Abstract Session Viewer

PB

Code: PB	Time Slot/Poster Number: 018	Session: Dynamics & Computation, Poster
Spin-Lattice Relaxation Study of Reorientational Dynamics of Imidazolium-Based Ionic Liquids: Effect of Methylation at the 2-position of Imidazolium Ring
Takatsugu Endo; Mamoru Imanari; Hiroko Seki; Keiko Nishikawa
Chiba University, Chiba, Japan
Abstract
Ionic liquids (ILs) are salts with low melting point and have some outstanding properties as solvents. It is known that a proton at the 2-position of imidazorium ring plays an important role in the interaction between a cation and an anion, and methylation at the 2-position changes physical properties of ILs significantly. In this study, we have compared reorientaional dynamics of 1-butyl-2,3-dimethylimidazolium bromide to that of 1-butyl-3-methylimidazolium bromide to clarify the effect of the methylation at the 2-position by measuring spin-lattice relaxation times (T1) of 13C. The results show that the methylation affects not only the motion of the base of the butyl group but also all the other 13C motion in the cation.

Code: PB	Time Slot/Poster Number: 019	Session: Dynamics & Computation, Poster
Cardinal Series Filter for NMR Signals
Dimitri Bytchenkoff; Stéphane Rodts; Teddy Fen-Chong
Ecole Nationale des Ponts et Chaussées, Champs-Sur-Marne, France
Abstract
We designed a digital low pass filter suitable for processing truncated NMR signals. In developing our algorithm we were inspired by the Bayesian approach to solving inverse problems. Our method consists in fitting raw NMR data with a finite series of truncated cardinal sine functions and requires nothing but the signal being band-limited. We devised sensible and, in practice, hardly restrictive rules for setting parameters of the filter and applied it to various computer-simulated and experimentally measured data sets to demonstrate its filtering performance and tolerance towards signal truncations. We believe that our method can be particularly useful for processing data collected in numerous NMR experiments in which relevant information is extracted direct from the signal in the time domain.

Code: PB	Time Slot/Poster Number: 020	Session: Dynamics & Computation, Poster
Dynamic activation of an allosteric regulatory protein
Shiou-Ru Tzeng; Charalampos G. Kalodimos
Rutgers University, Piscataway, NJ
Abstract
Allosteric regulation is efficient to control protein activity. Cyclic AMP (cAMP) switches catabolite activator protein (CAP) from the 'off' state (inactive), which binds DNA weakly and non-specifically, to the 'on' state (active), which binds DNA strongly and specifically. In contrast, cAMP binding to a single CAP mutant, CAP-S62F, fails to elicit the active conformation; yet, cAMP binding to CAP-S62F strongly activates the protein for DNA binding. NMR and thermodynamic analyses show that despite the fact that CAP-S62F-cAMP(2) adopts the inactive conformation, its strong binding to DNA is driven by large conformational entropy originating in enhanced protein motions induced by DNA binding. The results provide strong evidence that changes in protein motions may activate allosteric proteins that are otherwise structurally inactive.

Code: PB	Time Slot/Poster Number: 021	Session: Dynamics & Computation, Poster
Protein Proton-Proton Dynamics from Solution NMR measurements of Spin Flip Rates
Daniel Weaver; Erik Zuiderweg
University of Michigan, Ann Arbor, Michigan
Abstract
Proton spin-flip rates (kappa) are measured with ηzκ -TROSY. κ detects nano-pico second time scale dynamics, which in turn reports on configurational entropy. We define an interproton order parameter Q as the ratio of experimental κ rates to theoretical κ rates. The mean Q is 0.81 ± 0.02 for calmodulin and 0.89 ± 0.02 for peptide-bound calmodulin. The increase in Q confirms the known high entropic cost of this process. Excellent data is also obtained for the nucleotide-binding domain of Hsc70 (44 kDa). The median Q is found to be 0.86 ± 0.03 for this protein, with values as low as 0.3 for some areas, revealing major dynamical phenomena in large proteins.

