Abstract Session Viewer

PJ

Code: PJ	Time Slot/Poster Number: 155	Session: Liquids Methods: Biomolecules, Poster
Protein Resonance Assignments by Hydrogen Deuterium Exchange and Chemical Shift Correlation
Wendy K Nkari; Hsiau-Wei Lee; Adam W Barb; James H Prestegard; Lu Meng; Kelley W Moremen
University of Georgia, Athens, GA
Abstract
NMR studies of proteins >30 kDa, or those requiring non-bacterial expression, are extremely challenging when relying on uniform isotopic enrichment and standard triple resonance techniques because they face problems of resonance overcrowding and signal loss during coherence transfer. However, sparse labeling where single or few amino acids are labeled, overcomes resonance overcrowding and, if suitable assignment strategies exist, they can provide valuable structural and functional data. Our assignment strategy relies on, correlating hydrogen-deuterium amide exchange rates from peaks of a native protein with its denatured form, and assigning the denatured spectrum using mass spectrometry sequenced peptides, denatured state NOEs, and literature chemical shift predictions. This methodology has been applied to Galectin-3 (16 kDa), DCSIGN (30 kDa) and Fucosyltransferase-III (37 kDa).

Code: PJ	Time Slot/Poster Number: 156	Session: Liquids Methods: Biomolecules, Poster
Nonuniform sampling for improving resolution in multidimensional NMR
Mehdi Mobli⁴; Alan S. Stern²; Wolfgang Bermel³; Glenn F. King⁴; Mark W. Maciejewski¹; Adam D. Schuyler¹; Jeffrey C. Hoch¹
¹Univ of Connecticut Health Ctr, Farmington, CT; ²Rowland Institute at Harvard, Cambridge, MA; ³Bruker BioSpin GmbH, 76287 Rheinstetten, Germany; ⁴The University of Queensland, St. Lucia, 4072, Australia
Abstract
Resolution in pulsed NMR depends on the largest evolution time sampled. For multidimensional experiments, the achievable resolution is frequently limited by available experiment time. Nonuniform sampling (NUS) in the time domain enables collection of data at long evolution times, without the requirement of sampling all intervening samples at the Nyquist interval. Different alternatives to Fourier transformation can be used to compute spectra from NUS data, including maximum likelihood (MLM) or Bayesian methods, multiway decomposition (MWD), back-projection reconstruction (BPR), and maximum entropy reconstruction (MaxEnt). Although many promising NUS strategies have been developed, the question of how to design an optimal sampling strategy remains unsolved. We describe recent progress in our lab in the application of nonuniform sampling in multidimensional NMR.

Code: PJ	Time Slot/Poster Number: 157	Session: Liquids Methods: Biomolecules, Poster
Characterization of an Enzyme Reaction by STD NMR Spectroscopy
Thomas Hackl; Bernd Meyer
University of Hamburg, Department Chemistry, Hamburg, Germany
Abstract
The enzyme germacren D synthase, a terpene synthase, was chosen as a model system for the investigation of hydrophobic protein ligand interactions by STD NMR. Because the interaction to the substrate farnesyl diphosphate can only be observed during catalytic transformation a modified pulse sequence was used acquiring STD spectra for short time intervals. The setup allows observation of substrate and product binding in a single experiment. At the same time the progress of the reaction can be obtained from the off-resonance spectra allowing a kinetic analysis. A closed form solution of the Michaelis-Menten equation, based on Lambert W function, was applied to derive the kinetic parameters.

Code: PJ	Time Slot/Poster Number: 158	Session: Liquids Methods: Biomolecules, Poster
NMR Detection and Structural Analysis of a Phosphoethanol-amine-Modified Glycosyl Diradylglycerol in the Polar Lipids of Clostridium tetani
Anthony A Ribeiro
Duke University, Durham, NC
Abstract
1D and 2D 1H, 31P and 13C NMR spectroscopy has identified a novel Phosphoethanolamine-Modified Glycosyl Diradyl- glycerol in the Polar Lipids of the pathogenic bacterium, Clostridium tetani, the cause of tetanus, which is widely prevalent among humans.

Code: PJ	Time Slot/Poster Number: 159	Session: Liquids Methods: Biomolecules, Poster
Applying 4-D Sparse Sampling and FFT-CLEAN Processing to NMR Studies of Large Proteins
Brian E. Coggins; Jonathan Werner-Allen; Cheng-Yu Chen; Bruce R. Donald; Pei Zhou
Duke University, Durham, NC
Abstract
One of the significant challenges in NMR studies of large proteins is spectral crowding. For several years, we have been developing techniques to address this using nonuniform sampling and FT processing, including sparse sampling based on concentric shells and the CLEAN algorithm for suppression of low-level undersampling artifacts, allowing high resolution 4-D experiments to be run quickly. We now report the application of this to several projects, including structural studies of the 23 kDa Ssu72 and sequential assignment of a 60 kDa protein. In the former, we assessed the quantitative accuracy of CLEAN, and obtained good correlations between peak volumes before and after. We will discuss these results in detail, as well as practical guidelines for applying these methods.

Code: PJ	Time Slot/Poster Number: 160	Session: Liquids Methods: Biomolecules, Poster
Structural basis of interactions between the CUE Domain of an Ubiquitin Ligase gp78 and K48-linked Polyubiquitin
Shan Liu; Andy Byrd
National Cancer Institute, Frederick, MD
Abstract
The human ubiquitin ligase, gp78, plays a critical role in the endoplasmic reticulum associated degradation (ERAD) pathway to regulate misfolded proteins. The CUE domain of gp78 is crucial to the enzyme activity, but its detailed function has not been clearly determined. Here we report the solution structures of a unique construct of the gp78 CUE domain, and investigate interactions of gp78 CUE with K48-linked di-ubiquitin and tetra-ubiquitin. gp78 CUE displays different on-off rates and binding affinities towards individual subunits of polyubiquitin and variant lengths of polyubiquitin, reflecting a complex mode of binding. This complexity is examined here with plausible interaction mechanisms which provide insights into the structure-function relationship of the gp78 CUE domain in the ERAD pathway.

Code: PJ	Time Slot/Poster Number: 161	Session: Liquids Methods: Biomolecules, Poster
Transferred RDCs, Induced by an artificial Lanthanide Binding Peptide
Martin J. Haehnke; Harald Schwalbe
Center for Biomol. Magnetic Resonance, Frankfurt, Germany
Abstract
A lanthanide-binding peptide tag with nanomolar affinity to trivalent lanthanide ions is attached to a short peptide that binds to ubiquitin. The alignment induced by the anisotropic magnetic susceptibility of the bound lanthanide ion when exposed to a strong magnetic field arises residual dipolar couplings not only in the peptide but also in the coordinated protein and may therefore be used to investigate protein-ligand complexes. Utilizing the UIM1 sequence from hepatocyte growth facor-regulated tyrosine kinase substrate and loaded with one equivalent of Terbium (III) we were able to measure residual dipolar couplings as well as paramagnetic relaxation enhancement and paramagnetic pseudocontact shifts on ubiquitin.

Code: PJ	Time Slot/Poster Number: 162	Session: Liquids Methods: Biomolecules, Poster
Heteronuclear Decoupling and Broadband Excitation Pulses by Multiply Modulated RF-Fields
Arthanari Haribabu²; Gerhard Wagner²; Navin Khaneja¹
¹Harvard University, Cambridge, MA; ²Harvard Medical School, Boston, MA
Abstract
We present the method of multiply modulated rf-fields, as a technique to perform heteronuclear decoupling in solution. The decoupling is understood by performing a sequence of coordinate transformations, each of which demodulates a component of the oscillating field to a static component, which progressively averages the chemical shift and dipolar interaction. We show that by increasing the number of modulations in the decoupling field, we improve decouplng performance. Each new modulation frame improves the ratio of the spread of the chemical shift frequencies and the effective rf-field. We finally describe how this principle can be used as a general technique to design broadband decoupling and excitation pulses which can be made immune to rf-inhomogeneity and strength of couplings

Code: PJ	Time Slot/Poster Number: 163	Session: Liquids Methods: Biomolecules, Poster
Preservation of CaM-lobes Linker Fast Dynamics in holo-CaM/peptide Complex from The Plasma Membrane Calcium Pump
Nenad Juranic¹; Slobodan Macura¹; John T. Penniston²; Elena Atanasova¹; Emanuel E. Strehler¹; Franklyn G. Prendergast¹
¹Mayo Foundation, Rochester, MN; ²Massachusetts General Hospital, Boston, MA
Abstract
In the holo-CaM/peptide complex from the plasma membrane calcium pump, which has CaM wrapped around the 28-residue helical peptide (PDP # 2kne), we observed low order parameter of the linker region, with a profile similar to the free holo-CaM profile. Yet, the rotational correlation time as well as the backbone RDC values are consistent with a rigid relative disposition of the CaM lobes. Molecular dynamics simulations on the whole complex (2kne) substantiated that finding, and also showed that fast sub-nanosecond local motions are sufficient to account for the low order parameter of the linker. This finding suggests that a low order parameter of the central linker does not exclude the possibility of two-lobes, wrap-around, binding of holo-CaM.

