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Nucleotide‐binding domain 1 of cystic fibrosis transmembrane conductance regulator: Production of a suitable protein for structural studies

Author:
Duffieux, Francis, Annereau, Jean‐Philippe, Boucher, Julien, Miclet, Emeric, Pamlard, Olivier, Schneider, Michael, Stoven, Véronique, Lallemand, Jean‐Yves
Source:
European journal of biochemistry 2000 v.267 no.17 pp. 5306-5312
ISSN:
0014-2956
Subject:
adenosinetriphosphatase, cystic fibrosis, fluorescence, genes, histidine, multiple drug resistance, mutation, nuclear magnetic resonance spectroscopy, patients, structure-activity relationships, transporters, tryptophan
Abstract:
Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). This protein belongs to the large ATP‐binding cassette (ABC) family of transporters. Most patients with cystic fibrosis bear a mutation in the nucleotide‐binding domain 1 (NBD1) of CFTR, which plays a key role in the activation of the channel function of CFTR. Determination of the three dimensional structure of NBD1 is essential to better understand its structure–function relationship, and relate it to the biological features of CFTR. In this paper, we report the first preparation of recombinant His‐tagged NBD1, as a soluble, stable and isolated domain. The method avoids the use of renaturing processes or fusion constructs. ATPase activity assays show that the recombinant domain is functional. Using tryptophan intrinsic fluorescence, we point out that the local conformation, in the region of the most frequent mutation ΔF508, could differ from that of the nucleotide‐binding subunit of histidine permease, the only available ABC structure. We have undertaken three dimensional structure determination of NBD1, and the first two dimensional 15N‐1H NMR spectra demonstrate that the domain is folded. The method should be applicable to the structural studies of NBD2 or of other NBDs from different ABC proteins of major biological interest, such as multidrug resistance protein 1 or multidrug resistance associated protein 1.
Agid:
103615