Abstract Optimization of aqueous solutions of the integral membrane protein (IMP) OmpW for NMR structure determination has been monitored with micro-coil NMR, which enables the acquisition of NMR spectra using only micrograms of protein and detergent. The detergent 30-Fos (2-undecylphosphocholine) was found to yield the best 2D [15N, 1H]-TROSY correlation NMR spectra of [2H, 15N]-labeled OmpW. For the OmpW structure determination we then optimized the 30-Fos concentration, the sample temperature and long-time stability, and the deuteration level of the protein. Some emerging guidelines for reconstitution of β-barrel integral membrane proteins in structural biology are discussed.
Content Type Journal Article
Category Communication
Pages 1-5
DOI 10.1007/s10858-012-9658-x
Authors
Pawel Stanczak, Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Qinghai Zhang, Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Reto Horst, Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Pedro Serrano, Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Kurt Wüthrich, Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
The application of micro-coil NMR probe technology to metabolomics of urine and serum
The application of micro-coil NMR probe technology to metabolomics of urine and serum
Abstract Increasing the sensitivity and throughput of NMR-based metabolomics is critical for the continued growth of this field. In this paper the application of micro-coil NMR probe technology was evaluated for this purpose. The most commonly used biofluids in metabolomics are urine and serum. In this study we examine different sample limited conditions and compare the detection sensitivity of the micro-coil with a standard 5 mm NMR probe. Sample concentration is evaluated as a means to leverage the...
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03-09-2011 04:19 AM
Solid-State NMR on a Large Multidomain Integral Membrane Protein: The Outer Membrane Protein Assembly Factor BamA.
Solid-State NMR on a Large Multidomain Integral Membrane Protein: The Outer Membrane Protein Assembly Factor BamA.
Solid-State NMR on a Large Multidomain Integral Membrane Protein: The Outer Membrane Protein Assembly Factor BamA.
J Am Chem Soc. 2011 Mar 1;
Authors: Renault M, Bos MP, Tommassen J, Baldus M
Multidomain proteins constitute a large part of prokaryotic and eukaryotic proteomes and play fundamental roles in various physiological processes. However, their structural characterization is challenging because of their large size and...
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03-03-2011 12:34 PM
Solid-State NMR on a Large Multidomain Integral Membrane Protein: The Outer Membrane Protein Assembly Factor BamA
Solid-State NMR on a Large Multidomain Integral Membrane Protein: The Outer Membrane Protein Assembly Factor BamA
Marie Renault, Martine P. Bos, Jan Tommassen and Marc Baldus
http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/0/jacsat.ahead-of-print/ja109469c/aop/images/medium/ja-2010-09469c_0004.gif
Journal of the American Chemical Society
DOI: 10.1021/ja109469c
http://feeds.feedburner.com/~ff/acs/jacsat?d=yIl2AUoC8zA
http://feeds.feedburner.com/~r/acs/jacsat/~4/9XN1qiW-S-I
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03-02-2011 02:01 AM
[NMR paper] How to prepare membrane proteins for solid-state NMR: A case study on the alpha-helical integral membrane protein diacylglycerol kinase from E. coli.
How to prepare membrane proteins for solid-state NMR: A case study on the alpha-helical integral membrane protein diacylglycerol kinase from E. coli.
Related Articles How to prepare membrane proteins for solid-state NMR: A case study on the alpha-helical integral membrane protein diacylglycerol kinase from E. coli.
Chembiochem. 2005 Sep;6(9):1693-700
Authors: Lorch M, Faham S, Kaiser C, Weber I, Mason AJ, Bowie JU, Glaubitz C
Several studies have demonstrated that it is viable to use microcrystalline preparations of water-soluble proteins as...
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12-01-2010 06:56 PM
[NMR paper] Bead-linked proteoliposomes: a reconstitution method for nmr analyses of membrane protein-ligand interactions.
Bead-linked proteoliposomes: a reconstitution method for nmr analyses of membrane protein-ligand interactions.
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J Am Chem Soc. 2005 Aug 31;127(34):12021-7
Authors: Yokogawa M, Takeuchi K, Shimada I
Structural information about the interactions between membrane proteins and their ligands provides insights into the membrane protein functions. A variety of surfactants have been used for structural analyses of membrane...
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12-01-2010 06:56 PM
[NMR paper] NMR study of a membrane protein in detergent-free aqueous solution.
NMR study of a membrane protein in detergent-free aqueous solution.
Related Articles NMR study of a membrane protein in detergent-free aqueous solution.
Proc Natl Acad Sci U S A. 2005 Jun 21;102(25):8893-8
Authors: Zoonens M, Catoire LJ, Giusti F, Popot JL
One of the major obstacles to membrane protein (MP) structural studies is the destabilizing effect of detergents. Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep MPs water-soluble under mild conditions. In the present work, we have explored the...
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11-25-2010 08:21 PM
[NMR paper] Membrane protein-lipid interactions in mixed micelles studied by NMR spectroscopy wit
Membrane protein-lipid interactions in mixed micelles studied by NMR spectroscopy with the use of paramagnetic reagents.
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Chembiochem. 2004 Apr 2;5(4):467-73
Authors: Hilty C, Wider G, Fernández C, Wüthrich K
For solution NMR studies of the structure and function of membrane proteins, these macromolecules have to be reconstituted and solubilized in detergent micelles. Detailed characterization of the mixed...
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11-24-2010 09:51 PM
[NMR paper] Lipid-protein interactions in DHPC micelles containing the integral membrane protein
Lipid-protein interactions in DHPC micelles containing the integral membrane protein OmpX investigated by NMR spectroscopy.
Related Articles Lipid-protein interactions in DHPC micelles containing the integral membrane protein OmpX investigated by NMR spectroscopy.
Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13533-7
Authors: Fernández C, Hilty C, Wider G, Wüthrich K
Intermolecular nuclear Overhauser effects (NOEs) between the integral outer membrane protein OmpX from Escherichia coli and dihexanoylphosphatidylcholine (DHPC) provided a detailed...
Micro-coil NMR to monitor optimization of the reconstitution conditions for the integral membrane protein OmpW in detergent micelles
Linear Mode
