|
Code:
PR
|
Time Slot/Poster Number:
351
|
Session:
Solids: Bio-Applications, Poster
|
Analysis of Binding Sites of Curcumin in the Fibrils of 42-Residue Amyloid Beta-Protein (Abeta42) Using Solid-State NMR
|
| Yuichi Masuda1; Masashi Fukuchi1; Tatsuya Yatagawa1; Kazuyuki Takeda1; Kazuhiro Irie2; K. Takegoshi1
|
1Graduate School of Science, Kyoto University, Kyoto, Japan; 2Graduate School of Agriculture, Kyoto University, Kyoto, Japan
|
| Abstract |
Aggregation of 42-residue amyloid-beta protein (Abeta42) plays a crucial role in the pathogenesis of Alzheimer’s disease. Since curcumin, the yellow pigment in the rhizome of turmeric, interacts with the aggregates (fibrils) of Abeta42 and dissolve them, binding sites of curcumin in the Abeta42 fibrils were analyzed by solid-state NMR using dipolar assisted rotational resonance (DARR). To improve the quality of 2D spectrum, covariance processing was applied to the 2D data. In the 2D DARR spectrum at a mixing time of 500 ms, clear cross peaks were observed between the carbons at positions 17-21 of the Abeta42 fibrils and those of curcumin, indicating the existence of the curcumin molecules binding to the intermolecular beta-sheet at positions 17-21 of the Abeta42 fibrils.
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Code:
PR
|
Time Slot/Poster Number:
352
|
Session:
Solids: Bio-Applications, Poster
|
Orientation and Depth of Guanidinium-Containing Molecules in Lipid Membranes by Solid-State NMR
|
| Yongchao Su1; Tim Doherty1; William F. DeGrado2; Mei Hong1
|
1Iowa State University, Ames, IA; 2University of Pennsylvania, Philadelphia, PA
|
| Abstract |
The orientation and depth of insertion of two guanidinium-containing molecules in lipid bilayers have been determined using 2D anisotropic correlation NMR in aligned bicelles and intermolecular distance experiments under magic-angle spinning. 19F NMR shows that the antimicrobial arylamide oligomer PMX30016 is found to slice into the bilayer like a knife, with the molecular plane nearly perpendicular to the bilayer surface. Fast uniaxial rotation at the membrane-water interface further destabilizes the lipid packing. 15N NMR of the Arg-rich voltage-sensing S4 helix of the potassium channel, KvAP, shows that the S4 helix spans the membrane in a tilted orientation, with one of the four Arg residues forming salt bridges with the lipid phosphates. This orientation is nearly identical to that of the S4 helix in the intact voltage sensor, suggesting that the folding of this important helix is mostly determined by protein-lipid interactions rather than protein-protein interactions.
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Code:
PR
|
Time Slot/Poster Number:
353
|
Session:
Solids: Bio-Applications, Poster
|
Is MgtC, a Potential Drug Target in Mycobacterium tuberculosis, Inhibited by MgtR? Peptides Interaction Study Using PISEMA
|
| Frantz L. Jean-Francois; Timothy A. Cross
|
NHMFL, Tallahassee, FL
|
| Abstract |
Solid state NMR is the most powerful technique to decipher protein dynamics within the membrane hydrophobic core. Recent results highlight the role of hydrophobic peptides that form a novel class of active molecules acting on membrane proteins through hydrophobic interactions. To further understand this new mechanism at a molecular level, the interaction between two potentially interacting peptides is studied. The studied system is made of the potential drug MgtR, one of these newly discovered hydrophobic peptides, and its potential target (MgtC) involved in the Tuberculosis latent state. These two peptides were chemically synthesized and reconstituted into DMPC/DMPG bilayers aligned on glass plates. The interaction is examined by using solid state NMR PISEMA on 15N specifically labeled peptides.
|
|
Code:
PR
|
Time Slot/Poster Number:
354
|
Session:
Solids: Bio-Applications, Poster
|
Membrane Topology of Human Alpha Defensin HNP-1 by Solid-State NMR
|
| Yuan Zhang; Mei Hong
|
Iowa State University, Ames, IA
|
| Abstract |
HNP-1 is a small disulfide-stabilized antimicrobial protein that disrupts the cell membranes of microbial organisms. To date no structural information of HNPs in the lipid membrane has been reported. We have carried out a solid-state NMR study of the membrane topology of HNP-1 in anionic lipid membranes. The secondary structure and the dynamics of the protein are similar between the membrane-bound state and the microcrystalline state, with the interesting exception of R25, which becomes more immobilized in the membrane. 1H spin diffusion from lipid chains to protein and from water to protein both support a transmembrane topology, where the hydrophilic surface of the protein is in contact with water in a pore and the hydrophobic surface facing the lipids. 13C-31P REDOR experiments indicate one out of 4 Arg’s is in close contact with the lipid phosphate groups. We propose R25 is responsible for phosphate interaction due to its preferential immobilization. The location of R25 in the dimer pore model is consistent with its close proximity to the lipid headgroups. These data provide the first high-resolution structural information of a mammalian defensin in lipid membranes. The mechanistic conclusion may be relevant to other defensins as well.