Code: PJ	Time Slot/Poster Number: 164	Session: Liquids Methods: Biomolecules, Poster
Progress in 13C direct detection for biomolecular NMR applications
Wolfang Bermel¹; Ivano Bertini²; Isabella C. Felli²; Klemens Kessler³; Rainer Kümmerle³; Roberta Pierattelli²
¹Bruker BioSpin GmbH, Rheinstetten, Germany; ²CERM, University of Florence, Sesto Fiorentino (FI), Italy; ³Bruker Biospin AG, Fällanden, Switzerland
Abstract
Recent progress in NMR instrumentation, such as the introduction of cryogenic technology in probes, and in the NMR methodology, such has the development of innovative experimental schemes, has greatly expanded the range of applications of 13C detected biomolecular NMR spectroscopy. Complete sequence specific assignment of a protein as well as the determination of several observables providing structural and dynamic information is now routinely possible. The impressive improvement in 13C sensitivity allows recording a 13C NMR direct detected 2D experiment (CON) in a few minutes and a 3D experiment in a few hours (CBCACON) on a 1 mM sample of ubiquitin. The experimental time of a multidimensional experiment can be further reduced with the introduction of non-uniform/sparse sampling techniques.

Code: PJ	Time Slot/Poster Number: 165	Session: Liquids Methods: Biomolecules, Poster
Temperature and pH effects on dynamic behavior and function of the innate immune receptor CD14
Seth Albright; Jagat Adhikari; Nitin U Jain
University of Tennessee, Knoxville, TN
Abstract
The structural basis for differential endotoxin pattern recognition by CD14 is unclear due to non-availability of high resolution structural data for the CD14-endotoxin complexes. Comparison of solution NMR structural data for binding interaction of CD14 with two bacterial ligands, Lipopolysaccharide and Lipoteichoic acid, indicates that specificity of endotoxin recognition is likely modulated by dynamic interaction of the carbohydrate moeties with charged residues in the hydrophilic rim of CD14. In order to further investigate the dynamic behavior of CD14, we have undertaken temperature and pH-dependence NMR studies of isotopically labeled CD14, results from which clearly demonstrate the dynamic contributions from several residues in CD14 and may provide a likely explanation for the broad specificity of ligand binding by CD14.

Code: PJ	Time Slot/Poster Number: 166	Session: Liquids Methods: Biomolecules, Poster
A New Look at the Heme Pockets of Ligated Hemoglobin
Yue Yuan; Virgil Simplaceanu; Nancy T. Ho; Chien Ho
Carnegie Mellon University, Pittsburgh, PA
Abstract
Recent studies by multinuclear NMR have shown that dynamics is an important factor, besides the structure, in the workings of the main oxygen transport agent, hemoglobin. Here, we re-visit the structural aspect in the characterization of the function of human hemoglobin, by investigating the relationship between ligand (oxygen) affinity, allosteric effectors (pH, temperature, IHP) and the structural changes of the heme pockets as detected by changes in the chemical shifts.

Code: PJ	Time Slot/Poster Number: 167	Session: Liquids Methods: Biomolecules, Poster
Observation of Excited-state Hoogsteen Base Pairs in Regular B-form DNA by NMR
Evgenia N Nikolova¹; Eunae Kim²; Abigail A Wise¹; Andricioaei Ioan²; Hashim M Al-Hashimi¹
¹University of Michigan, Ann Arbor, MI; ²University of California, Irvine, CA
Abstract
DNA structural dynamics at pico-to-millisecond timescales are perceived critical for biological processes, but the molecular nature of these transitions remains poorly characterized. We examine the motional signature of highly deformable CA/TG dinucleotide steps, various size adenine tracts known to assume alternative B-form conformations, and their juxtaposition using NMR carbon relaxation/relaxation dispersion. We uncover microsecond dynamics at various CA/TG doublets and characterize extensively their kinetic and thermodynamic profiles. Our findings including excited state chemical shifts are consistent with WC-to-Hoogsteen base-pair transitions at AT and GC sites, whereby the purine base undergoes an anti-to-syn reorientation. Further support for this hypothesis is provided by Targeted Molecular Dynamics and Conjugate Peak Refinement (CPR)-derived energy landscapes of anti-to-syn transitions, which yield comparable values to experiment.

Code: PJ	Time Slot/Poster Number: 168	Session: Liquids Methods: Biomolecules, Poster
Millisecond dynamics in the allosteric enzyme imidazole glycerol phosphate synthase (IGPS) from Thermotoga maritima
James M. Lipchock; Patrick Loria
Yale University, New Haven, CT
Abstract
IGPS is a 51 kDa heterodimeric enzyme comprised of two proteins, HisH and HisF, that catalyzes glutamine hydrolysis to produce NH3 in HisH and the cyclization of ammonia with N’-[(5’-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in HisF to produce IGP and 5-aminoimidazole-4-carboxamide ribotide (AICAR). PRFAR binding stimulates glutaminase activity by 5300-fold, despite the active sites being > 25 Å apart. This mechanism was investigated by solution NMR spectroscopy and CPMG relaxation dispersion experiments. In the absence of ligand, IGPS exhibits no μs-ms motions; however, ligand binding induces substantial backbone and sidechain dynamics as evidenced by dispersion and resonance broadening. These data provide evidence for a hybrid KNF/MWC model for allostery and potentially represents a unique example of entropically driven positive V-type allosteric regulation.

Code: PJ	Time Slot/Poster Number: 169	Session: Liquids Methods: Biomolecules, Poster
NMR Investigation of Integrin Signaling: Tale of Cytoplasmic Tails
Lalit Deshmukh; Vitaliy Gorbatyuk; Olga Vinogradova
University of Connecticut, Storrs, CT
Abstract
Cells in multicellular organisms have to continuously adjust and adapt in order to survive in a highly dynamic extracellular environment which requires a flow of information from extracellular matrix (ECM) to the cytoplasm. In metazoan, this process is controlled by Integrins, the ubiquitous heterodimeric glycoprotein adhesion receptors, which “integrate” the cells with their extracellular surroundings. We will be presenting the first 3D NMR structure of the full length integrin β3 cytoplasmic tail (CT) mono-phosphorylated at tyrosine-747. We will show that this phosphorylation causes significant conformational rearrangement in β3CT, surprisingly preventing the αIIb/β3 membrane-proximal clasp formation which is a key to maintain the receptor in the inactive state.

Code: PJ	Time Slot/Poster Number: 170	Session: Liquids Methods: Biomolecules, Poster
Detection of transient inter-chain interactions in the intrinsically disordered protein alpha-synuclein by NMR paramagnetic relaxation enhancement
Kuen-Phon Wu; Jean Baum
Rutgers University, Piscataway, NJ
Abstract
NMR paramagnetic relaxation enhancement experiments (PREs) have been applied to the intrinsically disordered protein alpha-synuclein, the primary protein in Parkinson’s disease, to directly characterize transient intermolecular complexes at neutral and low pH. At neutral pH, we observe weak N- to C-terminal inter-chain contacts that are driven by electrostatic interactions while at low pH, C- to C-terminal inter-chain interactions are significantly stronger and driven by hydrophobic contacts. Characterization of these first inter-chain interactions will provide fundamental insight into the mechanism of amyloid formation.

Code: PJ	Time Slot/Poster Number: 171	Session: Liquids Methods: Biomolecules, Poster
Reverse micelle encapsulation of membrane anchored proteins for solution NMR studies.
Kathleen Valentine¹; Ronald Peterson¹; Jamil Saad²; Michael Summers²; Xianzhong Xu³; James Ames³; Sarah Chung¹; Joshua Wand¹
¹Univ of Pennsylvania, Philadelphia, PA; ²HHMI and University of Maryland, Baltimore County, Baltimore, MD; ³University of California, Davis, Davis, California
Abstract
Perhaps 5-10% of proteins bind to the membranes via a post-translationally attached lipid such as myristate. High-resolution information about the nature of lipidated proteins is remarkably sparse, often because of inherent solubility problems. Reverse micelle encapsulation is used here to study two myristoylated proteins in their lipid-extruded states: myristoylated recoverin, which is a switch in the vision Ca+2 signaling pathway and the myristoylated HIV-1 matrix protein, which is postulated to be targeted to the plasma membrane through its binding to phosphatidylinositol(4,5)bisphosphate. Both proteins have been encapsulated in the lipid extruded state and high-resolution NMR spectra obtained. Both encapsulated proteins bind their activating ligands. This approach seems broadly applicable to such membrane anchored proteins that have eluded conventional structural characterization.

Code: PJ	Time Slot/Poster Number: 172	Session: Liquids Methods: Biomolecules, Poster
Structure and Topology of Insulin in a Membrane Environment
Vivekanandan Subramanian; Sathiah Thennarasu; Ayyalusamy Ramamoorthy
University of Michigan, Ann Arbor, MI
Abstract
Discrepancies in the regulation of human insulin, a 51-residue polypeptide hormone, are manifested in the disease states of type-2 diabetes and pancreatic cancer. Insulin’s indirect role in the regulation of membrane fluidity and membrane conductance, and the plasma membrane of human spermatozoa, suggest a receptor as well as non-receptor mediated interaction with the membrane. In an attempt to understand the role of lipids on the structure and dynamics of insulin, especially during amyloidogenesis, we studied the subtle variations in the secondary structure and their effect on the topology of insulin using NMR, Circular Dichroism, and fluorescence experiments. The ramifications of lipid-insulin and detergent-insulin interactions during the early stages of membrane mediated insulin self-association (amyloidogenesis) will be presented.

