Code: PK	Time Slot/Poster Number: 224	Session: Liquids: New Applications, Poster
Millisecond Timescale Dynamics of Human Liver Fatty Acid Binding Protein: Testing of Its Relevance to the Ligand Entry Process
Dong Long; Daiwen Yang
National University of Singapore, Singapore, Singapore
Abstract
The microsecond to millisecond timescale dynamics of FABPs was historically hypothesized to regulate the ligand entry/exit of the binding cavity. However, the validity of this hypothesis is still pending for verification. In the present study, the intrinsic slow dynamics of human liver fatty acid binding protein (hLFABP), as well as the kinetics of hLFABP-ANS interaction, was characterized in detail using NMR spectroscopy. The current result excludes the possibility that the intrinsic millisecond dynamics of hLFABP represents a critical conformational reorganization process required for ligand entry, but implies that it may represent the exchange between the apo-state and a state resembling the singly-bound conformation. Furthermore, the present result shows that the ligand-entry process could occur on the microsecond/sub-microsecond timescales.

Code: PK	Time Slot/Poster Number: 225	Session: Liquids: New Applications, Poster
Are Some Signal Points More Important Than Others?
Hasan Celik; A. J. Shaka; Vladimir A. Mandelshtam
UC Irvine, Irvine, CA
Abstract
It is common to use parametric spectral analysis methods to overcome Fourier time-energy uncertainty. They offer reliable applications only when signal to noise ratio is sufficiently high, otherwise, results become sensitive to noise, where this is a general problem for parametric approaches. Results of a sensitivity analysis are presented which characterize the variation of the spectral parameters of sample Filter Diagonalization Method(FDM) calculations with respect to the variations in the input data. Additionally, a general analytical expression for the sensitivity is expressed. Our results may be of interest in understanding performance of Linear Prediction, MaxEnt, FDM and other related methods in signal analysis and image processing.

Code: PK	Time Slot/Poster Number: 226	Session: Liquids: New Applications, Poster
Detection of ligand induced modulation of the extra-cellular surface of a G protein coupled receptor (GPCR) by heteronuclear 2D NMR
Luciano Mueller
Bristol-Myers Squibb, Princeton, NJ
Abstract
Reductive methylation of lysine residues permitted the introduction of 13C-labels in samples of 2 adrenergic receptor. Indirect detection of 13C enabled the monitoring ligand-induced modulations of the salt bridge between K305 & D192. This salt bridge contributes to the formation of a cavity on the extra cellular surface of the receptor. The extra-cellular surface adjacent to this NMR detectable K305 : D192 salt bridge forms cavities which may be amenable to the design of subtype-specific exosite modulators of 2 AR activity. This may prove an attractive venue for overcoming the challenge of designing subtype specific 2 AR modulators. Distinction between highly mobile and structurally restrained lysine methyls was achieved via saturation transfer filtered hc hmqc and hc hsqc experiments.

Code: PK	Time Slot/Poster Number: 227	Session: Liquids: New Applications, Poster
Improving 2D Experiments with Moving Tube NMR
Kevin J Donovan; Rachel Martin; A.J. Shaka
UC Irvine, Irvine, CA
Abstract
Moving Tube NMR is a technique that uses unique hardware and large sample volumes to achieve faster experiments with increased sensitivity. The essence of the moving tube experiment is to remove the relaxing sample segment after data acquisition is complete and replace it with a pre-equilibrated sample segment, effectively skipping the relaxation delay. Although the moving tube NMR technique has only been demonstrated on 1D carbon-13 spectra, it is applicable to many more experiments, as nearly all NMR experiments. This presentation will explore the application of moving tube NMR to some 2D NMR experiments, and also the potential of parallel 2D experiments, where different experiments (i.e. DQCOSY, NOESY) are conducted in parallel on different parts of the sample tube.

