Micro-coil NMR to monitor optimization of the reconstitution conditions for the integral membrane protein OmpW in detergent micelles
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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.
KeywordsStructural biology Integral membrane proteins Solubilization in detergent micelles E. coli outer membrane protein W
This work was supported by the Joint Center for Innovative Membrane Protein Technologies (JCIMPT-complexes, NIH Roadmap Grant P50 GM073197). K.W. is the Cecil H. and Ida M. Green Professor of Structural Biology at The Scripps Research Institute.
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