Code: PJ	Time Slot/Poster Number: 173	Session: Liquids Methods: Biomolecules, Poster
Deuterium Spin Probes of Backbone Dynamics in Proteins
Vitali Tugarinov; Devon Sheppard
University of Maryland, College Park, MD
Abstract
2H spin relaxation NMR experiments to study the dynamics of deuterated backbone alpha-positions, Da, are developed. It is shown that quantification of 2H relaxation rates at Da backbone sites and the derivation of associated order parameters of Ca-Da bond vector motions in small [U-15N,13C,2H]-labeled proteins is feasible. The utility of the developed methodology is demonstrated on a pair of small proteins - an 8.5-kDa ubiquitin at 10°C, 27°C, and 40°C and a variant of a 6.5-kDa protein GB1 at 22°C. In both proteins, the Da-derived parameters of the global rotational diffusion tensor are in good agreement with those obtained from 15N relaxation rates. Semi-quantitative solution state NMR measurements provide the average value of the quadrupolar coupling constant, QCC, for Da sites in proteins equal to ~174 kHz. Using the uniform value of QCC for all Da sites, we show that Ca-Da bond vectors are motionally distinct from the backbone amide N-H bond vectors, with 2H-derived squared order parameters of Ca-Da bond vector motions on average slightly higher than their N-H amide counterparts. Analogous measurements of longitudinal and transverse 2H spin relaxation rates of backbone amide deuterons (DN) in the [U-13C,15N]-labeled protein ubiquitin show that the utility of amide deuterons as probes of backbone order in proteins is compromised by substantial variability of DN QCC's from one amide site to another. However, using the dynamics parameters of 15N-2H bond vectors evaluated from 15N relaxation data, site-specific QCC values can be estimated directly from DN R1 and R2 rates providing useful information on hydrogen bonding in proteins. In agreement with previous indirect scalar relaxation-based measurements, the DN QCC values estimated directly from R1 and R2 2H relaxation rates correlate with the inverse cube of the x-ray structure-derived hydrogen bond distances in ubiquitin.

Code: PJ	Time Slot/Poster Number: 174	Session: Liquids Methods: Biomolecules, Poster
Characterization of Fast Local Dynamics in Biomolecules by Heteronuclear Double Resonance Methods
Simone Ulzega¹; Takuya Segawa¹; Mariachiara Verde¹; Fabien Ferrage²; Geoffrey Bodenhausen^{1, 2}
¹Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland; ²Ecole Normale Supérieure (ENS), Paris, France
Abstract
We have designed new Heteronuclear Double-Resonance (HDR) methods based on MLEV-32 and WALTZ-32 pulse sequences, applied simultaneously to two scalar-coupled spins, for the investigation of cross-relaxation of multiple-quantum (MQ) coherences. These methods can give access to correlated dynamic processes faster than those accessible to other methods. We have obtained analytical expressions, supported by experiments, to describe the effective relaxation of MQ coherences during HDR irradiation and the dependence of the effective MQ cross-relaxation rate on the applied radiofrequency (rf) amplitude, in the limit of fast exchange. Moreover, we have shown that the applied rf fields do not affect significantly the sample temperature. We present here applications of the HDR methods to the study of chemical-exchange-induced MQ cross-relaxation in proteins.

Code: PJ	Time Slot/Poster Number: 175	Session: Liquids Methods: Biomolecules, Poster
LpqH is the Enzymatic Target for LspA, a Drug Target for M. Tuberculosis
DaQun Ni^{1, 2}; QingZhe Ni²; Thach Van Can ^{1, 2}; Milton Truong^{1, 2}; Mukesh Sharma^{1, 2}; Timothy Cross^{1, 2}
¹NHMFL, Tallahassee, FL; ²Florida State University, Tallahassee, FL
Abstract
The lipoprotein LpqH (Rv3763) is an antigen in mycobacterium tuberculosis. Its N-terminal signal peptide is subject to LspA (Lipoprotein Signal Peptidase ) cleavage. Globomycin inhibits the activity of LspA. The uncleaved LpqH as a substrate of LspA was used to measure the activity and efficiency of LspA reconstituted either by detergent as protein-detergent micelles or by lipids as protein-liposome. NMR gNhsqc was used to monitor the folding of the four forms of LpqH in the lipoprotein processing process. The cleaved signal peptide was extracted from the reaction and quantified, that led to km and kcat parameters. These were further used to judge the efficiency of reconstituted LspA. Finally, the uniformly aligned LspA in lipid bilayer was run on ssNMR.

Code: PJ	Time Slot/Poster Number: 176	Session: Liquids Methods: Biomolecules, Poster
Site-specific backbone and side chain relaxation analysis using genetically encoded 15N/19F double labeled unnatural amino acids
Pan Shi; Chaowei Shi; Zhaoyong Xi; Changlin Tian
Univ. of Sci. & Tech. of China, Hefei, Anhui, CHINA
Abstract
Due to high gyromagnetic constant and high sensitivity to environmental change, 19F labeled amino acids can be applied to study protein’s structural and dynamics perturbation under different functional state. 19F labeling of proteins can be accomplished using unnatural amino acid tri-fluoromethyl-phenylalanine (tfmF) and artificial evolved orthogonal tRNA/RNA synthetase pairs. Using this method, a 19F labeled unnatural amino acid can be incorporated into a specific protein site. Here, we have synthesized 15N labeled tfmF, and this unnatural amino acid can be applied for site-specific backbone and side chain relaxation analysis of a protein. Structural and relaxation changes of SH3 domain from human vinexin protein was studied using 15N/19F-tfmF incorporated at several different sites, under the presence or absence of peptide ligand.

Code: PJ	Time Slot/Poster Number: 177	Session: Liquids Methods: Biomolecules, Poster
Phosphorylation and Pseudo-phosphorylation Shift Phospholamban Conformational Equilibrium toward Excited States as Revealed by NMR Spectroscopy
Martin Gustavsson; Nathaniel J. Traaseth; Raffaello Verardi; Larry R. Masterson; Gianluigi Veglia
University of Minnesota, Minneapolis, MN
Abstract
Phospholamban (PLN) is a 52-residue membrane-spanning protein that reversibly regulates SR Calcium ATPase (SERCA) and therefore has received much attention as a therapeutic target to treat heart failure. PLN is composed of a membrane-spanning helix connected to an amphipathic helix through a short loop and is in equilibrium between an ordered, membrane-associated T state and an excited, unfolded and membrane-dissociated R state. Using 13C-HSQC experiments of methyl groups combined with CPMG experiments and backbone chemical shifts we have quantitatively measured the population of ground and excited states for mutants of PLN in micelles and isotropic bicelles. We show how T and R states are in slow exchange with each other while simultaneously in fast exchange with a membrane-dissociated, unfolded state.

Code: PJ	Time Slot/Poster Number: 178	Session: Liquids Methods: Biomolecules, Poster
Solution structure of the RBD1,2 domains from human nucleolin
Sengodagounder Arumugam; M. Clarke Miller; James Maliekal; Paula Bates; John Trent; Andrew Lane
JG Brown Cancer Center, University of Louisville, Louisville, KY
Abstract
Nucleolin is a primarily nucleolar RNA packaging protein that has also been found in the cytoplasm and uniquely on the cell surface of cancer cells. It acts as a receptor for exogenous G-quartet DNA aptamer AS1411 that is currently in phaseI/II clinical trials for renal cell carcinoma and acute myelogenous leukemia. A high resolution three dimensional solution NMR structure of first two domains (RBD1,2) of the human nucleolin has been determined using 3D heteronuclear NMR techniques. Although the folding of the human RBD1,2 is similar to the homologous hamster RBD1,2 domain, differences are noted and will be discussed. Hence, this study would form structural basis for understanding the mechanism of binding of the DNA aptamer in detail.

Code: PJ	Time Slot/Poster Number: 179	Session: Liquids Methods: Biomolecules, Poster
New labels for RNA structure and dynamic characterization
Kwaku T Dayie; Chandar Thakur; Margaret Brown; Jacob Sama; Melantha Jackson; Gopal Narang
University of Maryland, College Park, MD
Abstract
NMR of RNA faces the perennial twin ills of signal overlap and decay as the molecules get bigger. We present efforts to make new isotopic labels that use the metabolic pathways of bacteria to produce custom labeled nucleotide monomers for downstream application in RNA synthesis for biophysical characterizations. This specific labeling provides a framework to work around common NMR problems of extensive 13C-13C coupling, overlap, and fast signal decay. This approach allows for higher resolution in structural analysis and accuracy in dynamic analysis.