Code: PK	Time Slot/Poster Number: 228	Session: Liquids: New Applications, Poster
Characterizing early misfolding events associated with light chain amyloidosis by relaxation dispersion NMR
Sujoy Mukherjee; Simon Pondaven; Christopher Jaroniec
Ohio State University, Columbus, OH
Abstract
Studies on the misfolding of a light chain, variable domain of an immunoglobulin (LEN) have been presented. Experiments probing fast timescale backbone dynamics show that the protein remains as a stable dimer at pH 6.5 with a largely rigid backbone that remains mostly rigid at pH 2 where it is known to form amyloid fibrils. These studies also revealed the existence of slow timescale conformational dynamics, usually associated with unfolding of proteins, which was studied in a residue specific detail. The protein exhibits significant conformational exchange in the complementarity determining regions (loops) 2 and 3 of the immunoglobulin that increases at low pH whereas the framework (β-strand) regions remains mostly rigid

Code: PK	Time Slot/Poster Number: 229	Session: Liquids: New Applications, Poster
Glycoprotein NMR: Methodology and Results, the De-coordination of Functional Dynamics in RNase
Lin Zhang; James M. Gibson; Chris B. Marotta; James G. Kempf
Rensselaer Polytechnic Inst., Troy, NY
Abstract
Using a simple, general approach to glycoprotein NMR, we elucidated dynamic factors in glycosylation-modulated function of RNaseA/B, the unmodified/glycosylated forms of a prototypical enzyme. Methods rely on transfer of (1H,15N) assignments from 15N-enriched, unmodified protein to the glycoprotein, which may not be expressed from E.coli and thus is studied at natural-abundance 15N. Critical dynamics are well-known in the unmodified enzyme, but little is known on the effects of glycosylation. We show that it modulates function by altering dynamics. Kinetics show 6 fold reduction in kcat, which, in RNaseA, is limited by an open/close motion coordinated among several residues. Here, Δ(ΔR2)glycos shows that dynamic sites exhibit both increased and decreased kex on glycosylation, a de-coordination accounting for decreased kcat.

Code: PK	Time Slot/Poster Number: 230	Session: Liquids: New Applications, Poster
Mutation effects on the structure and dynamics of the sarco(endo)plasmic reticulum Ca2+ ATPase nucleotide binding domain
Wazo Z. Myint; Qingguo Gong; Jinwoo Ahn; Rieko Ishima
University of Pittsburgh, Pittsburgh, PA
Abstract
The sarcoplasmic reticulum Ca2+ ATPase (SERCA) is an important protein in the regulation of cardiac muscle contraction. We apply NMR and other spectroscopic methods to characterize mutation effects on the structure and dynamics of the nucleotide-binding domain of Rabbit SERCA1a, a 244 residue long protein with independent ATP binding activity. A clinically discovered mutation that is related to Darier’s disease (E412G) as well as mutations on different moieties of the nucleotide-binding site (T441A, R560V, and C561A) were studied. We conclude that the reduction of the ATPase activity by E412G is most likely related to the stability of the domain whereas the other mutations may reduce activity due to differences in the microscopic environments at the ATP binding site.

Code: PK	Time Slot/Poster Number: 231	Session: Liquids: New Applications, Poster
15N−1H Scalar Coupling Perturbation: An Additional Probe for Measuring Structural Changes Due to Ligand Binding
Junhe Ma; James Gruschus; Nico Tjandra
Laboratory of Molecular Biophysics, Bethesda, MD
Abstract
Chemical shift perturbation mapping of backbone amides is one of the most widely employed techniques in biomolecular NMR, providing residue-by-residue information on interaction interfaces, ligand binding, and chemical modification sites, even for samples where poor solubility, short lifetime, or large size precludes more sophisticated experimental approaches. Significant changes can also occur in the amide one-bond 15N−1H scalar coupling constants due to ligand binding. For Gln Binding Protein (GlnBP) the largest changes (>1 Hz), measured using signal intensity J-modulation, are seen near the site of glutamine binding, though significant perturbations also occur distant to the site reflecting allosteric effects. The coupling constant perturbations correlate with structural changes, especially changes in backbone amide hydrogen bonding.