|
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Code:
PR
|
Time Slot/Poster Number:
355
|
Session:
Solids: Bio-Applications, Poster
|
Novel Analysis of Kinked Helices from PISEMA Data
|
| Dylan Murray1, 2; J.R. Quine1, 2; Timothy Cross1, 2
|
1The Florida State University, Tallahassee, FL; 2The National High Magnetic Field Laboratory, Tallahassee, FL
|
| Abstract |
We have devised a method by which the geometry of a kinked helix can be determined from solid state NMR PISEMA experimental data. First we derive the mathematical description of the kink assuming two ideal helical segments and a single pair of non-ideal torsion angles. Next we relate the experimentally observed helix orientation angles determined from a PISA wheel analysis to the geometry of the kink. Then, assuming a PISA wheel can be determined for each segment of the kinked helix, the kink angle between the two helices can be determined within a small degeneracy. We apply the results to existing experimental data for the M2 proton channel.
|
|
Code:
PR
|
Time Slot/Poster Number:
357
|
Session:
Solids: Bio-Applications, Poster
|
Structure Determination Method for Biomineral-associated Protein Using Combined Solid-state NMR and Computational Structure Prediction
|
| Jason T Ash1; David Masica2; Moise Ndao1; Jeffrey Gray2; Gary Drobny1
|
1The University of Washington, Seattle, WA; 2Johns Hopkins University, Baltimore, MD
|
| Abstract |
Protein-biomineral interactions are paramount to materials production in biology, including the mineral phase of hard tissue. Unfortunately, the structure of biomineral-associated proteins cannot be determined by X-ray crystallography or solution NMR. We report here a method that combines solid-state NMR (ssNMR) and ssNMR-biased computational structure prediction in an iterative fashion to determine the structure of human salivary statherin interacting with hydroxyapatite, the predominate mineral phase of tooth enamel. Computation and experiment converge quickly on a small ensemble of related structures. In addition, the algorithm is able to investigate crystal-face binding specificity by identifying lattice geometries most compatible with ssNMR constraints. This is, to our knowledge, the first high-resolution structure of a biomineral-adsorbed protein determined with experimentally biased structure prediction.
|
|
Code:
PR
|
Time Slot/Poster Number:
358
|
Session:
Solids: Bio-Applications, Poster
|
Lipid Dynamics of DPPC-d62, POPC-d31, POPG-d31, and POPE-d31 in Calf Lung Surfactant Extract
|
| R. Suzanne Farver; Joanna R. Long
|
University of Florida, Gainesville, FL
|
| Abstract |
Calf Lung Surfactant Extract (CLSE) is an endogenous pulmonary surfactant commonly administered to premature infants with respiratory distress syndrome. We are investigating and comparing CLSE to a fully synthetic, SP-B(1-25)-containing lipid formulation. 31P and 2H static solid-state NMR was used to determine if the lipid phase behavior seen in CLSE is similar to that found in our synthetic system. Comparison of the synthetic lung surfactant to a more native form of lung surfactant may reveal similar characteristics or point to a relative oversimplification. Data for both the synthetic and endogenous lung surfactant systems will be presented.
|
|
Code:
PR
|
Time Slot/Poster Number:
359
|
Session:
Solids: Bio-Applications, Poster
|
Solid-State NMR Spectroscopic Characterization of the Endogenous Ubiquinone in the Disulfide Bond Generating Membrane Protein DsbB
|
| Ming Tang; Lindsay Sperling; Deborah Berthold; Anna Nesbitt; Robert Gennis; Chad Rienstra
|
University of Illinois at Urbana-Champaign, Urbana, IL
|
| Abstract |
Ubiquinone (Coenzyme Q) plays an important role in the mitochondrial respiratory chain, and also protects cellular membranes from peroxidation as an antioxidant in the reduced form. De novo disulfide bond generation in the E. coli periplasm is involved with the complex system consisting of DsbA, DsbB and ubiquinone. The periplasmic oxidase DsbA is responsible for the disulfide bond formation of substrate proteins, while the membrane protein DsbB is in charge of reoxidizing DsbA by transferring electrons from DsbA to ubiquinone. However, the structure of the charge-transfer complex of DsbB and UQ and the structural changes of DsbB upon DsbA binding are still unclear. We applied advanced multidimensional solid-state NMR methods to provide high-resolution atomic-level structural information of UQ in the membrane protein DsbB. The chemical shifts obtained by solid-state NMR spectroscopy suggested the formation of charge-transfer complex.
