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

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 ?F508, 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.
Type de document :
Article dans une revue
European Journal of Biochemistry, Wiley, 2000, 267 (17), pp.5306-5312. 〈10.1046/j.1432-1327.2000.01614.x〉
Liste complète des métadonnées

https://hal-polytechnique.archives-ouvertes.fr/hal-00954905
Contributeur : Denis Roura <>
Soumis le : mardi 11 mars 2014 - 13:18:35
Dernière modification le : jeudi 10 mai 2018 - 02:07:30

Lien texte intégral

Identifiants

Collections

Citation

Francis Duffieux, Jean-Philippe Annereau, Julien Boucher, Emeric Miclet, Olivier Pamlard, et al.. Nucleotide-binding domain 1 of cystic fibrosis transmembrane conductance regulator: Production of a suitable protein for structural studies. European Journal of Biochemistry, Wiley, 2000, 267 (17), pp.5306-5312. 〈10.1046/j.1432-1327.2000.01614.x〉. 〈hal-00954905〉

Partager

Métriques

Consultations de la notice

174