Code: PB	Time Slot/Poster Number: 022	Session: Dynamics & Computation, Poster
Spin Relaxation Studies of the Dynamics of Leu5-Enkephalin in Aqueous Solution and in the Presence of Model Membrane Surfaces
Michael M. Fuson; Christopher L. Bennett; Colin. H. Murphy
Denison University, Granville, OH
Abstract
Leu5-enkephalin is a pentapeptide (YGGFL) believed to adopt conformers stabilized by membrane surfaces before binding to opiate receptors. Using 13C NMR spectroscopy, we have measured the coupled relaxation of methylene groups (13CH2) at several locations in the peptide in aqueous solution and in the presence of model membrane surfaces (isotropic DMPC/DHPC bicelles) and over a range of temperatures. From these experiments, we have calculated rotational diffusion constants for three sites in the two environments using several motional models. For example, for the leucine Cβ methylene the anisotropy of motion is low at all temperatures in aqueous solution, while increasing significantly with temperature in the presence of bicelles.

Code: PB	Time Slot/Poster Number: 023	Session: Dynamics & Computation, Poster
Probing the Folding Mechanism of PBX-HD using NMR and DSC
Patrick J. Farber; Hariyanto Darmawan; Anthony Mittermaier
McGill University, Montreal, CANADA
Abstract
It has been suggested that many marginally stable proteins fold non-cooperatively, with no significant barriers to separate the energy landscape into distinct thermodynamic states. Here we present an approach for studying the cooperativity of rapid protein folding with a combination of DSC, NMR relaxation dispersion experiments, and an analysis of the temperature dependence of amide 1H and 15N chemical shifts. We show that the native form of the protein undergoes two-state exchange with a small population of the thermally-denatured form, well below the melting temperature. This result directly demonstrates the coexistence of distinct folded and unfolded forms, and establishes that folding of PBX-HD is cooperative.

Code: PB	Time Slot/Poster Number: 024	Session: Dynamics & Computation, Poster
Anisotropic Collective Motions in Crystalline Proteins
Józef R. Lewandowski¹; Loïc Salomon²; Hans Jürgen Sass³; Julien Sein¹; Guillaume Bouvignies²; Stephan Grzesiek³; Martin Blackledge²; Lyndon Emsley¹
¹Université de Lyon/CNRS/ENS-Lyon/UCB-Lyon 1, Lyon, France; ²Protein Dynamics and Flexibility, IBS J-P. Ebel, Grenoble, France; ³Biozentrum, University Basel, Basel, Switzerland
Abstract
We present examples of Anisotropic Collective Motion analysis including the analysis of protein GB1 based on spin-lattice relaxation data at multiple magnetic fields. We compare the ACM of the β-sheet in GB1 resulting from the analysis of spin-lattice relaxation rates of a crystalline sample to solution RDCs. In addition we present a number of developments in protein dynamics methodology including measurements of site-specific 13C spin-lattice relaxation in microcrystalline [U-13C,15N]-GB1.

Code: PB	Time Slot/Poster Number: 025	Session: Dynamics & Computation, Poster
Dynamics of the Nucleotide Binding Domain of Na+/K+ ATPase by NMR Spectroscopy
Dan Meng; Roberto Salinas; Lei Bruschweiler-Li; Rafael Bruschweiler
Flordia State University, Tallahassee, FL
Abstract
Na+/K+ ATPase, a ubiquitous membrane P-type pump of eukaryotes, transports Na+ and K+ across the plasma membrane. A series of crystal structures of Na+/K+ ATPase and the solution structures of the nucleotide binding domain (NBD) in apo and ATP bound states are available. However, the detailed mechanism of the pump function is still not clear. Here we use relaxation studies to describe the dynamics of NBD in the ATP free and bound states. Fast backbone dynamics of NBD (ps-ns) is determined by measurements of the 15N longitudinal (T1) and transversal (T2) relaxation rates, and heteronuclear NOE (1H-15N) at 800 MHz. Lipari-Szabo model-free analysis is used to extract mobility parameters associated with the internal motions of N-H bond vectors.

Code: PB	Time Slot/Poster Number: 026	Session: Dynamics & Computation, Poster
Probing the Internal Dynamics of Thrombin by NMR
Brian Fuglestad¹; Marco Tonelli²; Elizabeth Komives¹
¹University of California, San Diego, San Diego, CA; ²National Magnetic Resonance Facility at Madison, Madison, WI
Abstract
Thrombin, a procoagulant serine protease, binds thrombomodulin and switches activity to an anticoagulant. Previous H/D exchange studies by mass spec have shown that there is a reduction of dynamics in the active site loops as well as a beta strand that runs from the trombomodulin binding site to the thrombin active site. Higher resolution NMR experiments will provide a more complete picture of the dynamics change that leads to the change in thrombin activity. Resonance assignments must first be made and are in progress, already showing line broadening in the beta strand consistent with exchange in this region.

