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Side chain NMR assignments in the membrane protein OmpX reconstituted in DHPC micelles

Hilty, Christian ; Fernández, César ; Wider, Gerhard ; Wüthrich, Kurt

In: Journal of Biomolecular NMR, 2002, vol. 23, no. 4, p. 289-301

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    Titre
    • Side chain NMR assignments in the membrane protein OmpX reconstituted in DHPC micelles
    Auteur
    • Hilty, Christian. Eidgenössische Technische Hochschule Zürich, Institut für Molekularbiologie und Biophysik, CH-8093, Zürich, Switzerland
    • Fernández, César. Eidgenössische Technische Hochschule Zürich, Institut für Molekularbiologie und Biophysik, CH-8093, Zürich, Switzerland
    • Wider, Gerhard. Eidgenössische Technische Hochschule Zürich, Institut für Molekularbiologie und Biophysik, CH-8093, Zürich, Switzerland
    • Wüthrich, Kurt. Eidgenössische Technische Hochschule Zürich, Institut für Molekularbiologie und Biophysik, CH-8093, Zürich, Switzerland
    Type de document
    • Postprint
    Langue
    • Anglais
    Publié dans
    • Journal of Biomolecular NMR, 2002, vol. 23, no. 4, p. 289-301. Kluwer Academic Publishers
    Autre version électronique
    Identifiant OAI-PMH
    • oai:doc.rero.ch:309729
    Summary
    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 60kDa micelles in aqueous solution. Using previously established techniques, OmpX was uniformly 2H,13C,15N-labeled with selectively protonated Val-γ1,2, Leu-δ1,2and Ile-δ1methyl 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 500MHz. 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 δ1-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 100kDa, 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