proteins. The ability to perform all these roles depends on the mechanical properties of the MTs, which are based on the MT lattice structure, and modulated by molecular motors and MT associated proteins. Our previous simulations (1,2) showed that the mechanical response of a tubulin dimer and an MT protofilament (PF) is highly sensitive to the orientation and point of application of external forces. Here, we focus on the effect of varying the length and degree of complexation of an MT PF. In particular, we studied a complex of MT PFs, consisting of three PFs joined together by lateral contacts. We found that the flexibility of PFs, as determined by the spring constant in the linear regime of bending, increases with PF length. Our most striking result is that the addition of the lateral interfaces, which represent the weakest interactions in the MT lattice, has a significant effect on the mechanics of the system. Namely, the energy required to depolymerize the PF decreases dramatically compared to the energy required to depolymerize an isolated PF. This is due to crowding effects similar to the behavior of protein folding in the cellular environment (3,4), and is manifested as a high degree of cooperativity between the lateral and longitudinal interfaces in the full MT cylinder (5). Ultimately, our results are relevant for understanding the mechanism of severing enzymes such as spastin and katanin (6).
  (1) Joshi, H. et al.; Biophys. J. 2010, 98:657-666
  (2) Theisen, K.E., et al.; J. Phys. Chem. B 2012, 116:8545-8555
  (3) Cheung, M.S. et al.; Proc. of the Nat. Acad. of Sci. 2005, 102:4753-4758
  (4) Pincus, D.L. and Thirumalai, D.; J. Phys. Chem. B 2009, 113:359-368
  (5) Theisen, K.E., et al.; To be submitted to J. Chem. Phys.
  (6) Sharp, D.J. and Ross, J.L.; J. Cell Sci. 2012, 125:1-9
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• Self-assembly of di-/tri-phenylalanine peptides: insights from molecular dynamics simulations
  Cong Guo
  Fudan University
  
  Peptide nanostructures have attracted great attention lately due to their potential applications in nanotechnology. Among short peptide building blocks, the phenylalanine oligopeptides have been extensively studied. Recent experimental studies reported that diphenylalanine(FF)-based peptides self-assembled hollow nanovesicles and nanotubes, whereas the related triphenylalanine(FFF)-based peptides assembled into solid plate-like structures and nanospheres. The assembly mechanism and the detailed structural characteristics of FF/FFF nanostructures, as well as the molecular basis of structural differences between FF and FFF nanostructures remain poorly understood. Here we investigate the assembly processes of FF or FFF peptides by performing coarse-grained molecular dynamics simulations starting from random configurations. First, by simulating the assembly of FF peptides at different concentrations, we find that FF peptides assemble into hollow nanovesicles, nanotubes and other shapes of vesicle-like structures through the concentration-dependent pathways. At high concentrations, the self-assembly occurs through the formation, bending and closure of bilayers. At low peptide concentrations, it involves the fusion of small nanovesicles and bilayers. Second, we simulate the assembly of FF or FFF peptides at the same concentration and compare the two systems. We find that FFF peptides assemble into dry and closely packed nanospheres and nanorods, without the appearance of intermediate bilayers during the assembly process. Although the assembly of both FF and FFF peptides is mostly driven by side chain-side chain (SC-SC) aromatic stacking interactions, the relative contribution of main chain-main chain (MC-MC) interactions in FFF system are stronger than that in FF system. Thus FFF peptides are predominantly antiparallel-aligned and can form larger sizes of beta-sheet-like structures than the FF peptides do, which leads to the formation of solid FFF nanostructures. The different extent of competition between MC-MC and SC-SC interactions results in the different nanostructures formed by the two peptides. Our work provide molecular insights into the self-assembly mechanism and the molecular organization of short aromatic di-/tri-phenylalanine peptide assemblies, which would be helpful for the design of bioinspired peptide nanostructures.
  Reference:
  1. X. Yan, P. Zhu, J. Li, Chem. Soc. Rev. 39, 1877 (2010).
  2. C. Guo, Y. Luo, R. Zhou, G. Wei, ACS Nano 6, 3907 (2012).
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• DNA Topology and its Ramifications
  Junalyn Navarra-Madsen
  Texas Woman's University
  