Code: PB	Time Slot/Poster Number: 027	Session: Dynamics & Computation, Poster
MAS NMR Studies of Dynamics of Tyr Residues in HIV-1 Capsid (CA) Protein Assembly
Yun Han^{1, 2}; Guangjin Hou^{1, 2}; In-Ja L. Byeon^{2, 3}; Jinwoo Ahn^{2, 3}; Jason Concel^{2, 3}; Angela M. Gronenborn^{2, 3}; Tatyana Polenova^{1, 2}
¹University of Delaware, Newark, DE; ²Pittsburgh Center for HIV Protein Interactions, Pittsburgh, PA; ³University of Pittsburgh School of Medicine, Pittsburgh, PA
Abstract
In mature HIV-1 virions, the 25.6 kDa CA protein assembles into capsid cores that enclose the RNA viral genome. Previous studies of HIV-1 CA protein indicate that Tyr145 may play a pivot role in HIV-1 capsid assembly; however, the molecular mechanism including the dynamic behavior of this residue is unknown. We have recorded 15N chemical shift anisotropies and dipolar order parameters of selectively labeled 13C, 15N -Tyr HIV-1 CA protein assemblies of conical morphologies. Y145 exhibits reduced 15N chemical shift anisotropies, strong temperature dependence of the 15N and 13C isotropic chemical shifts, and large peak intensity variations in the 2D NCA spectra. These results are indicative of high backbone mobility of Y145 in the CA assemblies.

Code: PB	Time Slot/Poster Number: 028	Session: Dynamics & Computation, Poster
Dynafold: A provably optimal algorithm for protein structure determination from N-H and Cα-Hα Residual Dipolar Couplings collected in two alignments.
Rishi Mukhopadhyay; Homayoun Valafar
University of South Carolina, Columbia, SC
Abstract
Collection of Residual Dipolar Couplings from protein samples in multiple alignment media has become increasingly feasible. However, none of the algorithms for protein structure calculation from RDCs alone can guarantee the optimality of the results. A new algorithm, Dynafold, for the calculation of protein backbone structures from N-H and Cα-Hα RDCs collected in two alignment media is presented. Results have been collected for synthetic and experimental data. Synthetic data was generated for protein 2J01 with +/-1 Hz uniformly distributed error. Experimental data was taken from the BMRB for protein 1P7E and for protein 1BRF. For regions where RDC data could be collected, backbone RMSD was less than 1 angstrom, with all regions correctly oriented with respect to each other

Code: PB	Time Slot/Poster Number: 029	Session: Dynamics & Computation, Poster
NMR reveals backbone dynamics encode functional and inhibitory states in protein kinase A
Larry R Masterson; Gianluigi Veglia
University of Minnesota, Minneapolis, MN
Abstract
We present the amide backbone dynamics of Protein Kinase A (PKA) bound to a substrate and an inhibitor. A correlation exists between decreasing s-ms dynamics and decreasing catalytic efficiency. This dynamical encoding of functionality and inhibition appears to be synchronized at conserved loops, which toggle the enzyme between open and closed states. The rate constant for this motion is the same as the catalytic rate constant and likely rate-limiting to catalysis. We show that the active state of the enzyme is driven by favorable entropy upon substrate binding, while its inhibition is driven by favorable enthalpy upon inhibition. The internal entropic contribution to the events leading to catalysis could be a key to understanding the mechanism of PKA function.

Code: PB	Time Slot/Poster Number: 031	Session: Dynamics & Computation, Poster
Controlling Functional Motions in Ribonuclease A
Nicolas Doucet¹; J. Patrick Loria^{1, 2}
¹Department of Chemistry - Yale University, New Haven, CT; ²Molecular Biophysics & Biochem. - Yale University, New Haven, CT
Abstract
Understanding time-dependent fluctuations of a three-dimensional protein structure may allow us to control and design enzyme activity, which may have far-reaching implications for enzyme engineering and drug design. Using 15N-CPMG experiments on RNase A, we previously demonstrated the catalytic importance of long-range motional interactions implicating loop 1 and residue His48, both of which are linked to the active-site of the enzyme through an extensive network of noncovalent interactions. Here we present 15N-CPMG evidence pertaining to the control of exchange rates in this motional network using point mutants. We show that perturbations of noncovalent interactions at specific locations in RNase A do not completely abolish functional motions, suggesting that networks of coupled residue motions can be manipulated by mutagenesis.