Code: PK	Time Slot/Poster Number: 232	Session: Liquids: New Applications, Poster
Experience of extending Poisson-Gap sampling into multiple dimensions.
Sven G. Hyberts; Gerhard Wagner
Harvard Medical School, Boston, MA
Abstract
We have developed an algorithm for determining efficient schedules for non-uniform sampling (NUS) in one indirect dimension, applying a Poisson distribution of gap sizes. The approach is based on statistical facts and empirical findings about gap size distribution in non-uniform sampling of NMR data. The combination of Poisson-Gap sampling and FM reconstruction is presently evaluated for implementation in multiple dimensions. We compare the results when "weaving" the 1-dimensional schedule into multiple dimensions and modifications of the related Poisson-disk approach. Calculations are presently made on a workstation, equipped with four NVIDIA C1060 Tesla cards, each with 240 cores. Effectively, such workstation eliminates the need of a traditional cluster for FM reconstruction in multiple dimensions.

Code: PK	Time Slot/Poster Number: 233	Session: Liquids: New Applications, Poster
13C-direct detected NMR experiments for sequential resonance assignment of RNA
Christian Richter1; Helena Kovacs2, 3; Janina Buck1; Boris Fürtig1; Wolfgang Bermel2, 3; Harald Schwalbe1
1Center for Biomolecular Magnetic Resonance, Frankfurt, Germany; 2Bruker BioSpin AG, Fällanden, Switzerland; 3Bruker BioSpin AG, Rheinstetten, Germany
Abstract
In RNA, the low chemical shift dispersion of sugar protons severely complicates their assignment. The use of 13C-direct detected experiments can greatly facilitate the task. Modifications of the CC-TOCSY experiment with virtual decoupling were employed to enable the 13C-assignment in the ribose rings. In addition, the sequential connectivities were determined by means of a 2D 31P-correlated 13C-detected experiment. The experiments are performed demonstrated on a 14-mer and a 27-mer RNA hairpin. The current set of 13C-direct-detected experiments allowed a direct and unambiguous assignment of the majority of the hetero nuclei and the identification of the individual sugars and their sequential order. Thus, the 13C-direct-detected NMR methods are potentially useful to complement the traditional 1H-detected approach for the assignment of oligonucleotides.

Code: PK	Time Slot/Poster Number: 234	Session: Liquids: New Applications, Poster
PATHOGENESIS OF COXSACKIEVIRUS B6 ISOLATE BY 1H NMR SPECTROSCOPY
Syed Naved Akhtar1; Rishi Kumar Singh1; Pratima Tripathi2; Sarika Tiwari1; Raja Roy2; T. N. Dhole1
1Department of Microbiology, S.G.P.G.I.M.S., Lucknow, India; 2Centre of Biomedical Magnetic Resonance, Lucknow, India
Abstract
Dynamic studies of Coxsackievirus B6 infection in human RD cell and Vero Monkey kidney cells were performed at different time interval of infection by 1H NMR spectroscopy and microscopic studies. The intracellular concentrations of amino acids, myoinositol and choline were increased in RD cells at 12 hrs of infection whereas in Vero cells similar results were observed at 24 hrs of infection. Gradual decrease in amino acids and myoinositol concentration were observed at 36 and 48 hrs of infection in both cells this may be due to maximum pathogenesis at late hrs of infection. Progressive breakdown and consumption of all cellular components was observed as the infection increases. This study shows a possible NMR application in monitoring virus-cell interactions.

