Influence exercised by histidine-95 on chloride transport and the photocycle in halorhodopsin.
The anion pumping mechanism of halorhodopsin was studied using site-directed mutagenesis. Comparison of the amino acid sequence revealed that the B-C interhelix loop segment was highly homologous in all known halorhodopsins. Especially a basic residue, histidine-95, was conserved in all halorhodopsins. Using the expression-vector plasmid carrying the bop promoter, two His-95 mutants (H95R, H95A) were successfully expressed in Halobacterium salinarium. The expression levels of these halorhodopsin mutants were slightly lower than that for the wild-type halorhodopsin. In addition, these mutants were unstable under illumination compared with the wild-type. It suggested that His-95 is probably important for stabilizing the structure of halorhodopsin. The absorption maxima of these mutants are approximately 15 nm blue-shifted compared with the wild-type, suggesting that His-95 interacts with the retinal Schiff base. At low chloride concentrations, the light-induced chloride pumping activity of these mutants was more than 20 times lower than that for the wild-type. Only under physiological conditions, the chloride pumping activity was detected. Even at a high chloride concentration (1 M NaCl), the HR520 intermediate could not be detected for these mutants. These results clearly indicate that His-95 has a crucial role in the chloride transport of halorhodopsin.
Otomo
J
J
PRESTO, JRDC, Advanced Research Laboratory, Hitachi, Ltd., Saitama, Japan. jotomo@harl.hitachi.co.jp
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D004926:Escherichia coli; D006217:Halobacterium / Q000378:metabolism*; D025602:Halorhodopsins; D006639:Histidine; D007700:Kinetics; D008027:Light; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
D002712:Chlorides; D025602:Halorhodopsins; D011994:Recombinant Proteins; D006639:Histidine; D001436:Bacteriorhodopsins