A Comparative Study Of The Functional Roles Of W182/W183 In Bacteriorhodopsin And Archaerhodopsin 4

Bacteriorhodopsin(bR),a seven-transmembrane photoreceptor protein with retinal as the chromophore found in purple membrane of Halobacterium salinarum R1M1,functions as a proton pump.Archaerhodopsin 4(aR4),a proton pump found in the claret membrane of Halobacterium species xz515 from a Tibetan Salt Lake,is a dual-chromophore photoreceptor with bacterioruberin as the second chromophore,and it displays the opposite temporal order of proton release and uptake to bR.The conserved residue tryptophan 182(W182)and tryptophan 183(W183)were located in the intracellular side of the retinal binding pocket of bR and aR4 are the research object of this thesis.Choose Halobacterium Salinarum L33 and Halobacterium Salinarum MPK409 as protein expression host strain.Site-directed mutagenesis of W182 F and W182 A,W183F and W183 A,in combination with in-situ UV-visible absorption spectroscopy,light-induced transient absorption change spectroscopy,proton pump measurements,pH titration,dynamic light scattering(DLS)measurements and ATP formation rate measurements,is employed to elaborate the functional role of W182 and W183 in the bR and aR4 photocycle.The results suggest that all mutations cause the perturbation of the retinal binding pocket,changes in pKa of key residues,reduces in generation of photo-intermediates,proton pump capability weaken,the alteration of protein assembly state,and the decrease of ATP formation rate.W182 F mutant has more significant effects than W182 A mutant.The influences of W183 mutant is greater than W182 mutant.W183 mutant has a greater impact on the photocycle progress with the presence of bacterioruberin.