Side chain NMR assignments in the membrane protein OmpX reconstituted in DHPC micelles |
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Authors: | Hilty Christian Fernández César Wider Gerhard Wüthrich Kurt |
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Affiliation: | (1) Eidgenössische Technische Hochschule Zürich, Institut für Molekularbiologie und Biophysik, CH-8093 Zürich, Switzerland |
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Abstract: | Sequence-specific assignments have been obtained for side chain methyl resonances of Val, Leu and Ile in the outer membrane protein X (OmpX) from Escherichia colireconstituted in 60 kDa micelles in aqueous solution. Using previously established techniques, OmpX was uniformly 2H,13C,15N-labeled with selectively protonated Val- 1,2, Leu- 1,2and Ile- 1methyl groups. The thus labeled protein was studied with the novel experiments 3D (H)C(CC)-TOCSY-(CO)-[15N,1H]-TROSY and 3D H(C)(CC)-TOCSY-(CO)-[15N,1H]-TROSY. Compared to the corresponding conventional experimental schemes, the TROSY-type experiments yielded a sensitivity gain of about 2 at 500 MHz. The overall sensitivity of the experiments was further enhanced more than two-fold by the use of a cryoprobe. Complete assignments of the proton and carbon chemical shifts were obtained for all isopropyl methyl groups of Val and Leu, as well as for the 1-methyls of Ile. The present approach is applicable for soluble proteins or micelle-reconstituted membrane proteins in structures with overall molecular weights up to about 100 kDa, and adds to the potentialities of solution NMR for de novostructure determination as well as for functional studies, such as ligand screening with proteins in large structures. |
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Keywords: | cryoprobe isotope labeling membrane proteins side chain assignment TROSY-NMR |
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