|
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Code:
PR
|
Time Slot/Poster Number:
360
|
Session:
Solids: Bio-Applications, Poster
|
Is INEPT a inept sequence? It reveals the dances of a membrane protein embedded in bicelles
|
| Jiadi Xu; Ronald soong; Pieter Smith; Sang-Choul Im; Lucy Waskell; Ayyalusamy Ramamoorthy
|
university of michigan, Ann Arbor, MI
|
| Abstract |
Measurement of membrane protein dynamics is essential to understand the relationship between its structure and function. While these exciting biological molecules pose tremendous challenges to even solid-state NMR techniques, we demonstrate the power of the INEPT-based pulse sequences reveal the dynamical aspects of a membrane-associated cytochrome b5 using aligned DMPC:DHPC bicelles
|
|
Code:
PR
|
Time Slot/Poster Number:
361
|
Session:
Solids: Bio-Applications, Poster
|
Solid-State 13C NMR Study of Nanocrystalline Cellulose
|
| Clark Lemke1; Carl Michal1; Ronald Dong1; Wadood Hamad2
|
1University of British Columbia, Vancouver, Canada; 2FPInnovations – Paprican, Vancouver, Canada
|
| Abstract |
Nanocrystalline cellulose (NCC) shows very unique properties – in suspension, it spontaneously forms a chiral nematic phase and in high purity exhibits iridescence. While native cellulose has historically been extensively studied in solution and solid-state NMR with success, the physical structure of NCC particles is not fully known. In the past, broad peaks upfield of C4 and C6 have been labeled as surface/amorphous and one of our goals is to understand the origin of these peaks and to quantify the surface and amorphous components. In this study we used 2H{13C} REDOR, 13C{31P} REDOR and 13C CP/MAS NMR to investigate the structure of the NCC.
|
|
Code:
PR
|
Time Slot/Poster Number:
362
|
Session:
Solids: Bio-Applications, Poster
|
Molecular-Level Evidence of Protein Misfolding of Alzheimer’s β-amyloid in Solid-Phase Peptide Synthesis
by Sold-state NMR
|
| Songlin Wang; Yoshitaka Ishii
|
University of Illinois at Chicago, Chicago, IL
|
| Abstract |
Solid-phase peptide synthesis (SPPS) is a valuable technique in chemistry and biology to produce proteins/peptides of arbitrary sequences at high purity. Despite the effectiveness, synthesis yield of SPPS typically drops drastically for a longer sequence. Detailed molecular mechanism that prevents efficient reactions in SPPS for a longer peptide is still unknown to large extent. In this study, using 13C SSNMR we examine the possibility of detecting protein folding/misfolding in the course of SPPS, which has been suspected to interfere with the reaction in SPPS. Our 13C SSNMR data on Aβ(1-40) and a 16-residue fragment of Aβ(25-40) that are both bound to a resin support proved that even the short fragment is likely to misfold into β-sheet structure in organic solvent.
|
|
Code:
PR
|
Time Slot/Poster Number:
363
|
Session:
Solids: Bio-Applications, Poster
|
Structural and dynamical NMR studies of membrane-associated Cytochrome b5 – “an accomplice of Mother Nature’s blowtorch”
|
| Ronald Soong; Jiadi Xu; Ayyalusamy Ramamoorthy; Pieter Smith
|
University of Michigan, Ann Arbor, MI
|
| Abstract |
Microsomal cytochrome b5 (Cytb5) is a 16 kDa membrane anchored protein whose interactions with cytochrome P450 facilitate the regulation of drug metabolism. As no structure of the full-length-cytb5 has yet been solved, the mechanism by which it controls the activity of CytP450 remains enigmatic. A central difficulty of studying Cytb5 has been the inadequacy of current NMR methodologies to approach membrane proteins that belong to neither solution nor solid-state NMR domains. To address this problem, new NMR methodologies are being developed that allow for the structure determination of membrane protein comprised of regions with vastly different dynamics. Using various orientational constraints, key structural and dynamical features of this protein and its interaction with lipid bilayers are revealed.
|
|
Code:
PR
|
Time Slot/Poster Number:
364
|
Session:
Solids: Bio-Applications, Poster
|
Solution and solid-state NMR studies of natural and transgenic spider silk proteins
|
| Jérémie Leclerc1; Isabelle Cloutier1; Thierry Lefèvre1; Stéphane M. Gagné2; Michel Pézolet1; Michèle Auger1
|
1Department of Chemistry, Université Laval, Québec, Québec; 2Department of Biochemistry and Microbiology, Université Laval, Québec, Québec
|
| Abstract |
Spider silk is a biomaterial with astonishing properties that compete with the best synthetic man made materials such as Kevlar. The structure of silk proteins in solution was investigated as a function of temperature and pH by 1H and 13C NMR. Our results indicate that the protein conformation is unordered in solution and that the alanine residues adopt a polyproline II (ppII) structure. Another objective of this work was to analyze the secondary structure and dynamics of natural and transgenic silk proteins in fibers. Alanine and glycine dynamics increases as MaS < MiS < cocoon silk, showing that the whole fiber displays distinctive protein dynamics, chain packing and intermolecular interactions.