  This paper briefly surveys topological approaches utilized in modeling protein-DNA interactions; focuses on knot theoretical models of certain recombinases, topoisomerases and transposases; and explores the possibility of employing a more powerful knot and link invariant, quandle-coloring, to generalize the current models.
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• Persistently Differentially Expressed Genes in Mouse Erythroleukemia Cells Post Short Induction with DMSO
  Steven Pennington
  Oklahoma State University
  
  Determination is the process which a stem cell commits to differentiation. The process for how a cell goes through determination is not well understood. Determination is important for proper regulation of cell turn over in tissue and maintaining the adult stem cell population. Deregulation of determination or differentiation can lead to many diseases such as several forms of leukemia. Murine erythroleukemia (MEL) cells are a retrovirus-induced pluripotent cell line extensively studied regarding transcription affecting stages of differentiation to erythrocytes. Because known indicators of erythroid development can be used as biomarkers for determination ( SCHUTTE et al. 2012), irreversible changes in these expression profiles may identify candidate genes regulating determination. To identify candidate genes involved in determination we inducing MEL cells with DMSO for a short time and allowing them to grow for the deration of their differentiation time (about 8 days). Persistently differentially expressed genes are then identified with microarrays. Preliminary results indicate a large number of differential expressed genes, including several transcription factors specific to erythropoiesis such as GATA1 and others. Furthermore the data indicates several potential determination events in erythropoiesis.
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• A molecular dynamic insight into the cross seeding of Aβ and amylin peptides fibril like oligomers
  Workalemahu Berhanu
  University of Oklahoma
  
  Recent epidemiological data has shown that patients suffering from Type 2 Diabetes Mellitus (T2DM) exhibit an increased risk to develop Alzheimer<92>s disease (AD) and vice versa, and a molecular link between AD and T2DM has been suggested based on structural similarities of the amyloid peptides implicated in these diseases. There is a major overlap (90%) in the structural properties of amylin20<96>28 and Aβ 25<96>33. Furthermore, the U-shaped β-strand-turn-β-strand motif has been observed in both Aβ and Amylin fibril structures. Cross-sequence interaction between Aβ and amylin has been observed in vitro, leading to amyloid hetero-assembly. This cross interaction may provide the molecular explanation for the apparent link between AD and type II diabetes. Detailed structural information about the protein<96>protein contact sites could be difficult to determine experimentally and MD simulation can complement experiment and give an insight into an interface interaction. In this study we investigated conformational changes and the interaction of Aβ-amylin hetero-assembly using classical MD simulations on octamer oligomer of amylin, Aβ and a mixture of both peptides (four strands from each). We construct a model oligomer structures for the cross seeding of amylin and Aβ based on the atomic model of amylin and Aβ fibril U-bend conformation. The simulation of Aβ, amylin and the mixture of Aβ-amylin octamer oligomer revealed the oligomers retained the U-shaped β-strand-turn-β-strand motif in the three independent 300-ns simulations at 310 K, with the hydrophobic core in the β-sheet-β-sheet interface strongly contributing to the stability of the aggregates. This indicates β-sheets in the hydrophobic core and main chain hydrogen bonds are crucial in the self and cross seeding of Aβ and amylin. We found Aβ serves, as a good template for the growth of amylin and vice versa. Waters molecules were not initially inside the β-strand-turn-β-strand motif pore of the oligomers, but they permeate during the course the simulation in support of the commonly accepted toxicity of β-rich amyloid oligomers. The simulation suggests self-seeding and cross seeding is guided by the compatibility of the protein monomers with the conformation of the template seed. This cross interaction may provide the molecular explanation for the apparent link between AD and type II diabetes. The structural insights may also serve as a molecular guide for rational design of inhibitors that can target the toxic fibril like oligomers of Aβ and amylin oligomers.
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• Oklahoma Cyberinfrastructure Initiative
  Henry Neeman
  University of Oklahoma
  