Code: PB	Time Slot/Poster Number: 032	Session: Dynamics & Computation, Poster
Dynamics of the membrane anchored protein ARF-GTP
Yizhou Liu¹; Richard Kahn²; James Prestegard¹
¹university of Georgia, Athens, GA; ²Emory University School of Medicine, Atlanta, GA
Abstract
ARF is a GDP/GTP switch protein that regulates membrane vesicle formation. While ARF is soluble in its GDP-bound state, the GTP-bound ARF is anchored to the membrane through the myristoylated N-terminal helix. During our structural investigation of the bicelle-associated ARF-GTP, significant motion between the N-terminal helix and the C-terminal catalytic domain was noticed. The spatial distribution of this inter-domain motion was modeled by RDCs from multiple alignments and several sets of PREs. The resulting dynamic model of ARF-GTP provides insights into the modes of interactions between ARF and other proteins as they occur on the membrane surface.

Code: PB	Time Slot/Poster Number: 033	Session: Dynamics & Computation, Poster
Binding of Calcium is Sensed Structurally and Dynamically Throughout the Second Calcium-Binding Domain of the Sodium/Calcium Exchanger
Vincent Breukels; Geerten W. Vuister
Radboud University, Nijmegen, Netherlands
Abstract
The second Ca2+ binding domain is an important regulatory domain in the Na+/Ca2+ echanger. We report the effects of Ca2+ binding on the backbone relaxation rates and chemical shifts of the AD and BD splice variants of the second Ca2+ binding domain. To quantify the Ca2+-induced chemical shift changes, we also performed a comparative analysis of eight Ca2+-binding proteins that revealed large differences between different protein folds. The chemical shift and relaxation data together indicate that, in spite of the small structural changes, the Ca2+ binding event is felt throughout the molecule. The data suggests that the FG-loop plays an important role in connecting the Ca2+-binding event with the other cytosolic domains of the sodium-calcium exchanger.

Code: PB	Time Slot/Poster Number: 034	Session: Dynamics & Computation, Poster
Insight into the allosteric mechanism of Scapharca dimeric hemoglobin
Jennifer Laine¹; Brittany Morgan²; Francesca Massi¹
¹University of Massachusetts, Worcester , MA; ²Clark University, Worcester, MA
Abstract
Allosteric regulation is an essential function of many proteins that control a variety of different processes such as catalysis, signal transduction, and gene regulation. Structural rearrangements have historically been considered the main means of communication between different parts of a protein. Recent studies have highlighted the importance of changes in protein flexibility as an effective way to mediate allosteric communication. Scapharca dimeric hemoglobin (HbI) is the simplest possible type of allosteric system, with cooperative ligand binding between two identical subunits. In this study, we used NMR spin relaxation methods and H/D exchange experiments to investigate how changes in HbI flexibility contribute to cooperativity and how changes in protein dynamics are coupled to and correlated with structural changes upon ligand binding.

Code: PB	Time Slot/Poster Number: 035	Session: Dynamics & Computation, Poster
Structure-Independent Analysis of Distribution Radii for Disordered Groups in Macromolecules by Using 1H-1H Cross Relaxation Rates
Levani Zandarashvili; Junji Iwahara
University of Texas Medical Branch at Galveston, Galveston, Texas
Abstract
Very recently we have developed a formalism to determine distribution radii for disordered groups in macromolecules by using order parameters for long variable-length vectors such as those for paramagnetic relaxation enhancement (See the poster by Iwahara and Clore). Here we extend the formalism to 1H-1H cross relaxation rates for disordered groups in macromolecules. For a “diffusion in sphere” model, we derived the relationships between the distribution radii and 1H-1H cross relaxation rates. The theoretical relationships suggest that distribution radii for disordered groups can be determined from the 1H-1H NOE/ROE cross relaxation rates in favorable cases. We will present theoretical aspects of the formalism and actual data on 2H/15N-labeled ubiquitin.