|
|
Code:
PR
|
Time Slot/Poster Number:
365
|
Session:
Solids: Bio-Applications, Poster
|
Water-Protein Interactions of an Arginine-rich Membrane Peptide in Lipid Bilayers Investigated by Solid-state NMR Spectroscopy
|
| Shenhui Li; Yongchao Su; Wenbin Luo; Mei Hong
|
Iowa State University, Ames, IA
|
| Abstract |
The interaction of an arginine (Arg) residue with water in a transmembrane antimicrobial peptide, PG-1, is investigated by 2D heteronuclear correlation (HETCOR) solid-state NMR spectroscopy. Using 13C and 15N dipolar-edited 1H-15N MELODI-HETCOR experiments, we unambiguously assigned a water-guanidinium cross peak, indicating that water is in close contact with the membrane-inserted guanidinium. The temperature dependence of the Arg-correlated water 1H chemical shift is consistent with hydrogen bonding between water and guanidinium. Together with previous guanidinium-phosphate distance constraints, the HETCOR NMR data indicate that the Arg sidechains of PG-1 are stabilized by hydrogen bonding with both water and lipids.
|
|
Code:
PR
|
Time Slot/Poster Number:
366
|
Session:
Solids: Bio-Applications, Poster
|
J-couplings in Solid-State NMR:
Sensitivity and Resolution Improvement for Through-Bond Correlation Experiments
and Quantitative Measurement of 1JCC couplings in GB1.
|
| Ségolène Laage1; Guido Pintacuda1; Roberta Pierattelli2; Isabella Felli2; Ivano Bertini2; Jozef Lewandowski1; Lyndon Emsley1; Anne Lesage1
|
1Centre de RMN à Très Hauts Champs, Villeurbanne, France; 2CERM, University of Florence, Sesto Fiorentino, Italy
|
| Abstract |
We present our recent advances in the use of J-couplings for solid-state magic angle spinning NMR methods, and their applications to biological samples. We propose a new sequence, dubbed INADEQUATE-S3E, developed to improve the performance of through-bond correlation experiments in terms of sensitivity and resolution. This sequence limits the loss of sensitivity due to transverse dephasing and performs J-decoupling, leading to gains factors up to 2.5 in sensitivity and 50 Hz in resolution. We also measured for the first time JCC-couplings in a microcrystalline protein, possibly opening new access to structural and dynamical information. These methods were applied to a microcrystalline paramagnetic protein, the human superoxide dismutase (SOD), a dimeric Cu(II) enzyme of 32 kDa and microcrystalline protein GB1.
|
|
Code:
PR
|
Time Slot/Poster Number:
367
|
Session:
Solids: Bio-Applications, Poster
|
Probing peptides containing proline and pseudoproline : An NMR study in solution and solid state.
|
| S. Raghothama; Rajesh Sonti
|
Indian Institute of Science, Bangalore, India
|
| Abstract |
In designing secondary structure motif peptide sequences, use of proline residue play an important role. Such peptides will always have the problem of cis / trans isomerization, though trans is more favoured. In literature we see that Pseudoproline, an anlog of proline favours cis form through non bonded interactions. We have substituted one of the proline with psedoproline in our peptides containing di-proline segment in order to drive the conformation to cis form. Both solution and solid state NMR at natural abundance is used for analysis. It was interesting to see there were difference in the solution and solid-state studies. Energies involved being small, the number of intermolecular hydrogen bonds played a significant role to switch over between conformations.
|
|
Code:
PR
|
Time Slot/Poster Number:
368
|
Session:
Solids: Bio-Applications, Poster
|
Magnetization Transfer Pathways Between Backbone 15N Spins in Macroscopically Aligned Protein Samples under Mismatched Hartmann-Hahn Conditions
|
| Robert W. Knox; Alexander A. Nevzorov
|
North Carolina State University, Raleigh, NC
|
| Abstract |
Crosspeaks arising from proton-mediated 15N-15N magnetization transfer under mismatched Hartmann-Hahn conditions can be used for assignment and elucidation of intra- and intermolecular contacts in aligned protein samples. There are multiple possibilities for the transfer between the 15N spins in a protein backbone including adjacent (i.e. i and i+1) residues and long-range correlations. Using the coordinates of Pf1 phage (1ZN5), the magnetization transfer for each residue along the chain has been simulated for the (i, i+1) through (i, i+4) transfers. It has been found that the (i, i+1) crosspeaks prevail when the mismatch amplitude is increased by as much as 25%, which is also supported by our recent experiments. This allows one to distinguish between short- vs. long-range spin correlations.
|
|
Code:
PR
|
Time Slot/Poster Number:
369
|
Session:
Solids: Bio-Applications, Poster
|
Dehydration-induced Structural Changes in Bovine Cortical Bone Revealed by Solid-State NMR Spectroscopy
|
| Peizhi Zhu; Jiadi Xu; Michael Morris; Nadder Sahar; David Kohn; Ayyalusamy Ramamoorthy
|
University of Michigan, Ann Arbor, MI
|
| Abstract |
Understanding the structure and structural changes of bone, a highly heterogeneous material with a complex hierarchical architecture, continues to be a significant challenge even for high-resolution solid-state NMR spectroscopy. While it is known that dehydration affects mechanical properties of bone by decreasing its strength and toughness, the underlying structural mechanism at the atomic-level is unknown. Solid-state NMR spectroscopy, controlled dehydration were used to reveal structural changes of an intact piece of bovine cortical bone. The experiments revealed the slow denaturation of collagen due to dehydration while the trans-Xaa-Pro conformation in collagen remain unchanged. These results provide insights into the role of water molecules in the bone structure and on the relationship between the structure and mechanics of bone.
