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Code:
PL
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Time Slot/Poster Number:
245
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Session:
NMR & Crystallography, Poster
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A Model of the Solution Structure of Arginine Kinase by Combining NMR and X-ray Crystallography
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| Xiaogang Niu1; Lei Bruschweiler-Li1; Michael Chapman2; Rafael Brüschweiler1
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1Chemistry & Biochemistry, Florida State University, Tallahassee, FL; 2OHSU, Portland, OR
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| Abstract |
Arginine kinase (AK) is a 42 kDa protein that belongs to the phosphagen kinase family. Due to its large size, determination of the 3D structure of AK in solution by standard NMR methods is hard. Here we use the NMR residual dipolar couplings (RDCs) measured in two alignment media, the NMR chemical shifts and available crystal structures to determine the solution structure of apo-AK. The result shows that in solution this protein adopts a unique structure that corresponds to a superposition of the high-resolution X-ray crystal structures under different binding conditions. The recently refined crystal structure of the apo-state AK is largely consistent with the NMR model reflect the good accuracy of this approach.
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Code:
PL
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Time Slot/Poster Number:
246
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Session:
NMR & Crystallography, Poster
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Solution Structure for Human Cyclin Dependent Kinase-2 Associated Protein, p12DOC-1
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| asli ertekin1; Yuanpeng J. Huang1; Hsiau-Wei Lee2; James H Prestegard2; James Aramini1; Gaetano T Montelione1
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1Rutgers, The State University of New Jersey, Piscataway, NJ; 2Complex Carbohydrate Research Center, University o, Athens, GA
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| Abstract |
CDK2 associated protein-1 (CDK2-AP1) is a well known tumor suppressor protein, corresponding to gene doc-1 (deleted in oral cancer). It was first observed that the gene product was absent or reduced in the hamster oral cancer cells. This gene is also missing or down-regulated in many other cancer cell types. This ubiquitously expressed protein is the only known specific inhibitor for cyclin dependent kinase-2 (CDK2), making it an important part of cell cycle regulation at G1-to-S phase transformation. Here, the solution NMR structure of CDK2-AP1, a target of the cancer biology biomedical theme of the Northeast Structural Genomics Consortium, is presented for the first time.
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Code:
PL
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Time Slot/Poster Number:
247
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Session:
NMR & Crystallography, Poster
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Unusual Splitting of 31P Peaks in the CPMAS of Single Crystals of Ferroelectric RbH2PO4 at 900 MHz
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| Sneha Dugar1; Riqiang Fu2; Peter Gorkov2; William W. Brey2; Jason Kitchen2; Naresh S. Dalal1, 2
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1Florida State University, Tallahassee, FL; 2National High Magnetic Field Laboratory, Tallahassee, Florida
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| Abstract |
Hydrogen bonded compounds such as KH2PO4 (KDP) and RbH2PO4 (RDP) have been considered as model compounds where simple lattices exhibit the order/disorder phenomena. We have recently probed the RDP system by 31P variable temperature MAS measurements on a RDP crystal on the ultra-wide bore 900 MHz NMR magnet. It is found that the 31P isotropic resonance shows a clear splitting below the phase transition temperature at 148K. At 115 K, the two peaks become distinct with the intensity ratio of 1.2:1. But, for the powdered sample, the spectra showed just broadening with an apparent shoulder at this temperature. A Gaussian fit indicates the presence of two peaks. This is the first experimental observation of the ferroelectric domain effect.
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Code:
PL
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Time Slot/Poster Number:
248
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Session:
NMR & Crystallography, Poster
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CP/MAS with Crystals at 900 MHz: First Detection of Pretransitional Clusters Above a Paraelectric –Antiferroelectric Phase Transition
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| Riqiang Fu1; Ozge Gunaydin-Sen2; Naresh S. Dalal2
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1National High Magnetic Field Laboratory, Tallahassee, FL; 2Dept of Chem & Biochem Florida State University, Tallahassee, FL
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| Abstract |
Variable temperature 15N CP/MAS measurements at 21.1 T magnet using single crystals enable us to detect the long-sought-after clusters in the paraelectric phase of the model hydrogen-bonded compound NH4H2AsO4 at temperatures around its order-disorder antiferroelectric phase transition.
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Code:
PL
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Time Slot/Poster Number:
249
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Session:
NMR & Crystallography, Poster
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Characterization of a membrane anchored HIV MPER peptide with multi-dimensional NMR
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| Patrick N Reardon; Barton Haynes; Leonard Spicer
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Duke University Medical Center, Durham, NC
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| Abstract |
We have designed and expressed peptides containing the membrane proximal ectodomain region of HIV-1 with and without trimerization domains and membrane anchoring segments. Biophysical properties of these constructs are being studied by multi-dimensional NMR and equilibrium analytical ultracentrifugation. Of particular interest is the peptide anchored to detergent micelles using a transmembrane helix which is designed to mimic the corresponding viral gp41. Our studies of this peptide have been conducted in several detergent micelles and have found that the detergent LMPG yielded good spectra. In this detergent, we have successfully acquired 3D NMR datasets, including HNCA and HNCO. In addition, we have initiated studies of the construct in a trimeric form.
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Code:
PL
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Time Slot/Poster Number:
250
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Session:
NMR & Crystallography, Poster
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NMR Solution Structure and DNA Binding Model of the DNA Binding Domain of Competence Protein A
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| Carey Hobbs; Ben Bobay; Richele Thompson; John Cavanagh
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North Carolina State University, Raleigh , NC
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| Abstract |
Competence protein A (ComA) is a response regulator protein involved in the development of genetic competence in the Gram-positive, spore forming bacterium Bacillus subtilis, as well as the regulation of the production of degradative enzymes and antibiotic synthesis. The determination of the NMR solution structure of ComAC and the ComAC-DNA model allowed for comparison of the solved structures within the NarL family. Overlay of NarLC and DosRC in their DNA bound state with ComAC
revealed the greatest structural differences lie in α-helices 8 and 9, most likely due to the specific nature of these proteins binding their DNA targets in a slightly different manner. These observations suggest a basis for DNA binding specificity within the NarL family.