  The Oklahoma Cyberinfrastructure Initiative (OCII) encourages Oklahoma academics to use, on par with locals, centrally owned CI resources at the University of Oklahoma (OU) and Oklahoma State University (OSU): supercomputers, a Condor pool, storage resources, an emerging virtualized server pool, and Education, Outreach & Training (EOT) and Workforce Development activities. OCII capabilities and activities enable a tremendous span of research and education statewide: * Access to cyberinfrastructure. Currently, OCII has over 800 HPC users in Oklahoma: over 500 at OU, over 200 at OSU, and over 75 at 23 other Oklahoma academic, government and nongovernmental institutions, plus over 150 elsewhere. * Dissemination: The Oklahoma Supercomputing Symposium is an annual conference since 2002 -- over 2500 attendees from 101 academic in 27 US states and territories,; 119 companies, 35 government; 17 non-governmental.
  * Education: \"Supercomputing in Plain English\" (SiPE) workshop series: 11 talks about HPC, taught with stories, analogies and play rather than deep technical jargon have reached 248 institutions (academic, government, industry, nonprofit) in 47 US states and territories and 10 other countries.
  * Faculty & Staff Development: Workshops held at OU and OSU on advanced computing, computational chemistry and computational biology have been sponsored by a va riety of national organizations. * Outreach: SiPE overview talk (24 OK academic, including every public university in OK).
  * Proposal Support: Commitment letters for access to OCII resources; collaborations on projects.
  * Technology: Acquired or helped acquire technology (e.g., network upgrade, mini- or full supercomputer, high definition video camera) for that institution (14 OK academic).
  * Workforce Development: The Oklahoma Information Technology Mentorship Program provides \"A Day in the Life of an IT Professional\" presentations to courses across all of postsecondary education (and even secondary), plus other opportunities. Institution types have included career techs, community colleges, regional universities and PhD-granting universities. So far, 35 institutions have had 78 visits, plus 3 institutions have visited OU for a daylong series of presentations. Included in OCII\'s capabilities are deployments of WebMO, a web front end to a variety of chemistry applications (especially Gaussian), and OCII is in the process of deploying Galaxy, a similar web front end to a variety of bioscience applications.
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• Nitric Oxide Coupling Mediated by Iron Porphyrins
  Jun Yi
  University of Oklahoma
  
  The conversion of two molecules of nitric oxide (NO) to reduced nitrogen compounds such as the less toxic N2O is an essential step in the global nitrogen cycle. Importantly, binding of NO to iron porphyrins results in (por)Fe(NO)-containing products (H2por = porphyrin), some of which are biologically relevant, e.g., in mammalian blood pressure regulation. Fungal nitric oxide reductases (NORs) utilize monometallic heme active sites for NO reduction, while bacterial NORs (bacNORs) utilize active sites with dimetallic heme/non-heme-Fe centers to reduce NO to N2O. We use density functional theory calculations to examine the factors that control the fundamental N<96>N bond formation step during the NO coupling reaction mediated by a single iron porphyrin. The presence of an axial imidazole ligand, an extra electron, and importantly a proton, enhance this N<96>N bond formation; the proton addition has the greatest effect.
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• Nitric Oxide Coupling Mediated by Iron Porphyrins
  George Richter-Addo
  University of Oklahoma
  
  The conversion of two molecules of nitric oxide (NO) to reduced nitrogen compounds such as the less toxic N2O is an essential step in the global nitrogen cycle. Importantly, binding of NO to iron porphyrins results in (por)Fe(NO)-containing products (H2por = porphyrin), some of which are biologically relevant, e.g., in mammalian blood pressure regulation. Fungal nitric oxide reductases (NORs) utilize monometallic heme active sites for NO reduction, while bacterial NORs (bacNORs) utilize active sites with dimetallic heme/non-heme-Fe centers to reduce NO to N2O. We use density functional theory calculations to examine the factors that control the fundamental N<96>N bond formation step during the NO coupling reaction mediated by a single iron porphyrin. The presence of an axial imidazole ligand, an extra electron, and importantly a proton, enhance this N<96>N bond formation; the proton addition has the greatest effect.
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• Improvement of the treatment of loop structures in the UNRES force field
  Pawel Krupa
  University of Gdansk
  
