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Code:
WOA
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Time Slot/Poster Number:
08:30-08:55
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Session:
Liquids III Biomolecules
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NMR-based structural analysis of protein complexes in solution
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| Michael Sattler1, 2
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1Technische Universität München, Garching, Germany; 2Helmholtz Zentrum München, Neuherberg, Germany
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| Abstract |
Approaches for structural analysis of large protein complexes will be discussed.
First, an efficient protocol for determining the quaternary structure of multi-domain proteins and protein complexes in solution is presented. Specifically, we combine advanced solution state NMR methods with Small Angle X-ray and/or Neutron Scattering (SAXS/SANS) experiments. The use of solution techniques for these studies is important as many regulatory protein complexes involve weak and transient domain interactions with considerable dynamics.
Second, NMR structural analysis of the recognition of nuclear export cargo in a 150 kDa protein complex will be shown. By using state-of-the-art isotope-labeling and NMR methods (including PREs and 13C direct detection) structural analysis was feasible.
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Code:
WOA
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Time Slot/Poster Number:
08:55-09:10
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Session:
Liquids III Biomolecules
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Single-Scan 2D NMR Correlations by Multiple Coherence Transfers
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| Maayan Gal1; Lucio Frydman2
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1Harvard Medical School, Boston, MA; 2Weizmann Institute of Science, Rehovot, Israel
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| Abstract |
A new scheme for the acquisition of heteronuclear 2D NMR spectroscopy within a single scan is proposed. It is Similar to Mansfield’s “k-space walk” proposal, in the sense that it is based on repetitively transferring spin coherences back and forth between the two spin systems to be correlated. It is shown that if properly executed, these transfers enable the equivalent of a continuous sampling of the time-domain space supporting the heteronuclear 2D NMR spectrum. Details on how to execute the resulting “time-walk” experiments and examples comparing it against conventional and other single-scan 2D alternatives will be shown. Advantages and main drawbacks will be discussed.
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Code:
WOA
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Time Slot/Poster Number:
09:10-09:35
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Session:
Liquids III Biomolecules
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Bicelles and Peptide-Lipid Diskettes for Membrane Protein NMR
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| Mark Girvin
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Albert Einstein College of Medicine, Bronx, NY
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| Abstract |
Small MDRs are 8-TMH proteins of controversial structure, whose functional folding requires lipid bilayers. In initial bicelle studies, sample lifetimes were short, and only three Trosy-based experiments yielded complete data. Modifications led to dramatic improvements in sample stability, and the ability to acquire nearly complete triple resonance datasets. With these improvements, backbone resonance assignments were completed and 1H15N and 13Ca13CO RDCs were measured. Since these assemblies are still quite large, small helical peptide-lipid assemblies are being developed as an alternative to bicelles and larger nanodiscs. Samples in these peptide-lipid “nanodiskettes” show remarkable stability, and form considerably smaller particles with correlation times of ~35ns, where all standard Trosy-based experiments can be used.
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Code:
WOA
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Time Slot/Poster Number:
09:35-09:50
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Session:
Liquids III Biomolecules
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Cost-Effective and Comprehensive NMR Spectroscopy of Methyl Groups in Large Proteins
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| Renee Otten1; Byron Chu2; Karla D. Krewulak2; Hans J. Vogel2; Frans A.A. Mulder1
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1Groningen Biomolecular Sciences and Biotechnology, Groningen, Netherlands; 2Structural Biology, University of Calgary, Calgary, Canada
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| Abstract |
Bacterial expression using U-[1H,13C]-glucose and ∼100% D2O ensures the cost-effective enrichment of all methyl groups in a protein with 13C. In addition, this approach ensures a high level of deuteration along all methyl-bearing amino acid side chains, and high levels of the favorable CHD2 isotopomer for detection. Using a single, newly developed 3D C-TOCSY-CHD2 experiment, we were able to sequence-specifically assign 195 Ala, Val, Thr, Ile and Leu methyl groups (85%) in the 34 kDa protein FepB. The good overall sensitivity of the experiment is also maintained at 5°C, suggesting that comprehensive methyl group assignments are also possible for large molecular weight systems, including oligomeric proteins, supramolecular complexes and membrane proteins.
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