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Nucleotideâbinding domain 1 of cystic fibrosis transmembrane conductance regulator: Production of a suitable protein for structural studies
- Duffieux, Francis, Annereau, JeanâPhilippe, Boucher, Julien, Miclet, Emeric, Pamlard, Olivier, Schneider, Michael, Stoven, VÃ©ronique, Lallemand, JeanâYves
- European journal of biochemistry 2000 v.267 no.17 pp. 5306-5312
- adenosinetriphosphatase, cystic fibrosis, fluorescence, genes, histidine, multiple drug resistance, mutation, nuclear magnetic resonance spectroscopy, patients, structure-activity relationships, transporters, tryptophan
- Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). This protein belongs to the large ATPâbinding cassette (ABC) family of transporters. Most patients with cystic fibrosis bear a mutation in the nucleotideâbinding 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 structureâfunction relationship, and relate it to the biological features of CFTR. In this paper, we report the first preparation of recombinant Hisâtagged 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 ÎF508, could differ from that of the nucleotideâbinding subunit of histidine permease, the only available ABC structure. We have undertaken three dimensional structure determination of NBD1, and the first two dimensional 15Nâ1H 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.