  The UNited RESidue (UNRES) coarse-grained model of polypeptide chains developed in our laboratory enables us to perform effectively millisecond-scale molecular-dynamics simulations of large proteins. It performs well in ab initio prediction of protein structure, as demonstrated in the last CASP10 community-wide experiment of blind prediction. However, the resolution of the simulated structure is too coarse, especially in loop regions, which results from insufficient specificity of the model of local interactions. To improve the representation of local interactions, in this work we introduced new side-chain backbone correlation potentials, derived from statistical analysis of loop regions of 4585 proteins. To obtain sufficient statistics, we reduced the set of amino-acid-residue types to five groups, based on statistical analysis of the amino-acid properties. The new correlation potentials are expressed as one-dimensional Fourier series in the virtual-bond-dihedral angles involving side-chain centroids. The weight of these new terms was selected based on a trial-and-error method, in which Multiplexed Replica Exchange Molecular Dynamics (MREMD) simulations were run on selected test proteins. The obtained value of 0.57, for which average RMSD score was the lowest, is close to the value of RT factor at room temperature. The obtained conformational ensembles were analyzed in detail by using the Weighted Histogram Analysis Method (WHAM) and Ward’s minimum-variance clustering. This analysis showed that the RMSD dropped by 0.5 Å on average, compared to simulations without the new terms and in some test-proteins RMSD value decreased up to 33 %.
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• Simulation of Viral Capsid Self-Assembly
  Jef Wagner
  University of California Riverside
  
  Across the virus world the protein shell surrounds the genome take on a great many shapes, including icosaheadra, cylinders, cones, and irregular spheroidal particles. We have developed a coarse grained physical model for the viral capsid growth, treating each protein as a single triangular shaped monomer that binds edge to edge. The model has only three parameters: the ratio of the bending to stretching moduli, the preferred curvature, and the minimum angle inducing the formation of pentamers. We present a survey of the capsid shapes over the full range of parameter space.
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• Off-Lattice Montel Carlo Simulation on Nanophotothermal Therapy of Cancer cells by Laser Heating Single-walled Carbon nanotubes (SWNTs)
  Feng Gong
  University of Oklahoma
  
  A novel off-lattice Monte Carlo model was developed to simulate the nanophotothermal therapy of cancer using near-infrared radiation and single-walled carbon nanotubes (SWNTs). Wherein, a heterogeneous biological systems were built with normal tissue, cancer cell, as well as randomly orientated and distributed SWNTs. Meanwhile, heat transfer was monitored and modeled as the results of random movements of discrete thermal walkers containing same energy amount. Thermal boundary resistance, the resistance to heat flow at the interface between any two phases, was taken into account among the normal tissue, cancer cell and SWNTs. Both temperature increase of the cancer cell and effective thermal conductivity were calculated with different volume fraction of SWNTs, laser irradiation time and thermal boundary resistances. Results demonstrated the cancer cell temperature increased as the thermal boundary resistance between tissue and cancer cell increased, but decreased with the rise of thermal boundary resistance between cancer cell and SWNTs as well as the resistance between tissue and SWNTs. Besides, longer laser irradiation time and bigger volume fraction of SWNTs both induced higher temperature of the cancer cell. However, effective thermal conductivity decreased as the thermal boundary resistance between tissue and cancer cell increased, but increased with the rise of thermal boundary resistance between cancer cell and SWNTs as well as the resistance between tissue and SWNTs.. These intriguing results have definitely paved a way for theoretical instruction on the photothermal therapy of cancer using near-infrared radiation and highly thermal conducive carbon nanotubes.
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• The Investigation of Oligomeric Assembly of a Small Heterotetrameric Protein
  Fatih Yasar
  Hacettepe University
  