Code: PJ	Time Slot/Poster Number: 180	Session: Liquids Methods: Biomolecules, Poster
NMR spectroscopic investigation of mobility and hydrogen bonding of the chromophore in the binding pocket of phytochrome proteins
Marco Röben¹; Janina Hahn¹; Eva Klein¹; Tilman Lamparter²; Georgios Psakis³; John Hughes³; Peter Schmieder¹
¹Leibniz-Institut für molekulare Pharmakologie, FMP, Berlin, Germany; ²Karlsruhe Institute of Technology, Karlsruhe, Germany; ³Justus Liebig Universität, Giessen, Germany
Abstract
Phytochromes are a member of the photosensory protein family. They feature a covalently bound bilin chromophore. Upon irradiation Z/E-photoisomerization occurs at the C15 double bond. Crystal structures gave insight into possible interactions sides of the chromophore with its protein surrounding. Still, various points are unclear regarding the phytochrome photocycle, e.g. the stabilization or mobility of the chromophore inside the binding pocket. We looked at the mobility of the bilin moiety in diﬀerent environments. Comparing the linewidth of the free chromophore in solution and diﬀerent protein environments, we found that the chromophore has diﬀerent properties regarding the mobility within the binding pocket. Furthermore we were able to identify protons of various amino acid side chains involved in hydrogen bonds.

Code: PJ	Time Slot/Poster Number: 181	Session: Liquids Methods: Biomolecules, Poster
GSD (Global Spectrum Deconvolution) in Metabolomics: Absolute Quantification and GSD Binning
Silvia Mari¹; Valeria Mannella¹; Giacomo Quilici¹; Giovanna Musco¹; Carlos Cobas²; Felipe Seoane²; Stanislav Sykora³
¹San Raffaele Scientific Institute, Milano, Italy; ²Metrelab Research, Santiago de Compostela, Spain; ³Extra Byte, Castano Primo, Italy
Abstract
NMR spectroscopy is an important metabolomic tool, but quantitative handling of the extremely complex spectra of biosamples presents a large number of practical problems. A novel algorithm known as GSD (Global Spectrum Deconvolution) overcomes many of the obstacles and even permits an automatic treatment of the data. We present the first controlled tests of the applicability of GSD to the multivariate analysis of complex metabolite mixtures (cell culture media). We also take advantage of GSD to implement the quantitative referencing strategy known as PULCON and bring it to a stage where one can perform absolute quantification even of metabolites whose peaks fall into crowded spectral areas. Again, controlled experimental tests of the GSD – PULCON method are presented.

Code: PJ	Time Slot/Poster Number: 182	Session: Liquids Methods: Biomolecules, Poster
Coupled Conformational Exchange, Ligand Binding and Oligomerization: NMR Line Shape and Isothermal Titration Calorimetry Analysis
Evgenii Kovrigin
Medical College of Wisconsin, Milwaukee , WI
Abstract
Biomacromolecules frequently exhibit conformational transitions coupled to ligand binding and/or formation of oligomers. Combination of NMR line shape analysis, relaxation dispersion measurements, Isothermal Titration Calorimetry (ITC) and other biophysical techniques is capable of resolving parameters of coupled transitions. Here I explore combined use of NMR line shape analysis and ITC titrations for complex binding models (LineShapeKin Simulation, http://lineshapekin.net) to simulate spectral and ITC patterns in a wide range of experimental conditions. Analysis of multiple models helps an experimentalist propose possible molecular mechanisms by simple inspection. To simultaneously fit experimental NMR line shapes, relaxation dispersion, ITC, stopped-flow and other data a new software package, BiophysicsLab, is being developed and will be available from http://biophysicslab.net.

Code: PJ	Time Slot/Poster Number: 183	Session: Liquids Methods: Biomolecules, Poster
Role of the N-terminal domain of Nephila clavipes Spidroin 1 in spider dragline silk self-assembly
Stuart Parnham
MUSC, Charleston , SC
Abstract
Very little is known about the structure and dynamics of MaSp-N domains and, more importantly, their contribution to spidroin function and/or assembly. NMR relaxation studies reveal that the 14 kDa MaSp-N acts as finely tuned sensors of ionic environment and dimerizes. Lower ionic strength promotes more stable association between dimer subunits (Figure 1) and we hypothesize that the dimerization has functional significance to the self-assembly process that leads to fiber formation The structure of the MaSp1-N monomer (stabilized by 500mM NaCl), an orthogonal bundle with five helices is well defined by a total of 1794 NOE distance restraints (265 long range, \|i-j\| ≥ 5). The backbone rmsd to the mean structure is 1.39 Å.

Code: PJ	Time Slot/Poster Number: 184	Session: Liquids Methods: Biomolecules, Poster
Advanced Cell Adaptation for Enhanced, Simplified Expression of Perdeuterated Proteins
Xinji Guo; James G. Kempf
Rensselaer Polytechnic Inst., Troy, NY
Abstract
High-yield expression of perdeuterated proteins is a common need in NMR spectroscopy. For large proteins (>20-30 kDa), deuteration levels to >90% are essential for NMR characterization of structure and dynamics. In addition, the low sensitivity of NMR demands this be achieved without major sacrifice to yield. We demonstrate a simple and effective approach to high-yield, perdeuterated expression, applied to a particularly complex system - human adult hemoglobin (Hb A). Cells were adapted for proliferation in a deuterated minimal environment. The method improved Hb A yield and deuteration levels of protein expressed from shake-flask cultures by 150% and 17%, respectively, compared to results from more traditional techniques. A discussion was also included of the improved spectra quality due to enhanced deuteration.

Code: PJ	Time Slot/Poster Number: 185	Session: Liquids Methods: Biomolecules, Poster
Investigation of Protein-Solute Interactions and Solute-Based Changes in the Protein-Inhibitor Interactions for HIV-1 Protease.
Mandy Blackburn; Gail Fanucci
University of Florida, Gainesville, FL
Abstract
A major drug target in AIDS therapy is HIV-1 protease (HIV-1PR), which performs a critical step in the maturation of viral particles. HIV-1PR contains two beta-hairpin turns, “the flaps”, which cover the active site. In order for substrates or inhibitors to bind, the flaps must undergo a large segmental motion, which can be experimentally characterized via pulsed EPR distance measurements. However, these experiments are preformed at cryogenic temperatures, which means a cryoprotectant is added to the sample. Solutes can potentially affect the structure and conformational changes of the protein. Here, we present data from 15N HSQC experiments investigating the exchange dynamics of protein-solute interactions as well as changes in the exchange dynamics protein-inhibitor interactions in the presence of solutes.

Code: PJ	Time Slot/Poster Number: 186	Session: Liquids Methods: Biomolecules, Poster
Investigation of the photostationary state of light-sensing proteins and photochromic compounds by NMR spectroscopy
Matthias Dorn¹; Sabine Seedorff^{1, 2}; Marcel Jurk¹; Peter Schmieder¹
¹Leibniz-Institut für Molekulare Pharmakologie, FMP, Berlin, Germany; ²Technische Universität Berlin, Berlin, Germany
Abstract
A varyity of NMR-spectroscopic investigations require uniform illumination of the sample within the magnet especially when photoactive molecules in their photostationary state (PSS) will be studied. For this purpose we constructed an inexpensive fiber-coupled light source (FCLS) based on LED’s as light sources. No modifications of the spectrometer or the samples are necessary. Influences of the optical fiber whithin the sample on magnetic field homogenity and spectral line width are both minimal. We used the FCLS to characterize photoswitchable cyclic peptides for suitabillity in biological applications and to investigate light-induced conformational changes of a blue-light photoreceptor (YtvA) from Bacillus subtilis.

Code: PJ	Time Slot/Poster Number: 187	Session: Liquids Methods: Biomolecules, Poster
Clean Absorption Mode (CAM) NMR Data Acquisition: General Theory and Applications
Arindam Ghosh; Yibing Wu; Yunfen He; Thomas Szyperski
State University of New York at Buffalo, Amherst, NY
Abstract
Phase-sensitive, pure absorption mode NMR signal detection is key for achieving high spectral resolution because dispersive signal components decay much slower than their absorptive counterparts. Recently, we developed‚ ‘Clean Absorption Mode‘ (CAM) NMR data acquisition based on ‘mirrored time domain sampling’ of indirect evolution periods. The CAM acquisition scheme eliminates, without application of a phase correction, dispersive signal components and can be employed to all indirect dimensions of multidimensional experiments, including G-matrix FT and Reduced-dimensionality projection experiments. In order to pave the way for future developments, we present a general theory of phase-sensitive mirrored time domain sampling. Applications include optimized CAM data acquisition and evaluation of r.f. pulse seqeunce performance.