Code: PB	Time Slot/Poster Number: 036	Session: Dynamics & Computation, Poster
Nuclear Magnetic Resonance (NMR) Studies of NADH Oxidase (NOX), a 46kDa Enzyme
Teresa Miletti
McGill University, Montreal, Canada
Abstract
Being thermophilic, NADH oxidase(NOX), a 46kDa homodimer, is stable at high temperature, but exhibits low activity below 70°C, the optimum physiological temperature of the organism. At 65°C, NOX catalytic activity increases ~7-fold compared to ambient temperature1. NMR was used to investigate the temperature dependence of NOX structure and dynamics. NOX was successfully expressed in E.coli, purified to homogeneity, and verified to be catalytically active. Backbone assignments were obtained as well as HSQC spectra from 25ºC to 70ºC. Residues with great chemical shift movement between 50ºC and 70ºC were mapped onto NOX molecular structure. Order parameters at these two temperatures were also derived from NMR relaxation experiments. These measurements suggest that temperature-dependent structural and/or dynamic changes occur in this domain.

Code: PB	Time Slot/Poster Number: 037	Session: Dynamics & Computation, Poster
Investigation of the dynamical properties of water in elastin by deuterium double quantum filtered NMR
Cheng Sun; Gregory Boutis
Brooklyn College of The City University of New Yor, Brooklyn , NY
Abstract
The anisotropic motion of tightly bound waters of hydration in bovine nuchal ligament elastin has been studied in our laboratory by deuterium double quantum filtered (DQF) NMR. The experiments have allowed for a direct measurement of the degree of anisotropy within pores of elastin over a time scale ranging from 100μs to 30ms, corresponding to a spatial displacement ranging from 0.2 to 7μm. We studied the anisotropic motion of deuterium nuclei in D2O hydrated elastin over a temperature of -15C to 37C and in solvents with varying dielectric constants. Our experimental measurements of the residual quadrupolar interaction as a function of temperature support an already existing notion of hydrophobic collapse near 20C.

Code: PB	Time Slot/Poster Number: 038	Session: Dynamics & Computation, Poster
Analytical derivatives of spin dynamics simulations
Ilya Kuprov; Christopher Rodgers
University of Oxford, Oxford, United Kingdom
Abstract
We report analytical equations for the derivatives of spin dynamics simulations with respect to pulse sequence and spin system parameters (couplings, shieldings, tensor orientations, pulse widths, phase shifts etc). The resulting derivatives may be used in fitting, optimization, performance evaluation and stability analysis of spin dynamics simulations and experiments.

Code: PB	Time Slot/Poster Number: 039	Session: Dynamics & Computation, Poster
Dynamics of Lysine Side-Chain NH3+ Groups in a Protein Studied by 15N Relaxation
Alexandre Esadze; Junji Iwahara
University of Texas Medical Branch, Galveston, TX
Abstract
We have analyzed the dynamics of lysine side-chain NH3+ groups in a protein by using heteronuclear 1H-15N NMR spectroscopy. Under our current experimental conditions, 1H-15N cross peaks from six NH3+ groups out of seven lysine residues in human ubiquitin are clearly observed, and they were assigned by using lysine-specific 1H/13C/15N triple resonance spectra. Although both NH3+ and methyl groups are AX3 spin systems, the heteronuclear relaxation analysis of NH3+ groups is more difficult due to their rapid hydrogen exchange. We designed and performed 15N relaxation experiments that are not affected by the scalar relaxation arising from the rapid hydrogen exchange. We will present the timescale of bond rotations for the NH3+ groups and order parameters for their symmetric axes.

Code: PB	Time Slot/Poster Number: 040	Session: Dynamics & Computation, Poster
Coupling NMR and Isothermal Titration Calorimetry to Identify Binding Cooperativity from an Induced Folding Event in Aminoglycoside N6'-Acetyltransferase-Ii
Lee Freiburger; Karine Auclair; Anthony Mittermaier
McGill University, Montreal, Canada
Abstract
Aminoglycoside-N6'- Acetyltransferase-Ii (AAC(6')-Ii is an antibiotic resistance enzyme which has some interesting biophysical characteristics. AAC(6')-Ii is a dimeric protein which contains two active sites each on separate monomers. There has been evidence of subunit cooperativity during binding of its substrates. We used Isothermal titration calorimetry to understand a hitherto unknown physical process which is responsible for the cooperativity during binding. Through the use of nuclear magnetic resonance (NMR) spectroscopy we gained information at the atomic resolution. Through coupling both ITC and NMR we were able to develop a model which explains the observed cooperativity which occurs during binding through both thermodynamic and structural processes.