|
|
Code:
PR
|
Time Slot/Poster Number:
370
|
Session:
Solids: Bio-Applications, Poster
|
Gaining Additional High-Resolution Structural Insights into Bone – A Solid-State NMR Investigation Utilizing Paramagnetic Doping
|
| Neil Mackinnon; Jiadi Xu; Peizhi Zhu; Michael D. Morris; David Kohn; Ayyalusamy Ramamoorthy
|
University of Michigan, Ann Arbor, MI
|
| Abstract |
The inherent hierarchal structural complexity of bone is fundamental to its properties as a supportive material, and this complexity challenges traditional spectroscopic techniques at understanding the structure-function relationship. Bone is composed of both inorganic and organic materials, with collagen making the largest contribution to the organic phase and a calcium mineral making up the inorganic phase. In order to probe the inorganic/organic interface of bone, we have employed the paramagnetic ion Cu(II) and MAS experiments. Monitoring the 1H T1 relaxation properties in the presence and absence of Cu(II), in addition to careful attention to 13C linewidths, we demonstrate the utility of paramagnetic probes in furthering the understanding of the structural properties of complex, heterogeneous materials.
|
|
Code:
PR
|
Time Slot/Poster Number:
371
|
Session:
Solids: Bio-Applications, Poster
|
Long-range polarization transfer in protein MAS NMR spectroscopy: Effects of backbone and side chain dynamics
|
| Marvin J. Bayro1; Neil Birkett2; Christopher Dobson2; Robert Griffin1
|
1Massachusetts Institute of Technology, Cambridge, MA; 2University of Cambridge, Cambridge, UK
|
| Abstract |
We have examined temperature effects on backbone-backbone correlations in PDSD and band-selective RFDR experiments in proteins. At temperatures below the sample freezing point, we observe an improvement in polarization transfer obtained with BASE RFDR, but not with PDSD. The enhancement can be interpreted in terms of dynamics in the protein backbone, while the selectivity of this effect may be attributed to the difference in polarization transfer mechanisms. In addition, we have identified interference effects due to aromatic side chain motions and illustrate cases in which the efficiency of long-range aromatic-aliphatic transfers are improved significantly. We describe the above observations in the context of distance measurements for the structure determination of protein PI3-SH3 in amyloid fibril form.
|
|
Code:
PR
|
Time Slot/Poster Number:
372
|
Session:
Solids: Bio-Applications, Poster
|
Magic-Angle Spinning Solid-State NMR Studies of Large Membrane Proteins and Membrane Protein Complexes
|
| Lindsay J Sperling; Ming Tang; Myat Tun Lin; Anna E. Nesbitt; Deborah A. Berthold; Robert B. Gennis; Chad M. Rienstra
|
University of Illinois at Urbana-Champaign, Urbana, IL
|
| Abstract |
Here we use magic-angle spinning solid-state NMR (MAS SSNMR) techniques to study the structure, dynamics, and other mechanisitic details of a 41 kDa membrane protein complex DsbA-DsbB, as well the 144 kDa membrane protein cytochrome bo3 oxidase. To address the challenges of studying such a large system, we have developed and optimized spectroscopic methods to improve sensitivity and resolution of our experiments on DsbA (21 kDa). Full chemical shift assignments have been performed using 2D, 3D and 4D MAS SSNMR experiments of uniformly and selectively labeled sample preparations at varying magnetic fields from 500 MHz to ultra-high magnetic fields. These sensitivity improvements are key to site specifically elucidating key functional understandings of large membrane proteins.
|
|
Code:
PR
|
Time Slot/Poster Number:
373
|
Session:
Solids: Bio-Applications, Poster
|
Studying structure and topology of OMPs in lipid bilayers using solid-state NMR
|
| Marie Renault; Martine Bos; Jan Tommassen; Marc Baldus
|
Utrecht University, Utrecht, Netherlands
|
| Abstract |
Outer membrane proteins (OMPs) carry out a multitude of biological tasks and have structurally been well characterized by X-ray crystallography and solution-state NMR. We have investigated such proteins using solid-state NMR that offers the opportunity to probe protein topology and dynamics in a functional membrane setting. For the eight-stranded antiparallel β-barrel protein PagL, we find a well-defined protein structure and no evidence for conformational exchange as regular seen in micelle-based solution-state NMR. Further experiments were performed to probe the overall protein topology in membranes as well as to identify the protein region involved in catalytic activity. Progress to conduct similar studies in larger (multidomain) OMPs will be shown.
|
|
Code:
PR
|
Time Slot/Poster Number:
374
|
Session:
Solids: Bio-Applications, Poster
|
Long-term-stable ester vs conventional ester lipids in oriented solid-state NMR– altered information in studies of antimicrobial peptides?