Code: PK	Time Slot/Poster Number: 235	Session: Liquids: New Applications, Poster
Measurement of HIV-1PR-inhibitor Binding Affinity and NMR Chemical Shifts with Active and Inactive Constructs
Xi Huang; Angelo Veloro; Mandy Blackburn; Gail Fanucci
Department of Chemistry, University of Florida, Gainesville, FL
Abstract
Subtype polymorphisms among HIV-1 protease (HIV-1PR) variants have been shown to alter protease-inhibitor complexes stabilities with FDA-approved inhibitors determined from differential scanning calorimetry (DSC). The work presented here utilizes 1H NMR titration experiments to estimate protein-ligand binding affinity with both active and inactive constructs. Dissociate constants are determined by fitting the changes in line width and signal intensity of the proton signal of the different inhibitors. Also, to further understand the binding modes of inhibitors with different variants, 2D 13C/15N filtered TOCSY and NOESY experiments are performed to explore the NMR chemical shifts of bound inhibitors. The chemical shifts will be used as parameters for AF-QM/MM calculations to determine the structures of the bound inhibitors.

Code: PK	Time Slot/Poster Number: 236	Session: Liquids: New Applications, Poster
WE DON’T NEED ANOTHER SOLVENT-SUPPRESSING PRESATURATION SEQUENCE ... EXCEPT (EXPONENTIALLY CONVERGING ERADICATION PULSE TRAIN) FOR WATER-SIGNAL SUPPRESSION IN BIOMASS-TO-FUEL INVESTIGATIONS
Wenjia Zhang; Annalise R. Pfaff; EmmaLou T. Satterfield; Klaus Woelk
Missouri S&T, Rolla, MO
Abstract
Selective presaturation is a common technique to suppress large solvent signals that otherwise overpower and disturb the analysis of low-concentration compounds. For biomass-to-fuel (BTF) conversion studies, we optimized many prominent solvent-signal suppression sequences but found that they are very sensitive to changes in the water’s longitudinal relaxation times. Because our BTF reactions are conducted with different salt concentrations and at different pH values, a new presaturation sequence (EXCEPT = EXponentially Converging Eradication Pulse Train) was developed, which is extremely robust with respect to varying relaxation times. It consists of 20 selective inversion pulses with exponentially decreasing interpulse delays. Simulations and practical examples show how EXCEPT suppresses water signals (suppression factor <10 4) within an order of magnitude proton relaxation range.

Code: PK	Time Slot/Poster Number: 237	Session: Liquids: New Applications, Poster
Insight into the Reverse-Phase Ion-Pair Separation of Heparin Derived Oligosaccharides by HR-MAS NMR
Christopher Jones; Szabolcs Beni; Cynthia Larive
University of California, Riverside, Riverside, CA
Abstract
Reverse-phase ion-pairing HPLC and UPLC (RPIP-HPLC) has proven to be a useful chromatographic technique for the separation of heparin and heparan sulfate derived oligosaccharides. However, there is some debate as to the exact mechanism that governs the separation itself. To gain further insight into this mechanism the presented research utilizes the results of HR-MAS saturation transfer difference and diffusion NMR experiments to examine the relative strength and types of interactions that occur between the stationary phase, ion-pairing reagents, and the heparin disaccharides. Use of this knowledge can allow for the development of more efficient and high-resolution RPIP-HPLC methods for the study of heparin and heparin sulfate. This knowledge should also be applicable to the separation of other classes of analytes.

Code: PK	Time Slot/Poster Number: 238	Session: Liquids: New Applications, Poster
Investigation into the Structure and Dynamics of the Bacterial Cell Division Regulator Protein MinE by Solution NMR
Houman Ghasriani1; Thierry Ducat1; Chris T. Hart1; Fatima Hafizi1; Ali Al-Baldawi1; Nina Chang1; Patrik Lundström3; Natalie K. Goto1, 2
1Department of Chemistry, University of Ottawa, Ottawa, Canada; 2Biochemistry, Microbiology, and Immunology, University of Ottawa, Canada; 3Medical Genetics, Biochemistry and Chemistry, University of Toronto, Canada
Abstract
In gram-negative bacteria, the concerted action of three proteins, called MinC, MinD and MinE, regulates the spatial positioning of the cell division plane. MinC and MinD form a complex at the cell membrane and inhibit cell division while MinE counters this inhibition selectively at the midpoint of the cell. This occurs through interactions between MinD and an N-terminal anti-MinCD domain of MinE, the structural details of which are not yet known. In this study we present the results of a solution NMR study of the structure and dynamics of MinE from Neisseria Gonorrhoeae that will help to shed light on the mechanism of MinE function.