Code: PB	Time Slot/Poster Number: 041	Session: Dynamics & Computation, Poster
Calibration of an experimental dynamical proxy for conformational entropy and illumination of its role in molecular recognition by calmodulin
Michael Marlow; Jakob Dogan; Kendra Frederick; Kathleen Valentine; Joshua Wand
Univ of Pennsylvania, Philadelphia, PA
Abstract
The physical basis for high affinity interactions involving proteins involves a range of energetic contributions. Among these are changes in protein conformational entropy, which cannot yet be reliably computed. We have recently employed changes in dynamics as a proxy for changes in conformational entropy of calmodulin upon association with domains from regulated proteins. This approach warrants a more quantitative foundation. Here we explicitly calibrate an “entropy meter” employing a dynamical proxy based on NMR relaxation and find that changes in the conformational entropy of calmodulin are a significant component of the energetics of binding. The distribution of motion at the binding interface is surprisingly non-complementary. These observations promote modification of our understanding of the energetics of protein-ligand interactions.

Code: PB	Time Slot/Poster Number: 042	Session: Dynamics & Computation, Poster
Certification of Molecular Dynamics Trajectories with NMR Chemical Shifts
dawei li; rafael bruschweiler
Florida State University, Tallahassee, FL
Abstract
Molecular dynamics ensembles of proteins generated by different force fields (AMBER ff99, ff99SB, ff03) have been quantitatively assessed based on their back-calculated Cα, Cβ, and C' NMR chemical shifts in comparison with experiment. The new force fields ff99SB and ff03 display better overall performance than the older ff99. A substantial improvement is found for ensemble averaged chemical shifts over individual snapshots for all three force fields, consistent with other NMR data. Since NMR chemical shifts are available for a vast number of proteins, this novel strategy opens up the possibility to quantitatively certify molecular dynamics simulations on a very large scale.

Code: PB	Time Slot/Poster Number: 043	Session: Dynamics & Computation, Poster
NMR Pulse Sequence Sketcher - a graphical tool to create NMR pulse sequences via the web browser.
Evgeny Fadeev
University of California, Irvine, CA
Abstract
Pulse Sequence Sketcher is a graphical tool for creation of NMR pulse sequences that works through the web browser and is a new addition to the spectroscopist tool chest hosted at NMR Wiki. In the beginning of the session user is presented with an empty canvas and is prompted to define hardware channels involved in the experiment. With those definitions in place user can pick up icons representing RF, gradient pulses and delays - drag and place them on the canvas. Users actions are transmitted to the web server via asynchronous HTTP requests driven by the in-browser javascript runtime. The web server component generates code in Bruker on Varian formats and returns them as a response to the client.

Code: PB	Time Slot/Poster Number: 044	Session: Dynamics & Computation, Poster
Estimating Population Distribution of the Glycosidic Linkage through Maximum Entropy Analysis of Spectroscopic Data using Different Priors
Elin Säwén²; Tariq Massad²; Clas Landersjö²; Göran Widmalm²; Peter Damberg¹
¹Tallinn University of Technology, Technomedicum, Tallinn, Estonia; ²Stockholm University, Stockholm, Sweden
Abstract
The characterization of the conformational preferences of flexible molecules is a fundamental problem in chemistry. Inference about the conformational ensemble is challenging as the ensemble may be arbitrary complex. In the poster the analysis of the glycosidic linkage of the disaccharide α-D-Manp-(1→2)-α-D-Manp-OMe is investigate through the joint analysis of a J-couplings, cross-relaxation, optical rotation, an explicit solvent force-field simulation and a database survey. In the process improved empirical Karplus relations where the effects of central and peripheral electronegative substituents as well as uncertainty and flexibility in the reference torsion angles are accounted for. The importance of the background information and the Karplus relations is apparent.

Code: PB	Time Slot/Poster Number: 045	Session: Dynamics & Computation, Poster
Algorithmic and Methodology Improvements of REDCRAFT
Mikhail Simin; Homayoun Valafar
University of South Carolina, Columbia, SC
Abstract
The increasing use of RDC data in recent years has necessitated the development of more effective analysis tools. Our lab has presented a viable approach for structure determination named REDCRAFT. The original shortcomings of REDCRAFT have motivated the development of a number of improvements, including structure refinement using Levenberg-Marquardt, and filtering based on provided order tensor (OT) estimates. This poster will present a number of examples that demonstrate the new capabilities of REDCRAFT. We present a back-bone RMSD between the structure 1a1z and a structure predicted by REDCRAFT without and with OT filtering, showing a structural backbone difference and successful structure determination respectively.