  Understanding the functional and structural properties of protein is the one of the main tasks for biomolecular studies. Many strategies are proposed and studied both experimentally and theoretically. One of them is known oligomerization because it was reported that while many proteins are inactive in the monomer state, these are the active dimers or higher-order oligomers. As an oligomeric proto-type, homo and heterotetrameric proteins were chemically synthesized with 21-residue peptides that provide the mixed alpha/beta structure. Hence, these small protein complexes can be used as a model to get insight to self-assembly of protein and protein-protein interactions. In present work, we have studied chemically synthesized heterotetrameric protein titled BBAheT1. This small protein consisting of 84 residues in total is derived by computer-aided design based on the structure of BBAT2. We performed multiplexed replica exchange molecular dynamics simulations with the UNRES force field to determine the folding and association pathway of this heterotetramer in solution.
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• Estrogenic Activity of PCB-30<92>s CYP2D6 and CYP3A4 metabolites: an integrated computational and cell-based approach
  Jason Harris
  University of Tennessee
  
  Endocrine disruption via interference with the estrogen pathway is affected by the cytochrome P450 (CYP450) metabolism of environmental compounds. In this study, in silico and experimental approaches were integrated to show that PCB-30 (2,4,6-trichlorobiphenyl) is estrogenically activated following oxidation by the CYP450 enzymes 3A4 and 2D6. Potential oxidation sites on PCB-30 and subsequent binding affinities for the P450-oxidized metabolites were evaluated using a combined knowledge-based and structure-based approach (SMARTCyp combined with molecular docking). Hydroxylation of PCB-30 was predicted to occur at the 3, 3\', or 4 phenol positions with the hydroxylated metabolites having a stronger binding affinity to the estrogen receptor than the parent molecule and the 4-hydroxy-PCB-30 exhibiting the strongest affinity towards the estrogen receptor. Experimental validation of these computational predictions was performed using CYP2D6 and CYP3A4 microsomal reaction mixtures (MRMs) to generate hydroxylated metabolites of PCB-30 which were then screened by bioassay measurements using a bioluminescent yeast reporter system expressing the alpha human estrogen receptor (hER-á). In agreement with the computational approaches, CYP2D6 and CYP3A4 MRMs generated metabolites with significantly higher hER-á activity than PCB-30. These results indicate that an integrated computational and experimental approach can be used to predict the estrogenic effects of environmentally and pharmaceutically relevant compounds and their metabolites.
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• Monte Carlo Simulation of A Polyelectrolyte with Smeared Charge Distribution
  Sahin Uyaver
  Istanbul Commerce University
  
  Using Monte Carlo simulations the behavior of a polyelectrolyte chain having smeared charge distribution is studied in different solvent conditions. Standard Metropolis is applied to the system with three different Monte Carlo moves, local move, pivot move and reptation move. Equilibrium quantities are obtained and the results are checked with the scaling laws. The effect of smeared charge distribution is discussed with the comparison of the other charge distribution. The polyelectrolyte chain is simulated in good, poor and theta solvent conditions and the solvents effects to the dramatic changes in the conformations of the chain are analyzed.
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• Solvent Effects and Dynamic Averaging of 195Pt NMR Shielding in Cisplatin Derivatives
  Kiplangat Sutter
  Department of Chemistry, SUNY Buffalo
  
  Solvent and dynamic effects on the 195Pt NMR chemical shifts of cisplatin and three cisplatin derivatives in aqueous solution were computed by combining ab initio molecular dynamics (aiMD) simulations with all-electron relativistic density functional theory NMR shielding tensor calculations. Structural analyses support the presence of a solvent-assisted “inverse” or “anionic” hydration. The aiMD-averaged chemical shifts agree with experiment to within about 5% on average (1% of the PtII chemical shift range)
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• Origin of the pH-dependent conformational dynamics of AcrA
  Zhi Yue and Jana Shen
  Department of Pharmaceutical Sciences, School of Pharmacy University of Maryland, Baltimore
  
