Résumé :
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Communication n° 87. Dystrophin is the protein genetically deficient in Duchenne Muscular Dystrophy (DMD). Its C-and N-terminal ends interact with several cytoskeletal and membrane proteins, thus establishing a link between the cytoskeleton and the extracellular matrix. However, the rod domain partners remain largely unknown. After the first work of DeWolf (1997) showing that membrane phospholipids biophysical properties are modified by the presence of the 2nd repeat of the rod domain, we undertaken to further describe precisely this interaction. The repeat was produced as a recombinant protein in E. Coli. After refolding and using tryptophan fluorescence emission and quenching techniques, we show that the repeat interacts strongly with phospholipids including the anionic phosphatidylserine and that hydrophobic and electrostatic interactions are involved (J Biol Chem, 2003, 278, 5993-6001). Further, ultrafiltration was used to determine the affinity constant of the 2nd repeat with the lipids ; the interaction is shown to be cooperative and the kD is about 130 microM in presence of salts while it decreased to 15 microM in absence of salts. This indicates that several residues are involved in the interaction and that electrostatic forces are involved in the first steps of the binding and that hydrophobic forces maintain the binding. Finally, we undertaken the study of the interaction of membrane phospholipids with whole the rod domain. For that purpose, several proteins comprising several rod domain repeats are produced, namely, ROD1 (repeat 1-3), ROD2 (repeat 4-9), ROD3 (Repeat 11-15), ROD4 (repeat 16-19) and ROD5 (repeat 20-24). Protein are produced as recombinant proteins on a pGEX vector with a GST tag in E. Coli. They are purified on affinity column and further cleaved by thrombin to remove the GST tag. Tryptophan fluorescence properties are used as direct reporters for studies of folding and unfolding of the proteins in comparison with circular dichroism techniques. Quenching of the tryptophan fluorescence with acrylamide and nitroxyl-labelled fatty acids is used to assess the interaction with phospholipids. We will be able to show the map of interaction of the rod domain with the phospholipids.
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