Titre : | Expression of different ZZ regions of dystrophin in E.coli or eukaryotic cells for structural analyses (abstract : congrès international de Myologie, 2005) |
contenu dans : | |
Auteurs : | Congrès international de myologie 2005 (International Congress of Myology 2005; 9-13 mai 2005; Nantes, France) ; Thuries L ; Winder SJ ; Savarin P ; Le Saint N ; Clocheau S ; Chea V ; Toma F ; Curmi PA |
Type de document : | Article |
Année de publication : | 2005 |
Pages : | p. 92 |
Langues: | Anglais |
Mots-clés : | actine ; colloque ; dystrophie musculaire de Duchenne ; dystrophine ; Escherichia coli ; mutation génétique ; spectroscopie RMN |
Résumé : |
Communication n° 472 Introduction : Mutations in the dystrophin gene, which result either in the absence or alterations of dystrophin, may cause Duchenne muscular dystrophy. An understanding of the molecular pathogenesis of this disease depends partly on detailed knowledge of the precise structure of dystrophin. High resolution structural data are nowadays available for different regions of dystrophin, the N-terminus actin binding domain, and the C-terminal WW-EF domains. Some single amino-acids substitutions without deletion of the protein have been reported in the ZZ region of the cystein-rich domain (amino acids residues 3080 to 3360) that yield to Duchenne muscular dystrophy phenotype. Aims : To elucidate the NMR solution structure of the wild type cysteine-rich domain and the effects of the mutations responsible for the Duchenne muscular dystrophy phenotype [D3335-H, C3340-Y or R3345-T]. To analyse the influence of these mutations on the interaction with beta-dystroglycan. Methods : Attempts to produce in E.coli different constructs of the cysteine-rich domain have hitherto yielded insoluble aggregates improper to NMR spectroscopy. We have designed small constructs centred on the ZZ region to restrict the expression products to the region of interest and try to increase solubility. In parallel, we have constructed eukaryotic secretion vectors to evaluate another folding pathway. Results: In E.coli, expression of the small constructs in presence of zinc yields partly soluble products whose folding is under investigations. Expression of soluble products in the eukaryotic system will be quantified. Conclusions : We have designed expression vectors to produce in E.coli or eukaryotic cells soluble forms of the ZZ region of dystrophin, a region affected by single amino-acid substitution responsible for Duchenne muscular dystrophy. We expect that these expressed domains will be proper for structure determination by NMR spectroscopy. |