Dynamic Factors of BacteriorhodopsinRegulation Key Points in Helices B, C and F

Résumé

The main objective of this work is the analysis of the structural and functional role of some amino acids in Bacteriorhodopsin. - Characterization of the motif Thr90-Pro91 in the centre of Helix C of Bacteriorhodopsin. Because Thr90 interacts with Asp115 through a hydrogen bond and with the retinal through hydrophobic interaction, the mutants T90V and D115A were taken into account. The following objectives were settled: Design and construct the Bacteriorhodopsin mutants P91A, T90A,T90V and D115A by site directed mutagenesis. Express the mutants in the purple membrane of Halobacterium salinarum. Analyze the role of Thr90 and Pro91 in the properties of the protein and in the proton pumping function in particular through the study of the mutants. Infer a role to the motif Thr90-Pro91 in Bacteriorhodopsin. - Analyze the role of the other two Prolines located in transmembrane helices in BR, and to extend this role to other Transmembrane Proteins. The subsequent objectives were proposed: Design and construct the Bacteriorhodopsin mutants P50A and P186A by site directed mutagenesis. Express the mutants in the purple membrane of Halobacterium salinarum. Characterize the role of Pro50, and Pro186 in Bacteriorhodopsin properties and proton pumping through the analysis of the mutants. Infer a role to Helix-embedded prolines of Bacteriorhodopsin in particular and to transmembrane helices of proteins in general.