Code: PB	Time Slot/Poster Number: 046	Session: Dynamics & Computation, Poster
Studying Dynamics of Class A β‑lactamases with the use of a TEM-1/PSE-4 Chimera
Sebastien Morin¹; Christopher Clouthier^{1, 3}; Sophie Gobeil^{1, 4}; Joelle N. Pelletier^{1, 3}; Stéphane M. Gagné^{1, 2}
¹PROTEO, Quebec, Canada; ²Biochemistry and Microbiology, U. Laval, Quebec, Canada; ³Chemistry, U. de Montréal, Montréal, Canada; ⁴Biochemistry, U. de Montréal, Montréal, Canada
Abstract
Class A β‑lactamases are involved in resistance to antibiotics, a persistent phenomenon in medicine and agriculture. We previously investigated the dynamics of TEM-1 and PSE-4 β‑lactamases using NMR to try to understand why these are so efficient (kcat/KM nearly diffusion limited). Now also with the aim of obtaining insights into the evolutionary potential of these enzymes, we study an artificially engineered, catalytically active chimera of the class A TEM-1 and PSE‑4 β-lactamases: cTEM-17m. Kinetics data toward different β‑lactam substrates, backbone chemical shifts, 15N spin relaxation and CPMG relaxation dispersion data are presented for cTEM-17m, confirming the presence of previously observed features in TEM-1 and PSE-4.

Code: PB	Time Slot/Poster Number: 047	Session: Dynamics & Computation, Poster
Structural insights into protein recognition dynamics using a combined NMR/MD approach: Investigation of ubiquitin interactions with a UIM domain.
Nikolaos Sgourakis; Mayank Patel; Angel Garcia; George Makhatadze; Scott McCallum
Rensselaer Polytechnic Institute, Troy, NY
Abstract
We are presenting a combination of NMR relaxation experiments and microsecond-timescale MD simulations to study conformational exchange processes in protein-protein interactions. To illustrate the power of this approach, we focus on interactions between ubiquitin with a Uibiquitin-Interacting Motif (UIM). Analysis of CPMG relaxation dispersion and T1ρ relaxation measurements suggests the presence of two types of exchange processes, one directly related to the UIM interface, the other being induced to distal parts of the protein upon UIM binding. We perform all-atom MD simulations at the µsec timescale starting from the NMR ensemble in order to obtain a plausible structural model for the observed dynamics. Comparative analysis of our results indicates the structural basis of molecular recognition for this model domain-interaction system.

Code: PB	Time Slot/Poster Number: 048	Session: Dynamics & Computation, Poster
Molecular structure refinement by direct atomic coordinate fitting to magnetic resonance spectra
Matthew Krzystyniak¹; Gareth Charnock¹; Dimitri Svistunenko²; Ilya Kuprov¹
¹University of Oxford, Oxford, United Kingdom; ²University of Essex, Essex, United Kingdom
Abstract
We report an attempt to streamline structure determination of irregular (non-protein) molecules by introducing a direct structure fitting procedure, wherein the atomic coordinates are iterated directly against the experimental data (using DFT and spin dynamics simulations to get theoretical spectra directly from atomic coordinates).