Code: PJ	Time Slot/Poster Number: 188	Session: Liquids Methods: Biomolecules, Poster
Site-specific labeling of nucleotides using an E.coli mutant strain (K10 -1516)
Chandar Thakur; Kwaku Dayie
University of Maryland College Park, College Park, MD
Abstract
Growing an E.coli mutant strain (K10-1516: oxidative pentose phosphate pathway disabled) on 13C-2-glycerol with 13C-sodium formate in the growth media adds labels to new positions in both purine and pyrimidine bases. This approach promises to further extend the utility of these alternate site specific labels for making labeled nucleotides to study interesting biologically relevant RNA. Growth studies, metabolic pathway and NMR studies will be presented

Code: PJ	Time Slot/Poster Number: 189	Session: Liquids Methods: Biomolecules, Poster
Structural consequence of RS domain phosphorylation
Shengqi Xiang¹; Sina Möhlmann²; Ralf Ficner²; Stefan Becker¹; Markus Zweckstetter¹
¹Max Planck Institute for Biophysical Chemistry, Goettingen, Germany; ²Georg-August-University Goettingen, Goettingen, Germany
Abstract
Serine/arginine-rich (SR) protein family plays multiple functions in RNA splicing which contain an essential arginine/serine-rich (RS) domain. A number of studies have shown that the serine residues within the RS domains of SR proteins are extensively phosphorylated. This phosphorylation appears to change the ability of SR proteins to function in splicing. Here we use APSY (Automated projection spectroscopy) NMR and other routine NMR methods to study the conformations of RS repeated peptide and RS domain. It suggests that both in RS domain and isolated peptide, RS repeated stretch is in random coil status, but once the serines are phosphorylated, the RS stretches get structured.

Code: PJ	Time Slot/Poster Number: 190	Session: Liquids Methods: Biomolecules, Poster
Conformational Heterogeneity of Human Pancreatic Glucokinase in the Absence and Presence of Glucose
Mioara Larion¹; Roberto Kopke Salinas^{1, 2}; Lei Bruschweiler-Li^{1, 2}; Rafael Bruschweiler^{1, 2}; Brian G. Miller¹
¹Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, 32306; ²National High Magnetic Field Laboratory, Florida State University,Tallahassee, FL, 32310
Abstract
Human pancreatic glucokinase is a monomeric enzyme that displays kinetic cooperativity. To explain this behavior two mechanisms have been proposed. The mnemonic mechanism assumes the existence of one thermodynamically favored enzyme conformation in the unliganded state, while the ligand-induced slow transition (LIST) model requires two enzyme conformations. To investigate if either of these mechanisms sufficiently describes glucokinase kinetic cooperativity, transient state and NMR methods were employed. A quadruple exponential change in intrinsic protein fluorescence was observed upon exposure to glucose. Global analysis of glucose binding revealed a kinetic pathway comprised of two apo states and four binary complexes. 15N-1H TROSY spectra of glucokinase show significant broadening of cross-peaks suggesting the enzyme samples multiple conformations.

Code: PJ	Time Slot/Poster Number: 191	Session: Liquids Methods: Biomolecules, Poster
Solution NMR Studies of the Nick Repair Process by the Chlorella virus DNA Ligase-Adenylate (ChvLig-AMP)
Andrea Piserchio^{1, 4}; Pravin Nair²; Stewart Shuman²; Ranajeet Ghose³
¹The City College of New York, New York, NY; ²Sloan-Kettering Institute, New York, NY; ³The Graduate Center of the City University of New, New York, NY; ⁴The New York Structural Biology Center, New York, NY
Abstract
DNA ligases are enzymes that specifically recognize and seal 3’-OH/5’-phosphate nicks in duplex DNA. Chlorella virus ligase (ChvLig) is a minimized pluripotent ATP–dependent ligase consisting of two domains. Here we present a study of ChvLig-AMP (adenylated form) using solution NMR spectroscopy. These studies reveal that the interdomain orientation of the DNA-free enzyme is flexible, and, on average, is intermediate between the DNA-bound and DNA-free X-Ray crystal structures. We also proved that inorganic phosphate binds at the nick recognition site, remodels spatially and dynamically the catalytic region and induces long-range dynamical and structural changes in the entire system. We inferred that phosphate anions mimic some of the conformational transitions that occur when the ligase–adenylate interacts with the nick 5’-phosphate.

Code: PJ	Time Slot/Poster Number: 192	Session: Liquids Methods: Biomolecules, Poster
Measuring Apparent Transverse Relaxation Rates in Homonuclear J-Coupled Systems
Takuya Segawa¹; Nicolas Aeby¹; Bikash Baishya¹; Geoffrey Bodenhausen^{1, 2}
¹Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland; ²Ecole Normale Supérieure (ENS), Paris, France
Abstract
Measuring transverse relaxation rates in systems with homonuclear scalar couplings is a challenge because the envelopes of spin echoes obtained with trains of refocusing pulses are normally modulated. These echo modulations can be quenched by cumulative pulse errors that arise after many refocusing pulses with moderate rf amplitudes, as long as some narrow “recoupling” conditions are avoided. This method was applied to a system with a large number of coupled protons. Resonances of overlapping protons were analyzed by magnetization transfer to neighboring carbon-13 nuclei. The study of C alpha in the three-spin system of alanine enriched in carbon-13 showed that the variation of the carrier frequency can be an additional tool to obtain modulation-free echo decays.

Code: PJ	Time Slot/Poster Number: 193	Session: Liquids Methods: Biomolecules, Poster
IκBα Recognizes NF-κB in a Stepwise Process
Shih-Che Sue^{1, 2}; H, Jane Dyson¹
¹Department of Molecular Biology, The Scripps Research Institute, San Diego, California, USA; ²Institute of Bioinformatics and Structural Biology, National Tsing-Hua University, Hsin-Chu, Taiwan
Abstract
We present a study of the complex between NF-κB and its protein inhibitor IκBα, which consists of 6 ankyrin repeats. Structural instability in individual ankyrin repeats in free IκBα has been observed to relocate to other repeat regions upon binding of NF-κB. To understand the function of flexibility translocation, a DNA fragment containing the NF-κB binding sequence was titrated into the IκBα/NF-κB complex. The C-terminal PEST sequence is dissociated most rapidly, followed by the adjacent repeats 5 and 6. Repeats 1-4 remain bound to NF-κB even when DNA is present in excess. Recognition of NF-κB by IκBα starts at repeats 1-4 and propagates to repeats 5-6 and the C-terminal PEST, envisioned as a stepwise process.

Code: PJ	Time Slot/Poster Number: 194	Session: Liquids Methods: Biomolecules, Poster
Investigating the structure and dynamic properties of mitochondrial fission factors by NMR
Marijke Koppenol-Raab; Lora Picton; Robert Wells; Jonathan Lees; Ananya Majumdar; Blake Hill
Johns Hopkins University, Baltimore, MD
Abstract
Proper organelle function relies on maintenance of proper organelle morphology. For mitochondria and peroxisomes, the opposing processes of fission and fusion are partly responsible for regulating morphology. At least two proteins are known to cooperate in the fission process: the C-terminal transmembrane protein Fis1 and the dynamin-related GTPase Dnm1. However, little is known about how these proteins cooperate to effect fission. We are taking a structural biological approach to this problem and investigating the structure and dynamic properties of these molecules individually and together. We have found that Fis1 appears autoinhibited by a dynamic N-terminal arm. This inhibition is important in homodimerization and mechanoenzyme assembly. NMR investigations of mutations that affect autoinhibition support a new model for mitochondrial fission.

Code: PJ	Time Slot/Poster Number: 195	Session: Liquids Methods: Biomolecules, Poster
Narrowing of Solution NMR Spectral Lines Broadened by Protein Conformational Exchange
Ying Li; Arthur Palmer
Columbia University, New York, NY
Abstract
A method to reduce line broadening caused by microsecond-to-millisecond time scale motions in protein molecules is presented. The method is based on scaling down isotropic chemical shifts by multiple radiofrequency pulses. The resolution and sensitivity enhancement for resonances broadened by conformational exchange is demonstrated on the protein ribonuclease A.

Code: PJ	Time Slot/Poster Number: 196	Session: Liquids Methods: Biomolecules, Poster
Structural Studies of Autoinhibitory Interactions between the PDZ2 and C-terminal Domains of NHERF
Hong Cheng
Fox Chase Cancer Center, Philadelphia, PA
Abstract
NHERF is a protein adapter which is involved in the regulation of a number of cell receptors and channels. NHERF proteins contain two tandem PDZ domains and a C-terminal sequence that binds several members of the ERM family. Our NMR structure of PDZ2 reveals a fold similar to the PDZ1 crystal structure. Relaxation and transferred NOE studies show that 14 a.a. residues located in the end of the C-terminal domain are involved in the interaction with PDZ2. The residues from the C-terminal domain bind to the surface of a groove formed by the alpha-helices and beta-strands of PDZ2. These results provide a structural framework for understanding of the autoinhibitory interactions between the PDZ2 and C-terminal domains of NHERF.

Code: PJ	Time Slot/Poster Number: 197	Session: Liquids Methods: Biomolecules, Poster
NMR Structural Studies of a Membrane-Associated (>70kDa) Complex Between Cytochrome P450 and b5
Shivani Ahuja; Subramanian Vivekanandan; Ronald Soong; Kazutoshi Yamamoto; Jiadi Xu; Sang-Choul Im; Lucy Waskell; Ayyalusamy Ramamoorthy
University of Michigan at Ann Arbor, Ann Arbor,
Abstract
A series of solution and solid-state NMR experiments have been employed to obtain multiple structural constraints on the complex between mammalian cytochrome P450 (55.7 kDa) and cytochrome b5 (~16 kDa) necessary for oxidation of a variety of exogenous and endogenous compounds, including drugs, fatty acids, hormones and carcinogens. Using various labeling schemes, NMR pulse sequences and sample conditions we have been able to assign resonances of a full-length Cyt b5 in detergent micelles and the amino acids that form the interaction interface between the two redox partners in vitro.

