Bacteriorhodopsin, the single protein of the purple membrane in extremely halophilic bacteria, transduces light energy to chemical energy by pumping protons from inside to outside of the cell. The overall aim of this research is to study the structure, mechanism of action and biosynthesis of bacteriorhodopsin by using chemical, physicochemical, biochemical and genetic approaches. The primary structure and orientation of the protein in the purple membrane is now known. Attention will be focussed on the tertiary structure of the protein. There are presumed to be seven helices which traverse the membrane. Folding, interactions between individual helices, and polypeptidic environments around the retinal in different conformational states (e.g., light-adapted, dark-adapted, and the bleached form) of bacteriorhodopsin will be investigated. For studies of structure-function relationships, a major approach will involve the use of recombinant DNA carrying the gene for bacteriorhodopsin. This gene will be cloned and its expression will be studied in E. coli. If not expressed, synthetic E. coli promoters will be linked to the gene. Alternatively, coupled transcription and translation using a variety of systems will be investigated for production of bacteriorhodopsin in vitro. Site-directed mutagenesis of the cloned DNA will be used by using in vitro reconstitution of the proton pump which now has been worked out in this laboratory. In studies of biosynthesis of bacteriorhodopsin, the nature of the messenger RNA, the synthesis and insertion of the protein into membrane and different mechanisms of its regulation (by retinal, light and oxygen) will be studied.
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