  The tripartite efflux pump AcrA-AcrB-TolC is responsible for the intrinsic drug resistance of Escherichia coli. The periplasmic adaptor protein AcrA participates in the efflux process by forming a complex with both the outer-membrane channel TolC and the inner-membrane transporter AcrB. Recent crystallographic structures of AcrB revealed the mechanism of substrate transport driven by the proton motive force. However, the exact role of AcrA remains elusive. Here we present a computer simulation study of the pH-dependent conformational dynamics of AcrA. We employed a state-of-the-art technique, the continuous constant pH molecular dynamics based on a hybrid-solvent scheme and the pH replica-exchange sampling protocol. We found that the protonation of several titratable residues at the periplasmic pH disrupts the interacting network which in turn alters the local conformational flexibility. Our data are consistent with existing EPR experiments and offer new atomic-level insights which may contribute to the development of therapeutics for targeting the efflux pump.
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• Characterization of the Slow-Binding Inhibition by Acetopyruvate of the Dihydrodipicolinate Synthase from E. coli
  Lilian Chooback, William Karsten and Priscilla Seabourn
  Department of Chemistry, University of Central Oklahoma
  
  Dihydrodipicolinate synthase (DHDPS) catalyzes the first step in the biosynthetic pathway for production of L-lysine in bacteria and plants. The kinetic mechanism is ping pong with pyruvate binding to free enzyme and L-aspartate-b-semialdehyde (ASA) binding to the F enzyme form. The enzyme is feedback inhibited by the end-product L-lysine which reduces activity by ninety percent compared to the uninhibited activity. The enzyme has received interest as a potential drug target since the enzyme is not present in mammals. Acetopyruvate is a slow-binding inhibitor of DHDPS competitive versus pyruvate with an initial Kis of about 25 mM and a final inhibition constant of about 4 mM. The enzyme:acetopyruvate complex displays an absorbance spectrum with a lmax at about 303 nm and a longer wavelength shoulder. The rate constant for formation of the complex is 0.03 s-1. The enzyme forms a covalent enamine complex with the first substrate pyruvate and can be observed spectrally with a lmax at 275 nm. The spectra of the enzyme in the presence of pyruvate and acetopyruvate shows the initial formation of the enamine intermediate followed by the slower growing in of the E:acetopyruvae spectra with a rate constant of 0.005 s-1. The presence of lysine effects the E;acetopyruvate spectrum by decreasing the absorbance at the lmax and increasing the relative size of the long wavelength shoulder providing evidence for binding of L-lysine to the complex. The enzyme is proposed to form a covalent Schiff base between acetopyruvate and K161 on enzyme that subsequently deprotonates to form a resonance stabilized anion similar to the enamine intermediate formed with pyruvate.
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• Bacteriophage prohead RNA interacts with ATPase in biological nanomotor
  Xiaobo Gu
  Department of Chemistry and Biochemistry, University of Oklahoma
  
  The biological nanomotor in Bacillus subtillis bacteriophage phi 29 can package the 19kb DNA genome into the viral capsid against a load as high as 57pN with a maximum force exceeding 100 pN, thus filling the capsid to higher than 50% capacity and reaching internal pressures as high as 60 atm. The ATPase and pRNA form the core of this bionanomotor. Although the biomotors in phi29 and GA1 have the same function, the sequence similarity of ATPase and pRNA are as low as 53% and 12%, respectively. To better understand the mechanism of this unique RNA-dependent powerful nanomotor, the GA1 pRNA and its ATPase binding are studied and compared to the phi29 system. The ATPase gene was cloned into the pTBSG1 vector and overexpressed in E. coli BL-21 DE3 pRare cells. The ATPase binds specifically to the wild-type GA1 pRNA and the hairpin containing the GA1 bulge loop of UAAA. The optical melting experiments show that metal ions dramatically increase the melting temperatures of both GA1 and phi29 bulge loops through specific metal ion-RNA interactions. Nuclear magnetic resonance (NMR) is an effective technique to probe the tertiary structure and dynamics of the bulge loop. The 13C HSQC NMR spectrum shows that the bulge loop binds metal ions. The H1 -H8 NOESY walk suggest that the adenines in the bulge loop stack in the ensemble average conformation. The 13C HSQC and 13C relaxation studies reveal a wide range of base dynamics with the bulge loop and its closing pairs. Identifying the structural features and dynamics of pRNA and the interaction between pRNA and ATPase will contribute to understanding the mechanism of this biological nanometer.
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