Résumé :
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While dystrophin's native function is still largely unknown, it is well established that lack of functional dystrophin in muscle cell causes Duchenne muscular dystrophy (DMD). The native dystrophin is organised in four domains: a N-terminal actin binding domain, a potentially flexible rod domain of 24 spectrin-like repeats, a cystein rich domain and a C terminal beta-dystroglycan binding domain. In this study, we compared biophysical properties of the wild-type repeat 23 (R23) of human dystrophin rod domain and a double mutant encountered in a patient suffering from DMD, R23 E2910V-N2912D in helix C. Thermal denaturation analysis by circular dichroism showed a single transition at 67°C for the wild type repeat while a two transition behaviour with temperature of mid-denaturation of 44.5°C and 63°C was observed for the mutant repeat. This indicated that at 37 degrees, wild type repeat remains folded while the mutant repeat is one third of intensity unfolded. Urea unfolding analysed by tryptophan fluorescence showed a similar behaviour with two transitions for the mutant compared to a single transition of the wt. Refolding kinetics of urea denaturated proteins analysed by stopped flow spectrofluorimetry showed that the mutation induces a dramatic slowing down of refolding of the repeat with exponential rate constants of 50 s-1 and 18 s-1 for wt and mutated repeat, respectively. Molecular modelling indicated that the mutations located in helix C of the repeat, modified helix-helix interaction within the coiled coil structure. Taken together, these results show that the double mutation causing DMD changes surface properties, unfolding and refolding behaviours of R23. These data showing for the first time that a mutation in dystrophin rod domain induces unstability of the protein represent a contribution to the functional dissection of the dystrophin protein and emphasises the role of the rod domain and its putative behaviour in muscle cell.
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