Solution NMR studies of the integral membrane proteins OmpX and OmpA from Escherichia coli.
Membrane proteins are usually solubilized in polar solvents by incorporation into micelles. Even for small membrane proteins these mixed micelles have rather large molecular masses, typically beyond 50000 Da. The NMR technique TROSY (transverse relaxation-optimized spectroscopy) has been developed for studies of structures of this size in solution. In this paper, strategies for the use of TROSY-based NMR experiments with membrane proteins are discussed and illustrated with results obtained with the Escherichia coli integral membrane proteins OmpX and OmpA in mixed micelles with the detergent dihexanoylphosphatidylcholine (DHPC). For OmpX, complete sequence-specific NMR assignments have been obtained for the polypeptide backbone. The 13C chemical shifts and nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution, and in the collection of an input of conformational constraints for the computation of the global fold of the protein. For OmpA, the NMR assignments are so far limited to about 80% of the polypeptide chain, indicating different dynamic properties of the reconstituted OmpA beta-barrel from those of OmpX. Overall, the present data demonstrate that relaxation-optimized NMR techniques open novel avenues for studies of structure, function and dynamics of integral membrane proteins.
Fernández
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Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, CH-8093, Zürich, Switzerland. cesar@mol.biol.ethz.ch
Hilty
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Bonjour
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Adeishvili
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Pervushin
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Wüthrich
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D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D006867:Hydrolases; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C482905:OmpX protein, E coli; C078167:OMPA outer membrane proteins; D006867:Hydrolases