|
| Kresten Bertelsen; Brian Vad; Thomas Vosegaard; Niels Chr. Nielsen
|
Århus University, Århus, Denmark
|
| Abstract |
Recently ether lipids have been introduced as long-term stable alternatives to the more natural, albeit easier degradable, ester lipids in preparation of oriented lipid bilayers and bicelles for oriented-sample solid-state NMR spectroscopy. We report that ether lipids such as the frequently used 14-O-PC (1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine) may induce severe structural changes and altered interaction between peptides and lipids relative to conventional DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) bilayers. Significant changes in the structure/orientation were observed for the antimicrobial peptide alamethicin as observed by 15N and 2H solid state NMR spectroscopy on going from horizontal (flipped) to vertical orientation of the bicelles in which the peptides were incorporated.
|
|
Code:
PR
|
Time Slot/Poster Number:
376
|
Session:
Solids: Bio-Applications, Poster
|
Unstructured Conformation and High Dynamics of HIV TAT in Lipid Membranes from Solid-State NMR Spectroscopy
|
| Yongchao Su; Mei Hong
|
Iowa State University, Ames, IA
|
| Abstract |
A comprehensive solid-state NMR study has been carried out to investigate the conformation, dynamics and depth of insertion of a cell-penetrating peptide (CPP), HIV TAT, in the lipid bilayer. This Arg-rich peptide is found to adopt a highly dynamic random coil conformation in lipid bilayers. Backbone-phosphate distances and spin diffusion spectra indicate that TAT inserts into the membrane-water interface. Short guanidinium Cz to lipid 31P distances and relatively high guanidinium order parameters indicate strong guanidinium-phosphate interactions. Strong water cross peaks with polar sidechains are observed in 2D 13C-detected 1H spin diffusion spectra. These data indicate that Arg residues in TAT are stabilized by lipid phosphates and hydration water during translocation of the peptide across the lipid bilayer. The lack of intra- and inter-molecular hydrogen bonded conformation may serve to prevent these Arg-rich CPPs from permanently residing in and damaging the lipid membrane.
* To whom correspondence should be addressed. E-mail: yongchao@iastate.edu. Tel: (515) 294-5402. Fax: (515) 294-0105.
|
|
Code:
PR
|
Time Slot/Poster Number:
377
|
Session:
Solids: Bio-Applications, Poster
|
Effect of Temperature on the Cross-Relaxation between Amide Protons and Water Protons in Uniformly 15N, 13C, 2H labeled Ubiquitin
|
| Suvrajit Sengupta1; R. Andrew Byrd2; Kurt W. Zilm1
|
1Department of Chemistry, Yale University, New Haven, CT; 2Structural Biophysics Lab, National Cancer Inst., Fredrick, Maryland
|
| Abstract |
Water within a protein plays a vital role in protein structure, stability and function. Recently we have shown that there is significant dipolar cross-relaxation between the backbone amide protons and their surrounding water. This work investigates the dynamics of this dipolar cross-relaxation at different temperatures. 1D ssNMR experiments were performed on U–15N,13C,2H–ubiquitin. The amide–water cross-relaxation was observed in both directions. The characteristic relaxation rates, which are related to the auto and cross-relaxation rates of the amide and water protons, are found to vary from 30s-1 at 273K, to about 300s-1 at 232K. The implications on the dynamics of the cross-relaxation process will be discussed.
|
|
Code:
PR
|
Time Slot/Poster Number:
378
|
Session:
Solids: Bio-Applications, Poster
|
Spectral Editing in Solid-State Proteins using Homonuclear 13C J-Couplings: The Selection and Differentiation of Specific Amino Acid Types
|
| Ye Tian; Olivia Alley; Yachong Wang; Lingchao Zhu; Lingling Chen; Leonard J. Mueller
|
University of California, Riverside, Riverside, CA
|
| Abstract |
Recently we have demonstrated that homonuclear and heteronuclear 3D correlation experiments can be implemented using purely scalar-based transfers for the assignment of sidechain and backbone resonances. These J-based methods allow for incredible control and finesse over the spin dynamics. Here we make use of homonuclear 13C J-couplings to robustly and selectively edit out and label specific amino acids as part of J-based 2D and 3D correlation experiments. This selection, essentially a homonuclear version of INEPT, provides a mechanism for selecting specific amino acid types based on the number of attached γ- and/or δ- carbons. These experiments will be illustrated on several proteins, including GB1 and tryptophan synthase, under various MAS rates and decoupling conditions.