Code: PB	Time Slot/Poster Number: 049	Session: Dynamics & Computation, Poster
Backbone Dynamics and Global Effects of an Activating Mutation in Minimized Mtu RecA Inteins
Zhenming Du¹; Yangzhong Liu²; David Ban¹; Maria Lopez¹; Marlene Belfort³; Chunyu Wang¹
¹Rensselaer Polytechnic Institute, Troy, NY; ²University of Science and Technology of China, Hefei, Anhui, P.R.China; ³Wadsworth Center, New York State Dept. of Health, Albany, New York
Abstract
Inteins mediate an autocatalytic post-translational modification, called protein splicing, which has found many applications in biotechnology and protein engineering. A single valine-to-leucine mutation (V67L) can globally enhance splicing and related cleavage reactions in minimized Mtu RecA inteins. However, the V67L mutation causes little change in crystal structures. To test if protein dynamics contribute to activity enhancement in the V67L mutation, the conformations and dynamics of the minimized and engineered intein Ihh-V67CM and a single V67L mutant, Ihh-L67CM, have been studied by solution NMR. Chemical shift perturbations established that the V67L mutation causes global changes, including changes at the N- and C-termini of the intein, which are active sites for protein splicing. The single V67L mutation significantly slows down hydrogen exchange rates globally, indicating a shift to more stable conformations and reduction in the ensemble distribution. Whereas the V67L mutation causes little change for motions on the ps-ns timescale, the motions on the s-ms timescale affect a region involving the conserved F-block histidine and C-terminal asparagine, which are important residues for C-terminal cleavage. The V67L mutation is proposed to activate splicing by reducing ensemble distribution of the intein structure and by modifying the active sites.

Code: PB	Time Slot/Poster Number: 050	Session: Dynamics & Computation, Poster
REDCAT Improvements and XplorGUI Introduction
Christopher Schmidt; Rishi Mukhopadhyay; Homayoun Valafar
University of South Carolina, West Columbia, SC
Abstract
Recent improvements to REsidual Dipolar Coupling Analysis Tool (REDCAT) have increased the usability of the tool. These improvements include simultaneous multiple alignment media analysis, a more advanced selection feature for including and excluding data from analysis, as well as others. We will also be releasing REDCAT's source code in the hopes of aiding the community with the development of stronger tools. We also introduce XplorGUI, a graphical user interface for use with Xplor-NIH. This tool will aid in the creation and execution of template scripts for use with Xplor-NIH. This will serve to speed the tedious and time consuming process of creating scripts for common structure minimization through Xplor-NIH.

Code: PB	Time Slot/Poster Number: 051	Session: Dynamics & Computation, Poster
Conformational Dynamics and Ligand Specificity of PKC alpha C1B Domain
Mikaela Stewart; Tatyana Igumenova
Texas A&M University, College Station, TX
Abstract
C1 domains are independently folded modules of ~50 amino acids that associate with lipid membranes in response to binding diacylglycerol and tumor-promoting phorbol esters. To understand the origin of ligand specificity in C1 domains, we characterized the C1B domain of Protein Kinase C alpha and its diagnostic mutant, Y123W, using solution NMR methods. The mutation did not perturb the C1B structure or the sub-nanosecond dynamics of the protein backbone, but resulted in significant changes in the conformational dynamics, as measured by NMR relaxation-dispersion methods. The most dynamically perturbed region is the hinges of the ligand-binding loops. The data suggest that the conformational dynamics in C1 domains play an important role in the mechanism of ligand recognition and binding.

Code: PB	Time Slot/Poster Number: 052	Session: Dynamics & Computation, Poster
Graphical user interface for the construction and visualization of large spin systems
Gareth Charnock; Ilya Kuprov
University of Oxford, Oxford, United Kingdom
Abstract
We introduce a compact and clean XML-based format for spin system description, which is the result of extensive consultations with the spin dynamics simulation community. The resulting files are human-readable, easy to edit and easy to parse using standard XML libraries. We also describe the prototype of the graphical user interface, which was designed to facilitate the setting up of complicated spin systems and is capable of saving the results as input files for most major spin dynamics simulation packages.

Code: PB	Time Slot/Poster Number: 053	Session: Dynamics & Computation, Poster
The Two Zinc-Finger Domains in NC Protein Dynamically and Semi-independently Engage Sites on the SL1 RNA
Jeetender Chugh; Xiaoyan Sun; Hashim M Al-Hashimi
University of Michigan, Ann Arbor, MI
Abstract
We have used NMR spectroscopy to study the dynamics of NC and its interaction mode with SL1 RNA. NMR chemical shift titrations of 15N labeled NC with unlabeled RNA revealed surprising differential exchange kinetics on the two zinc fingers (F1, F2). The distinct exchange kinetics suggested that F1 and F2 in NC interact with SL1 sites semi-independently with F1 exhibiting weaker interaction compared to F2. We further explored domains motions in unbound-NC to shed light on their relative independence using NMR relaxation and CPMG dispersion experiments. These inter-domain motions help explain their independent mode of binding to NC which we hypothesize allow NC to efficiently localize on SL1 but retain weak interactions needed for its catalytic activity and turn over.