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Code:
PL
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Time Slot/Poster Number:
251
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Session:
NMR & Crystallography, Poster
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A Markov Random Field Algorithm for Protein Side-Chain Resonance Assignment
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| Jianyang Zeng1; Pei Zhou2; Bruce Donald3
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1Department of Computer Science, Duke University, Durham, NC; 2Department of Biochemistry, Duke Medical Center, Durham, NC; 3Depts of Comp. Sc. & Biochem., Duke Univ. Med. Ctr, Durham, NC
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| Abstract |
Traditional approaches for side-chain resonance assignment require TOCSY experiments, which usually perform poorly on large proteins due to the fast transverse relaxation of protonated carbons. To overcome this deficiency, we present a novel side-chain resonance assignment algorithm based on NOESY experiments, which does not require TOCSY experiments, and can save a significant amount of experimental cost and spectrometer time. We cast the side-chain assignment problem into a Markov Random Field (MRF) framework, and extend and apply combinatorial protein design algorithms, including dead-end elimination and A* search, to compute the optimal assignments that best interpret the NMR data. Tests on five proteins show that our algorithm can be successfully applied to automate side-chain resonance assignment and high-quality protein structure determination.
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Code:
PL
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Time Slot/Poster Number:
252
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Session:
NMR & Crystallography, Poster
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RDC-PANDA: a High-Resolution NMR Structure Determination Algorithm Starting with a Global Fold Calculated from Exact Solutions to the RDC Equations
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| Jianyang Zeng1; Jeffrey Boyles2; Chittaranjan Tripathy1; Lincong Wang1; Anthony Yan2; Pei Zhou2; Bruce Donald3
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1Department of Computer Science, Duke University, Durham, NC; 2Department of Biochemistry, Duke Medical Center, Durham, NC; 3Depts of Comp. Sc. & Biochem., Duke Univ. Med. Ctr, Durham, NC
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| Abstract |
We present a novel structure determination algorithm, called RDC-PANDA (RDC-based SSE PAcking with NOEs for Structure Determination and NOE Assignment), that exploits the global orientational restraints from RDCs to resolve ambiguous NOE assignments. Unlike traditional approaches that bootstrap the initial fold from ambiguous NOE assignments, we start by using RDCs to compute accurate secondary structure element (SSE) backbones at the beginning of structure calculation. RDC-PANDA was applied to NMR data for four proteins, and the results demonstrate the efficiency and accuracy of our algorithm, and show that RDC-PANDA can be successfully applied for high-resolution protein structure determination using only a limited set of NMR data by first computing RDC-defined backbones.
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Code:
PL
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Time Slot/Poster Number:
253
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Session:
NMR & Crystallography, Poster
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Solution Structure and DNA Binding of the Homeodomain of the Human Stem Cell Transcription Factor Nanog
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| Sarata Sahu; David Aceti; Justin Brumbaugh; James Thomson; John Markley
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University of Wisconsin, Madison, WI
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| Abstract |
The transcription factor Nanog is a key regulator in early mammalian development and a key determinant of pluripotency in embryonic stem cells. Nanog binds to promoter elements of hundreds of target genes and regulates their expression. The mechanism of this transcriptional regulation is not yet known. The only structure for this protein in the PDB is an X-ray crystal structure of the homeodomain of murine Nanog (mNanog), which was shown to be an octamer. We have determined the solution structure of the homeodomain of human Nanog (hNanog), which we show to be a monomer. We will compare the solution structure of human hNanog with the X-ray structure of mNanog and will present our studies hNanog interacting with DNA.
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Code:
PL
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Time Slot/Poster Number:
254
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Session:
NMR & Crystallography, Poster
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Structural refinement of N-terminal modules of factor H
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| Mateusz Maciejewski
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National Institutes of Health, NHLBI, LMB, Bethesda, MD
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| Abstract |
Factor H is a 155 kD molecule constructed of 20 CCP domains, that acts in the complement system of proteins as a cofactor in factor I mediated cleavage of C3b, which if left unregulated can opsonize self-cells for phagocytosis.
Residual dipolar coupling measurements were made on a construct of factor H protein consisting of three N-terminal CCPs, which had been shown to be the minimal functional unit to confer decay accelerating activity towards C3b. Those measurements were used to refine the relative orientation of domains, which is hard by other means due to scarcity of inter-modular contacts.
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Code:
PL
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Time Slot/Poster Number:
255
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Session:
NMR & Crystallography, Poster
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NMR structural investigations of pharmaceutical (co-)compounds
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| Mujeeb Khan; Gunther Brunklaus
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MPI for Polymer Research, Mainz, Germany
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| Abstract |
Enhancement of a drugs´ bioavailability is an ongoing challenge in pharmaceutical development. In the present work, we apply solid-state NMR to study hydrogen-bonding and structural features of powdered “co-crystallized” pharmaceutical compounds, selecting the abundantly used tablet excipient methyl paraben as one co-crystal former and considering poorly water-soluble APIs such as quinidine, an anti-malarial constituent of Cinchona tree bark. Notably, a means of almost quantitative separation of quinidine from a mixture with its stereoisomer quinine based on hydrogen-bond mediated molecular recognition is identified based on solid-state NMR data. In addition, NMR-structural features of other quinidine-based (co-)compounds, quinine and quinine hydrochloride are presented, thus highlighting the great potential of NMR-based “crystallography” of powdered samples.
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