|
|
Code:
PR
|
Time Slot/Poster Number:
379
|
Session:
Solids: Bio-Applications, Poster
|
Structural Studies of Alpha-Synuclein Fibrils by MAS SSNMR
|
| Gemma Comellas; Kathryn D. Kloepper; Andrew J. Nieuwkoop; Luisel R. Lemkau; Wendy S. Woods; Julia M. George; Chad M. Rienstra
|
University of Illinois at Urbana-Champaign, Urbana, IL
|
| Abstract |
MAS SSNMR offers unique access to insoluble protein systems such as alpha-synuclein (AS) fibrils, the main protein found in Lewy bodies, which are the pathological hallmark of Parkinson’s disease. To obtain atomic-resolution structural information of AS fibrils requires complete chemical shift assignments. Previous chemical shift assignments of the central region of AS fibrils have been reported by the Baldus group and our group. Here we further investigate the latter fibril state, and show that new multidimensional MAS experiments, sample preparation protocols and isotopic labeling schemes yield the resonance assignments for many additional sites. These assignments include a number of protein residues that are disordered (in a static or dynamic sense) and therefore escaped detection in dipolar experiments used previously.
|
|
Code:
PR
|
Time Slot/Poster Number:
380
|
Session:
Solids: Bio-Applications, Poster
|
Investigating Fungal Melanin Biosynthesis and Molecular Structure with Solid-State NMR
|
| Subhasish Chatterjee1; Susana Frases2; Rafael Prados-Rosales2; Hsin Wang1; Boris Itin3; Arturo Casadevall2; Ruth Stark1
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1The City College of New York, New York, NY; 2Albert Einstein College of Medicine, Yeshiva Unive, The bronx, New York, New York; 3New York Structural Biology Center, New York, New York
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| Abstract |
Solid-state NMR techniques have been used to elucidate the structural characteristics and biosynthetic pathways of fungal melanin, a biologically important but intractable polymer associated with functions including fungal virulence and anti-tumor drug resistance. 2D 13C-13C dipolar-assisted rotational resonance (DARR) experiments were used to delineate proximities between key aromatic structural moieties of the melanin pigment and cell-wall or cell-membrane constituents produced by Cryptococcus neoformans melanized in the presence of isotopically enriched L-dopa and/or D-glucose. Spectral fingerprints of melanins produced with equimolar mixtures of obligatory catecholamine precursors such as L-dopa and epinephrine suggested preferential incorporation of L-dopa in the final polymeric framework of the pigment and highlighted differences in the metabolic transformations associated with formation of the mature biopolymer.
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Code:
PR
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Time Slot/Poster Number:
381
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Session:
Solids: Bio-Applications, Poster
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The Effect of Bis(monoacylglycero)phosphate on the Thermotropic Phase Behavior of DPPC
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| Thomas Frederick; Philip Goff; Suzanne Farver; Joanna Long; Gail Fanucci
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University of Florida, Gainesville, FL
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| Abstract |
Bis(monoacylglycero)phosphate (BMP) is a unique anionic phospholipid found in the intraendosomal vesicular bodies, and is believed to play a role in the structural integrity of late endosomes, in the sorting/trafficking and degradation activities of glycosphingolipids. These studies characterized the effects of BMP on the acyl-chain dynamics and thermotropic phase behavior of model phosphatidylcholine lipid bilayers via solid-state 2H NMR, and compared them to the effects induced by its structural isomer dioleoyl phosphatidylglycerol (DOPG). Increasing the composition of either BMP or DOPG decreases both the main phase transition temperature and the breadth of the transition to the same extent. However, the acyl-chain order parameters revealed that BMP has a minimal effect on the acyl-chain order parameter as compared to DOPG.
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Code:
PR
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Time Slot/Poster Number:
382
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Session:
Solids: Bio-Applications, Poster
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The Structure of the Alpha-B-Crystallin Dimer and Oligomeric Organization Determined by Solid-State MAS NMR and Small-Angle X-Ray Scattering
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| Stefan Jehle1; Ponni Rajagopal2; Benjamin Bardiaux1; Stefan Markovic1; Ronald Kuehne1; Joseph R. Stout2; Victoria A. Higman1; Rachel E. Klevit2; Barth-Jan Van Rossum1; Hartmut Oschkinat1
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1Leibniz-Institut fuer Molekulare Pharmakologie, Berlin, Germany; 2University of Washington, Seattle, Washington
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| Abstract |
The small heat shock protein alpha-B-crystallin (aB) acts as an ATP-independent chaperone. Dysfunctions of human aB are associated with the occurrence of cataracts in the eye lens, multiple sclerosis, cardiomyopathies and Alzheimer’s disease. aB forms polydisperse supramolecular complexes with variable number of subunits (~24-32), which has confounded high-resolution structural studies for decades. Here, we provide a structural basis for the activation and inactivation of the aB oligomer applying solid-state MAS NMR spectroscopy and small-angle X-ray scattering. The basic building block is a curved dimer. The C-terminal IXI motif and the N-terminal residues S59-W60-F61 interact with other dimers. We observe a pH-dependent modulation of the interaction of the IXI motif which is associated with activation.