Code: PK	Time Slot/Poster Number: 239	Session: Liquids: New Applications, Poster
Effective Control of Spin Systems : Application of Optimal Control Theory
Manoj Nimbalkar1; Burkhard Luy1; Kyryl Kobzar2; Thomas Skinner3; Naum Gershenzon3; Wolfgang Bermel2; Begam Elavarasi4; Jorge Neves5; Haidong Yuan6; Robert Zeier1; Navin Khaneja7; Steffen Glaser1
1Technische Universität München, Garching, München, Germany; 2Bruker BioSpin GmbH, Silberstreifen 4, Rheinstetten, Germany; 3Department of Physics, Wright State University, Dayton, OH 45435,, USA; 4Department of Physics, IIT-Madras, Chennai 600036, India; 5Structural and Molecular Center, Brazilian Synchrotron Light Laborator,10000, CP619, Brazil; 6Department of Mechanical Engineering, MIT, Cambridge, Massachusetts 02139, UAS; 7Division of Engineering and Applied Science, Harvard University, Cambridge, , MA 02138
Abstract
A first topic will be the design and experimental implementation of numerically optimized RF pulses for robust broadband excitation, inversion and universal rotation pulses based on optimal control theory in multi-dimensional experiments such as 2D HSQC. Since these pulses designed for 1H and 13C nuclei are all of identical duration, they can be used directly as replacements for corresponding hard pulses in existing pulse sequences. The second topic will be the experimental implementation of time-optimal pulse sequences for creating spin order e.g. in the case of heteronuclear three spin systems with equal and unequal spin couplings. This type of sequences can be applied in multi-dimensional experiments to create desired coherence orders and for out and back transfer schemes.

Code: PK	Time Slot/Poster Number: 240	Session: Liquids: New Applications, Poster
13C NMR of polyolefins with a new high temperature 10 mm cryoprobe
Zhe Zhou1; David Redwine1; Rainer Kuemmerle2; James Stevens1; Jerzy Klosin1; Xiaohua Qiu1; Rongjuan Cong1; Yiyong He1; Nikki Montanez3; Gordon Roof1
1The Dow Chemical Company, Freeport, TX; 2Bruker, Fällanden, Switzerland; 3Kelly, Freeport, TX
Abstract
Recently, a HT 10 mm cryoprobe was developed. This probe provides a significant sensitivity enhancement for 13C NMR of polyolefins at a sample temperature of 120-135 °C. This greatly increases the speed of NMR studies of polymer stereo- and regio-errors, polymer chain ends, and diffusion of polymers. We compared the 13C NMR sensitivity of this probe with conventional probes. Then, we demonstrated its ability to perform 2D INADEQUATE in a relatively short period of time. The 2D INADEQUATE has been rarely used for polymer studies because of its inherently very low sensitivity. Here, the 2D INADEQUATE along with other 2D techniques were used to study the 13C NMR of 2,1-insertion regioerrors in poly(propylene-co-1-octene) and isotactic polypropylene.

Code: PK	Time Slot/Poster Number: 241	Session: Liquids: New Applications, Poster
Reverse Heteronuclear Saturation Transfer Difference NMR : Identifying receptors in complex mixtures.
James Longstaffe ; David McNally; Myrna Simpson; Andre J Simpson
University of Toronto, Toronto, Canada
Abstract
A general approach to identify binding receptors in complex mixtures is described. Reverse Heteronuclear Saturation Transfer Difference NMR spectroscopy is a powerful but simple technique derived from conventional Saturation Transfer Difference experiments. Rather than irradiating protons in the receptor molecule, a heteronucleus (i.e. 19F) in the small probe molecule is used instead. Saturation is passed from the irradiated probe molecule onto the binding macromolecule where is spreads via spin diffusion. The result of the RH-STD experiment is a 1H NMR spectrum showing only those macromolecular components in the mixture binding to the small probe molecule, thereby allowing for direct identification of receptors in complex matrices. We demonstrate the concept with human blood and soil.

