title: Characterization of the chimeric seven-transmembrane protein containing conserved region of helix C-F of microbial rhodopsin from Ganges River.
abstract: Proteorhodopsin (PR) is a light-driven proton pump that has been found in a variety of marine bacteria. Recently, many PR-like genes were found in non-marine environments. The goal of this study is to explore the function of rhodopsins that exist only as partial proteo-opsin genes using chimeras with marine green PR (GPR). We isolated nine partial genes of PR homologues using polymerase chain reaction (PCR) and chose three homologues of GPR from the surface of the Ganges River, which has earned them the name "CFR, Chimeric Freshwater Rhodopsin." In order to characterize the proteins, we constructed the cassette based on GPR sequence without helices C to F and inserted the isolated conserved partial sequences. When expressed in E. coli, we could observe light-driven proton pumping activity similar to proteorhodopsin, however, photocycle kinetics of CFRs are much slower than proteorhodopsin. Half-time decay of O intermediates of CFRs ranged between 143 and 333 ms at pH 10; their absorption maxima were between 515 and 522 nm at pH 7. We can guess that the function of native rhodopsin, a retinal protein of fresh water bacteria, may be a light-driven proton transport based on the results from chimeric freshwater rhodopsins. This approach will enable many labs that keep reporting partial PCR-based opsin sequences to finally characterize their proteins.
authors.affiliation: Department of Life Science and Institute of Biological Interfaces, Sogang University, Shinsu-Dong 1, Mapo-Gu, Seoul 121-742, South Korea.
authors.forename: Ah Reum
authors.initials: AR
authors.lastname: Choi
authors.forename: Se Jun
authors.initials: SJ
authors.lastname: Kim
authors.forename: Byung Hoon
authors.initials: BH
authors.lastname: Jung
authors.forename: Kwang-Hwan
authors.initials: KH
authors.lastname: Jung
mesh_terms: D005618:Fresh Water; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017494:Proton Pumps / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025604:Rhodopsins, Microbial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D017494:Proton Pumps; D011993:Recombinant Fusion Proteins; D025604:Rhodopsins, Microbial; C413548:proteorhodopsin; D012243:Rhodopsin