Code: PJ	Time Slot/Poster Number: 198	Session: Liquids Methods: Biomolecules, Poster
Protein Dynamics Assessed by Novel Methods using Dipolar Interactions
Beat Vogeli; Roland Riek
Swiss Federal Institute of Technology, ETH Zurich, Zürich, Switzerland
Abstract
To get a detailed understanding of the motions of a protein a lot of data must be combined. We demonstrate the conversion of NOEs into precise distances with high accuracy from triple- and double-labeled samples for backbone and side chains. NOEs between amide protons in perdeuterated ubiquitin enable the determination of HN-HN distances with an experimental random error of 0.07 Å. NOEs may also give insights into through-space dynamics. Motional effects on dipolar interaction between two atoms are assessed by RDCs. Then, motional correlations are detected by cross-correlated relaxation between two of these interactions. Evidence of correlated backbone motion and extension of the method into side chains is demonstrated.

Code: PJ	Time Slot/Poster Number: 199	Session: Liquids Methods: Biomolecules, Poster
Initial Characterization of RNA Binding by the Multifunctional Protein p54nrb
Mikaël Bédard^{1, 2}; Sébastien Morin^{1, 2}; Sami Aziz Ameur¹; Céline Bruelle^{1, 2}; Stéphane Gagné^{1, 2}; Michel Vincent^{1, 2}
¹Université Laval, Québec, Canada; ²PROTEO, Québec, Canada
Abstract
The multifunctional protein p54nrb is an RNA-binding protein involved in many nuclear processes including transcription, RNA processing and retention of hyper-edited RNA. Our previous data have shown that p54nrb is multiphosphorylated by CDK1 during mitosis. Recently, we found that the phosphorylation of p54nrb regulates its RNA binding ability in vitro. The specific phosphorylation of the threonin 15 that locates upstream a tandem of RNA recognition motif (RRM) is responsible for that RNA binding loss. Our main objective is therefore to explore the structural details of the RNA binding of p54nrb and the mechanisms by wich the phosphorylation shutdowns this ability by liquid state NMR.

Code: PJ	Time Slot/Poster Number: 200	Session: Liquids Methods: Biomolecules, Poster
NMR Studies of TRAP and Anti-TRAP
Joseph Sachleben
Dept. of Biochemistry and CCIC, Columbus, OH
Abstract
In Bacilli, the trp RNA-binding attenuation protein (TRAP) and its inhibitor Anti-TRAP (AT) are responsible for regulating transcription and translation of the genes required for tryptophan biosynthesis. TRAP is a small protein that assembles into an undecameric (11-mer) ring-shaped structure, while AT is a small zinc-binding protein that has been shown to homo-oligomerize to form dodecamer, AT12, from trimers, AT3. Measurements of 15N T1, T2, NOE, and relaxation dispersion measurements of apo, holo, and T28V TRAP, a mutant of TRAP that cannot bind tryptophan but is still capable of binding RNA, test if RNA binding is dynamically controlled in this system. Mass spectrometry and NMR measurements demonstrate that AT shows an N-formyl and pH dependent oligomerization from AT3 to AT12.

Code: PJ	Time Slot/Poster Number: 201	Session: Liquids Methods: Biomolecules, Poster
The Cold Denatured State of the C-terminal Domain of Protein L9 Is Compact and Contains Both Native and Non-native Structure
Bing Shan^{1, 2}; Sebastian McClendon³; Carla Rospigliosi³; David Eliezer Eliezer³; Daniel Raleigh¹
¹State University of New York at Stony Brook, Stony Brook, NY; ²National High Magnetic Field Laboratory and FSU, Tallahassee, FL; ³Weill Cornell Medical College, New York, NY
Abstract
A deeper understanding of cold denaturation, a general property of globular proteins, should provide insights into the origin of cooperativity of folding and the nature of partially folded states. Unfortunately, studies of protein cold denaturation have been hindered by the difficulty to experimental access of the cold denatured state without perturbing the structure of the protein. Here we take advantage of a destabilizing point mutation to conduct a detailed structural and dynamic characterization of the cold denatured state of the C-terminal domain of L9 under near native conditions. The cold denatured state of CTL9 contains significant native like secondary structure as well as non-native structure. The implications for protein folding and for studies of cold denatured states are discussed.

Code: PJ	Time Slot/Poster Number: 202	Session: Liquids Methods: Biomolecules, Poster
Structural Analysis of the C-Terminus of Lung Surfactant Protein B (SP-B)
Anna Kuznetsova; Reba Farver; Julieann Vanni; Vijay Antharam; Joanna Long
University of Florida, McKnight Brain Institute, Gainesville, FL
Abstract
We used solid state NMR to probe the structure and dynamics of the C-terminus of surfactant protein B(SPB59-80) in DPPC:POPG and POPC:POPG lipid vesicles. Helical SP-B59-80 penetrates deeply into 4:1 DPPC:POPG bilayers and binds more peripherally to 3:1 POPC:POPG bilayers. Deep penetration of SP-B59-80 within 4:1 DPPC:POPG bilayers requires the charged sidechains to "snorkel" to interact with charged phosphate headgroups and the actual torsion angles may deviate from those expected for canonical a-helix. We are using solution state NMR data to determine SP-B59-80 conformation in a highly curved lipid mimetic environment of detergent micelles and to compare to helical conformations of SP-B59-80 in lipid environments obtained with MAS ssNMR homonuclear recoupling experiments.

Code: PJ	Time Slot/Poster Number: 203	Session: Liquids Methods: Biomolecules, Poster
NMR-Structure of the VP16-Binding Domain of ARC92/MED25 and its Interaction with VP16 of Herpes Simplex Virus
Alexander Milbradt¹; Jim Zhen-Yu Sun¹; Madhura Kulkarni²; Koh Takeuchi¹; Ricard Rodriguez¹; Anders Naar²; Gerhard Wagner¹
¹Harvard Medical School/BCMP, Boston, MA; ²Department of Cell Biology, Harvard Medical School, Boston, MA
Abstract
The transcriptional mediator plays an important role in the fine regulation of gene transcription by interacting with transactivation domains of transcriptional activators on the one hand and the RNA polymerase II on the other hand. Here we present structural and functional studies of the VP16-binding domain of the mediator subunit ARC92/MED25. The interaction of the strong viral activator VP16 with the VP16-binding domain of ARC92 is crucial for activation of certain viral genes. Individual mutations on ARC92 abolish the interaction between the two proteins. We also present preliminary data from a fragment-based screen to develop small molecule inhibitors of the interaction.

Code: PJ	Time Slot/Poster Number: 204	Session: Liquids Methods: Biomolecules, Poster
Interaction of the Prolyl Isomerase Pin1 and a Multi-Phosphorylated Substrate
Alex Greenwood; Soumya De; Sarah Denial; Linda Nicholson
Cornell University, Ithaca, NY
Abstract
The phosphorylation-specific peptidyl-prolyl isomerase Pin1 is an important regulator of many cellular processes. Pin1 is comprised of two domains, a WW domain and a PPIase domain. The binding mode of a bivalent ligand to Pin1 was investigated by ITC and NMR titration experiments. Peptides derived from a known Pin1 substrate with two recognition motifs were synthesized. The binding of the mono- and bi-phosphorylated forms of these peptides to Pin1 or its individual domains was investigated. Nitrogen ZZ (NZZ) exchange spectroscopy was performed to measure the Pin1-catalyzed rates of cis-trans isomerization at each of the two sites. Preliminary results are suggestive of a bivalent binding mode, which has not previously been demonstrated for Pin1 with a biologically relevant ligand.

Code: PJ	Time Slot/Poster Number: 205	Session: Liquids Methods: Biomolecules, Poster
Quantitative 2D and 3D Γ-HCP Experiments for the Determination of the Backbone Angles α and ζ in Oligonucleotides
Senada Nozinovic¹; Christian Richter¹; Jörg Rinnenthal¹; Boris Fürtig²; Harald Schwalbe¹
¹Goethe-University/BMRZ, Frankfurt/M, Germany; ²MFPL, Vienna, Austria
Abstract
A heteronuclear NMR pulse sequence, the quantitative Γ-(HCP) experiment, for determination of the RNA backbone angles α(O3´i-1-Pi-O5´i-C5´i) and ζ(C3´i-O3´i-Pi+1-O5´i+1) in 13C labeled RNA is introduced. With the new pulse sequence, five different cross-correlated relaxation rates along the phosphodiester backbone can be measured in a quantitative manner. From conformational analysis projection angle restraints and torsion angle restraints for the two backbone angles α and ζ can be extracted. The restraints were incorporated into the structure calculation of a very high resolution structure of the model system RNA (Nozinovic et al., 2009). Comparison with the X-ray structure of the cUUCGg tetraloop confirms the high quality of the data and suggests that the method can significantly improve the quality of RNA structure determination.