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Code:
PR
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Time Slot/Poster Number:
383
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Session:
Solids: Bio-Applications, Poster
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Metal-Binding Structure and Sensitivity Enhancement in Solid-state NMR studies of
Cu2+ bound Alzheimer’s b-amyloid fibrils
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| Sudhakar Parthasarathy; Yoshitaka Ishii
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University of Illinois at chicago, Chicago, IL
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| Abstract |
In this poster, we will be discussing two separate topics. In the first topic we will examine the molecular details of Cu2+-binding on 40-residue Ab(1-40) fibrils by SSNMR and specifically answer the following questions (i) Is the Cu2+ binding site specific? (ii) If yes, what are the residues involved? (iii) Are there any major structural changes induced by Cu2+ binding? (iv) Is Met-35 in Ab fibrils oxidized. In the second topic, we discuss sensitivity enhancement in 13C SSNMR for Cu2+-Ab fibrils with paramagnetic assisted condensed data collection (PACC) method by taking advantage of short 1H T1 of this paramagnetic protein aggregates in an ultra high field (800 MHz) under very fast MAS at a spinning speed over 50 kHz.
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Code:
PR
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Time Slot/Poster Number:
384
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Session:
Solids: Bio-Applications, Poster
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Solid-State NMR Study of Lipid Storage Droplet Protein-1 in Lipoprotein Complex
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| Liying Wang; Estela L. Arrese; Penghui Lin; Lian Duan; Jose L. Soulages; Donghua H. Zhou
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Oklahoma State University, Stillwater, OK
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| Abstract |
Lipid storage droplet protein-1 (lsd-1) participates in the metabolism of neutral lipid triglycerides in fat cells. SSNMR is especially beneficial for lsd-1 study since samples can be prepared in native-like lipid environments and provides ways to study the structure and dynamics of lsd-1 in its membrane-bound functional state. Both 13C,15N- and 2H,13C,15N-enriched lsd-1 samples have been successfully expressed at tens of milligrams per liter of growth. Lsd-1/DMPG lipoprotein complexes were obtained after purification from inclusion bodies followed by reconstitution in DMPG liposome. Several 2D and 3D correlation spectra have been acquired and results show that lsd-1/DMPG gives good resolution (~0.5 ppm linewidth), and that SSNMR is the promising tool to elucidate the structure for lsd-1/DMPG.
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Code:
PR
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Time Slot/Poster Number:
385
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Session:
Solids: Bio-Applications, Poster
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Solid-state NMR Reveals the Insertion of Poly(amidoamine) Dendrimers into Biomembranes
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| Pieter E. Smith; Mark M. Banaszak Holl; Ayyalusamy Ramamoorthy; Jeffrey R. Brender
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University of Michigan, Ann Arbor, MI
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| Abstract |
PAMAM dendrimers are a versatile nanobiotechnology that has been utilized to mediate gene transfer in rodent cardiac grafts and to achieve highly specific targeting of chemotherapeutics to cancer cells, drastically improving the outcome of chemotherapeutic treatments. The interactions between dendrimers and lipids are of particular relevance to the exciting pharmaceutical applications of dendrimers. In this study, we used solid-state NMR to gain an understanding of the structural and dynamical properties of the lipid bilayer-generation 5 PAMAM dendrimer complex. Our results support a model of dendrimers’ partitioning into zwitterionic phosphocholine membranes. The knowledge gained about the interactions between dendrimers and phosphocholine membranes is expected to be useful in the design of dendrimer-based nanobiotechnologies.
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Code:
PR
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Time Slot/Poster Number:
386
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Session:
Solids: Bio-Applications, Poster
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High Resolution Backbone Structure of M2 Proton Channel from Influenza A Virus by Solid State NMR Spectroscopy
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| Mukesh Sharma1, 2; Myunggi Yi2; Emily Peterson3; Azlyn Velez2; Huajun Qin2; David Busath3; Huan-Xiang Zhou2; Timothy Cross1, 2
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1National High Magnetic Field Laboratory, Tallahassee, FL; 2Florida State University, Tallahassee, Florida; 3Brigham Young University, Provo, Utah
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| Abstract |
M2 proton channel that is expressed on the surface of the cell infected by influenza A virus. This tetrameric protein conducts protons across the viral membrane providing pH regulation of viral interior during the infection and replication process. We report three-dimensional structure of this tetrameric channel in lipid bilayer determined by solid-state NMR spectroscopy and refined by Molecular Dynamics Simulations in explicit lipid bilayer using experimental data.
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Code:
PR
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Time Slot/Poster Number:
387
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Session:
Solids: Bio-Applications, Poster
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Solid State 13C VT MAS NMR Study of Biomass Pretreatment with Sulfuric Acid
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| Benjamin Kohn; Gary Maciel
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Colorado State University, Fort Collins, CO
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| Abstract |
Solid State 13C MAS NMR was used to study a biomass pretreatment process in situ. In this process, poplar wood shavings are treated with 0.5 M sulfuric acid at 120 – 150 oC. Evidence to date indicates that the pretreatment process is 80% complete in ten minutes at 140 – 160 oC. For meaningful relaxation measurements, in situ experiments with natural abundance 13C biomass require several hours for adequate signal averaging. Thus, it is necessary to enrich the 13C content of the wood. This was accomplished by growing poplar trees in an atmosphere of 13CO2. Torch-sealed glass ampoules were used to contain the sample during MAS experiments.
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