Code: PK	Time Slot/Poster Number: 242	Session: Liquids: New Applications, Poster
O-Mannosyl Phosphorylation of Alpha-Dystroglycan Is Required for Laminin Binding
Takako Yoshida-Moriguchi1; Liping Yu1; Stephanie Stalnaker2; Sarah Davis1; Stefan Kunz3; Michael Madson4; Michael Oldstone5; Harry Schachter6; Lance Wells2; Kevin Campbell1
1University of Iowa, Iowa City, IA; 2University of Georgia, Athens, GA; 3University of Lausanne, Lausanne, Switzerland; 4Bio Logistics, Clear Lake, IA; 5The Scripps Research Institute, La Jolla, CA; 6The Hospital for Sick Children, Toronto, Ontario
Abstract
Alpha-dystroglycan (alpha-DG) is a cell-surface glycoprotein that acts as a receptor for both extracellular matrix proteins containing laminin-G domains and certain arenaviruses. Receptor binding is thought to be mediated by a posttranslational modification, and defective binding with laminin underlies a subclass of congenital muscular dystrophy. Using mass spectrometry- and nuclear magnetic resonance (NMR)-based structural analyses, we identified a phosphorylated O-mannosyl glycan on the mucin-like domain of recombinant alpha-DG, which was required for laminin binding. In this poster, we present the structural analysis by NMR spectroscopy of the O-mannosyl glycan. A combination of homo- and hetero-nuclear NMR techniques was used to assign the 1H, 13C, and 31P resonances and determine the structure of the phosphorylated O-mannosyl glycan.

Code: PK	Time Slot/Poster Number: 243	Session: Liquids: New Applications, Poster
Quantum Simulation of Many-body Quantum Systems in NMR
Xinhua Peng
University of Science and Technology of China, Hefei, China
Abstract
Quantum phase transitions describes an abrupt change in the ground state of a many-body system due to its quantum fluctuations. We consider a particularly interesting system with competing one-, two- and three-body interactions, whose ground state can be a product state, or it can exhibit genuine tripartite entanglement. We experimentally simulate such a system in an NMR quantum simulator and observe the different ground states. By adiabatically changing the strength of one coupling constant, we push the system from one ground state to a qualitatively different ground state. We show that these ground states can be distinguished and the transitions between them observed by measuring correlations between the spins or the expectation values of suitable entanglement witnesses.

Code: PK	Time Slot/Poster Number: 244	Session: Liquids: New Applications, Poster
Structure and Function of Transmembrane and Cytoplasmic Domains of SorLA, a Player in Alzheimer’s Disease
Xingsheng Wang; Qin Zhu; Richard L Gill, Jr.; Fang Tian
Penn State College of Medicine, Hershey, PA
Abstract
Alzheimer’s disease is characterized by appearance of amyloid plaques, which are mainly composed of aggregated amyloid-peptide (Aβ) derived from the amyloid precursor protein (APP). Alterations in APP cellular trafficking and localization impact its processing to Aβ. SorLA contains a transmembrane domain (TM) and a cytoplasmic domain (CT). Expression of SorLA is significantly reduced in AD brains. SorLA interacts with GGA, suggesting that SorLA controls APP trafficking between TGN and early endosomes. We reported that high quality TROSY spectra of TM and TM-CT of SorLA were obtained in DPC micelles, indicating the feasibility of their structural determinations. A peptide derived from the C-terminus of SorLA showed strong binding to the VHS domain of GGA in an NMR titration experiment.