Code: PJ	Time Slot/Poster Number: 206	Session: Liquids Methods: Biomolecules, Poster
Characterizing the N-glycan of the 55kDa IgG Fc fragment using 13C-labeled sugars
Adam Barb; James Prestegard
Complex Carbohydrate Research Center, UGA, Athens, GA
Abstract
Immunoglobulin G acts as either a pro- or anti-inflammatory mediator, depending on the composition of the N-glycans at Asn-297 of the Fc fragment. The effect of compositional changes on glycan conformation and dynamics is unknown. Here we present the preparation of near-homogeneous Fc-conjugated glycans using 13C-labeled galactose and 13C-N-acetylneuraminic acid. Assignments of observed resonances to either the alpha1-3 or alpha1-6 branch of the biantennary glycan were completed utilizing the branch specificity of the N-acetylneuraminic acid transferase ST6Gal-I. Resonances on the alpha1-3 branch of the glycan exhibit markedly narrower 1H linewidths than alpha1-6 resonances in a 13C-HMQC experiment. Data on the dynamics and the local structure of the glycan termini drawn from spin relaxation rates, NOEs, and RDCs will be presented.

Code: PJ	Time Slot/Poster Number: 207	Session: Liquids Methods: Biomolecules, Poster
NMR Investigations of the Solvent Effect on Polypeptide Structure
Soyoun Hwang; Qiang Shao; Yi Qin Gao; Christian Hilty
Texas A&M University, College Station, TX
Abstract
One of the major contributions that govern protein structure appears to be the solvation effect. Here, we present the results of two case studies using structure determination of short peptides and protein fragments in different solvent systems, in order to investigate the solvent effect on secondary structure formation. Firstly, the propensity for formation of alpha-helical and beta hairpin structure in a model peptide BBA5 was studied by NMR, and results were contrasted with molecular dynamics simulations. Secondly, peptides corresponding to individual transmembrane helices taken from an alpha-helical membrane protein, the disulfide bond forming protein B (DsbB), were determined. Comparison with the structure of full-length protein was used in order to test the two-stage model for membrane protein folding for DsbB.

Code: PJ	Time Slot/Poster Number: 208	Session: Liquids Methods: Biomolecules, Poster
NMR Studies of a G-protein Coupled Receptor, Cannabinoid Receptor-2 in Membrane Mimetic Environment
Elvis Tiburu¹; Sergiy Tyukhtenko¹; Lalit Deshmukh²; Olga Vinogradova²; Alexandros Makriyannis¹
¹Northeastern University, Boston, MA; ²University of Connecticut, Storrs, CT
Abstract
It has been shown that most rhodopsin-like G protein-coupled receptors promote receptor activation by altering the configuration of the TM helix 6 Pro-kink structure. The change in configuration results in the subsequent movement of the cytoplasmic end of TM helix 6 away from TM helix 3. To test whether this unique TM helix 6 structural variation is universal to all GPCRs, we performed high-resolution NMR studies on the amino acid sequence corresponding to TM helix 6 of the human cannabinoid receptor-2 in model membranes. The solution NMR structure revealed the existence of a functionally important Pro-based conformational switch. We are using a segmental approach to characterize other helices to identify structural domains that are important for GPCR function.

Code: PJ	Time Slot/Poster Number: 209	Session: Liquids Methods: Biomolecules, Poster
Study of the Electrostatic Interactions in the Binding of a Proline Rich Motif to an SH3 Domain
Erick Alejandro Meneses Ramirez¹; Anthony Mittermaier²
¹Ph.D student, Montreal, Canada; ²Assistant Professor, Montreal, Canada
Abstract
SH3 domain binds to a variety of physiological targets mediating cell signaling functions. SH3 binds to proline rich regions. This involves the interaction between a positively charged arginine in the ligand and negatively charged residues in the protein. This interaction leads us to investigate to what extent the electrostatic interactions mediate the binding process. We studied the SH3 domain from the Fyn tyrosine kinase and the proline rich peptide WSLARRPLPPLP. NMR and ITC experiments show that electrostatic contributions to association are shielded at an ionic strength of 500 mM NaCl. This effect is reflected in a 16-fold decrease in the binding constant. Surprisingly, the binding enthalpy becomes more favourable as the ionic strength is increased, even though the affinity decreases.

Code: PJ	Time Slot/Poster Number: 210	Session: Liquids Methods: Biomolecules, Poster
Saturation Transfer Difference (STD) NMR: A Powerful Bioanalytical Tool for the Design of Small Molecule Enzyme Inhibitors
David J McNally¹; Ian C Schoenhofen²; Scott Houliston²; Harold C Jarrell²; Nam H Khieu²; Dennis Whitfield²; Susan M Logan²; Andre J Simpson¹; Jean-Robert Brisson²
¹University of Toronto, Toronto, Canada; ²NRC Institute for Biological Sciences, Ottawa, Canada
Abstract
Microorganisms pose a continual health threat through antibiotic resistance. Pathways that produce glycan structures in bacteria are attractive therapeutic targets since many bacterial glycans are unique to prokaryotes. Helicobacter pylori and Campylobacter jejuni decorate their flagella with pseudaminic acid (Pse) which is required for motility, colonization and virulence. Agents that interfere with Pse biosynthesis therefore offer therapeutic potential. The initial enzyme of the CMP-Pse pathway, PseB, converts UDP-α-D-GlcNAc to UDP-2-acetamido-2,6-dideoxy-β-L-arabino-hexos-4-ulose and is a key therapeutic target. We used Saturation Transfer Difference (STD) NMR to characterize PseB inhibition by CMP-Pse as well as other potential inhibitors. Results from this study will be used to guide the development of small molecule inhibitors of the CMP-Pse pathway as therapeutic agents.

Code: PJ	Time Slot/Poster Number: 211	Session: Liquids Methods: Biomolecules, Poster
The measure of the fractal dimension of proteins by NMR, application to PolyProline and to a salivary intrinsically unstructured protein
Marc-André Delsuc¹; Matthieu Tanty¹; Franck Paté²
¹IGBMC, Illkirch, France; ²INRA-UMR SPO, Montpellier, France
Abstract
Disordered proteins do not have a definite structure, and only statistical properties can be used to describe their behavior. NMR is shown to allow the determination of the fractal dimension of a protein chain through the measure of translational diffusion. With this approach, we have found fractal dimension varying from 1.72 for denatured peptides to 2.56 for structured globular proteins. IB5, is a 70 amino-acid intrinsically disordered protein. It nevertheless efficiently and selectively binds polyphenols. The fractal dimension was measured on IB5 around 2.0 characteristic of a random walk chain. IB5 presents a pattern very different than observed for polyproline, and the simple PPII structure which as been proposed for this protein can be ruled out.

Code: PJ	Time Slot/Poster Number: 212	Session: Liquids Methods: Biomolecules, Poster
NMR Investigation of Visual Arrestin-a 46KDa Protein and its Interaction with IP6 and Calmodulin
Tiandi Zhuang; Sergey Vishnivetskiy; Vsevolod Gurevich; Charles Sanders
Vanderbilt University, Nashville, TN
Abstract
Arrestins terminate signaling by GPCRs through their preferential binding to the phosphorylated activated form of the receptors. Arrestins also function as a scaffold protein mediating the binding of other cofactors. Here we used NMR to partially assign the backbone residues of visual arrestin by incorporating sparse 3D NMR data, selective amino acid labeling, chemical shifts prediction from known crystal structure and PREs. The assigned residues are well distributed along the protein backbone and can be used as probes for the studying the interaction between this large protein and its cofactors. As an illustration, the interactions between visual arrestin and IP6 or calmodulin were studied and the residues involving in the binding were identified.

Code: PJ	Time Slot/Poster Number: 213	Session: Liquids Methods: Biomolecules, Poster
Time-resolved NMR-investigations of pH-induced structural change of the DNA i-motif
Anna Lena Lieblein¹; Boris Fuertig²; Janina Buck¹; Christian Richter¹; Harald Schwalbe¹
¹Center of Biomol. Magn. Resonance, Frankfurt, Germany; ²University of Vienna, Vienna, Austria
Abstract
The ends of chromosomes, the telomeres, consist of a guanine-rich strand and a cytosine-rich counter-strand. In a slightly acid environment, the cytosine-rich sequence forms two parallel stranded duplexes which are built by hemiprotonated C.C+ base pairs. Due to stacking of the base pairs the four stranded structure, the so-called DNA i-motif, is formed. NMR-spectroscopy allows us to analyze both structure and dynamics of biological molecules with atomic resolution. We investigate the pH-dependent folding of the DNA i-motif. With the help of a mixing-device the folding is induced and can be observed via time-resolved NMR-spectroscopy. For this reason we analyzed the appearance of the iminoproton-resonances at different times after mixing. We induced a pH-jump from pH 7 to 5.

Code: PJ	Time Slot/Poster Number: 214	Session: Liquids Methods: Biomolecules, Poster
Comparison of oligomerization propensity between Aβ and pyroglutamyl-Aβ by solution NMR
Shigeto Iwamoto¹; Takashi Saito²; Toshiyuki Kohno³; Takaomi C. Saido²; Hiroaki Terasawa¹
¹Facul. of Life Sci., Kumamoto Univ., Kumamoto, Japan; ²RIKEN BSI, Saitama, Japan; ³Mitsubishi Kagaku Inst. Life Sciences, Tokyo, Japan
Abstract
Small Aβ oligomers, intermediates in the formation of amyloid fibrils, may be potent neurotoxins in AD patients. N-terminal pyroglutamyl-Aβ peptides (Aβ (3pE)) have been reported to be resistant to degradation and prone to aggregation. Moreover, Aβ (3pE) predominantly comprises senile plaques in AD patients. In order to elucidate the role of Aβ (3pE) on the formation of small Aβ oligomers, we performed solution NMR analyses of Aβ (3pE) and Aβ. Our results indicate that Aβ (3pE) may play a critical role in the formation of Aβ oligomers by forming a β-sheet at the N-terminal regions. A possible mechanism for Aβ (3pE) oligomerization will be discussed.

Code: PJ	Time Slot/Poster Number: 215	Session: Liquids Methods: Biomolecules, Poster
Structure determination of HAR1F-RNA by NMR-Spectroscopy
Melanie Koschinat; Christian Richter; Jens Woehnert; Janina Buck; Harald Schwalbe
Center for Biomol. Magnetic Resonance, Frankfurt, Germany
Abstract
The human accelerated regions are a group of 49 segments in the human genome, which are ranked by their underlying fast mutation rate compared to homologous chimpanzee segments. Of the 49 HARs, 96% are in non-coding segments. Many of the HARs are associated with genes known to be responsible for human specific brain features and transcriptional regulation. The 118bp HAR1 region has with 18 substitutions the highest mutation rate in the homo sapiens genome. In contrast, the 49 segments are highly conserved throughout several vertebrates’ genomes. HAR1F is coexpressed with ‘Reelin’ in Cajal-Retzius cells which is known as a regulator in human cortical development. Detailed information about the structures of HAR1F and all other HARs are currently not available.

Code: PJ	Time Slot/Poster Number: 216	Session: Liquids Methods: Biomolecules, Poster
Solution Structure, Domain Motions, and RNA Recognition by RDE-4, an RNAi Initiator Protein in C. elegans
Sonu Kumar¹; M Udaya Kiran²; M Trivikram Rao¹; Shyam Bandari¹; Sai Chaitanya Chiliveri¹; Mandar V Deshmukh¹
¹Centre for Cellular and Molecular Biology, Hyderabad, India; ²Indian Institute of Chemical Technology, Hyderabad, India
Abstract
In C. elegans, Dicer associates with RDE-4 to execute silencing of exogenous genes through RNAi pathway. RDE-4 (~44 kDa) has two double stranded RNA binding domains and a C-terminal region, which induces dimerization. Due to high MW of WT RDE-4, two constructs of RDE-4: RDE-4DC (1-243) and RDE-4C (242-385) were designed. For RDE-4DC, we achieved ~ 92% backbone/side-chain (13C Me-ILV) resonance assignments. 15N backbone relaxation measurements indicate that both dsRDBs behaves independently. dsRNA titration with RDE-4DC, suggest that dsRNA preferentially binds dsRBD2. In contrast, very broad resonances observed in 15N/1H TROSY of RDE-4C denote a non-specific oligomerization or highly anisotropic structure. Global fold of RDE-4DC calculated using distance and oritational restraints and its correlation with WT RDE-4 will be presented.

Code: PJ	Time Slot/Poster Number: 217	Session: Liquids Methods: Biomolecules, Poster
Solution Structure of D6-HP: Modular Fragment of Villin Capable of Calcium-Dependent F-actin Bundling?
Danielle Pfaff¹; Fengli Zhang²; Serge Smirnov¹
¹Western Washington University, Bellingham, WA; ²Florida State University, NHMFL, Tallahassee, Florida
Abstract
We investigate via solution NMR the domain-domain and linker-domain interactions in Villin, a modular, gelsolin-like, calcium-sensitive, F-actin regulatory protein. Solution structures of Villin’s actin-bundling, 208-residue, modular D6-HP fragment and domain D6 in isolation have been determined. This analysis provides the opportunity to probe further both structure and function of Villin at the junction between its C-terminal headpiece domain and gelsolin-like core. We will provide the analysis of the site-specific structural properties of the inter-domain linker and domain-linker interfaces and the location of the Ca2+-binding site on D6-HP. Our data indicates that domain D6 in isolation is well folded, and domains HP and D6 folds in isolation resemble those in the context of the larger modular fragment D6-HP.

Code: PJ	Time Slot/Poster Number: 218	Session: Liquids Methods: Biomolecules, Poster
Quantitative Analysis of Related Multidimensional Spectra by Fast Maximum Likelihood Reconstruction (FMLR)
Roger Chylla
University of Wisconsin-Madison, Madison, WI
Abstract
Evidence is presented for the practicality of using fast maximum likelihood reconstruction (FMLR) to handle an important class of quantitative problems in multidimensional NMR spectroscopy of biomacromolecules. FMLR uses both linear and non linear least squares analysis to generate the simplest time-domain model whose FT spectrum most closely matches the similarly processed data. The results from the application of FMLR to a series of quantitative 2D HSQC experiments show that rapid, quantitatively useful reconstructions can be obtained in less than a half minute on a conventional off-the-shelf personal computer.

Code: PJ	Time Slot/Poster Number: 219	Session: Liquids Methods: Biomolecules, Poster
Dynamic Domains in Ras Isoforms
Casey O'connor; Evgenii Kovrigin
Medical College of Wisconsin, Milwaukee, WI
Abstract
Ras proteins perform diverse signaling functions in the cell ranging from gene expression to apoptosis and remain critical in cancer research. Conventionally thought of as a binary switch, mounting evidence over the past 15 years indicates at least two distinct states are in equilibrium for GTP-bound Ras. Measurements of nitrogen-15 relaxation dispersion in H and K isoforms of Ras bound to GTP mimic GppNHp were performed at two magnetic fields. Residues with pronounced relaxation dispersions were fit individually and as a group to the same kex and pa/pb. Four dynamic regions were identified which may provide a mechanism of motion coupling the effector interface to other structural elements in Ras proteins.

Code: PJ	Time Slot/Poster Number: 220	Session: Liquids Methods: Biomolecules, Poster
NMR Structural Studies Reveal Amyloid Prone Regions of SEVI and a Possible Mechanism for Amyloid Inhibition by EGCG
Nataliya Popovych; Jeffrey R. Brender; Ravi P. R. Nanga; Subramanian Vivekanandan; Ayyalusamy Ramamoorthy
University of Michigan, Ann Arbor, MI
Abstract
An amyloid peptide, called SEVI, increases HIV infection by four to five orders of magnitude. Atomic-level resolution structure of SEVI precursor PAP248-286 in detergent micelles will be presented. The binding of EGCG, a polyphenolic compound derived from green tea that suppress the activities of SEVI, with SEVI both in solution and in the presence of membrane will also be presented.

Code: PJ	Time Slot/Poster Number: 221	Session: Liquids Methods: Biomolecules, Poster
NMR analyses of a male-mice-specific peptidic pheromone ESP1
Makoto Hirakane¹; Sosuke Yoshinaga¹; Toru Sato²; Sachiko Haga²; Hiroko Kimoto²; Ichio Shimada³; Kazushige Touhara²; Hiroaki Terasawa¹
¹Facul. of Life Sci., Kumamoto Univ., Kumamoto, Japan; ²Grad. Sch. Agr. and Life Sci., Univ. Tokyo, Tokyo, Japan; ³Grad. Sch. Pharm. Sci., Univ. Tokyo, Tokyo, Japan
Abstract
Pheromones are species-specific chemical signals that regulate a wide range of social and sexual behaviours in many animals. The vomeronasal organ (VNO) mediates the pheromonal information via vomeronasal sensory neurons (VSNs) in mice. We identified a male-specific peptide ESP1 (exocrine-gland-secreting peptide 1) secreted into tear fluids that stimulates female's VSNs. In addition, ESP1 turns out to be a member of a multigene family (ESP family). The aim of this study is the elucidation of the mechanism to discriminate among individuals via the pheromone reception system. We report here a three-dimensional structure of ESP1 with solution NMR analyses. The structure-activity relationship of ESP1 based on the structural data and the mutational effects on the VSN-stimulating activity will be discussed.

Code: PJ	Time Slot/Poster Number: 222	Session: Liquids Methods: Biomolecules, Poster
A Novel NMR Resonance Assignment Approach Assisted by Predicted Local Structures: Application for Large Proteins
xingsheng wang; Brian Tash; John Flanagan; Fang Tian
Penn State College of Medicine, Hershey, PA
Abstract
We present a novel local structure assisted backbone resonance assignment strategy that uses orientational information derived from RDCs and supplemented with data from the most sensitive experiments. Local structural models come from consistent theoretical predictions. The assignment process begins with constructions of strings of linked resonances from HNCA sequential connectivity through exhaustive search (CA-links). CA-links are then ranked by the fitness of their experimental Cα chemical shifts and 15N-1H RDCs to the expected values. Finally, connecting CA-links from different fragments allow the identification and elimination of CA-links with resonances having multiple assignments. The proposed strategy was successfully applied for the assignment of resonances from structured regions of PDZ3 assisted with predicted models and proteins with experimental structures.