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.
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Acknowledgments
This work was supported by the Research Foundation of Korea grants (331-2008-1-C00242 and 2011–0012320) and the second stage of Brain Korea 21 graduate fellowship program for AR Choi, SJ Kim, and BH Jung. We thank Leonid Brown to critical comments on this research.
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Choi, A.R., Kim, S.J., Jung, B.H. et al. Characterization of the chimeric seven-transmembrane protein containing conserved region of helix C–F of microbial rhodopsin from Ganges River. Appl Microbiol Biotechnol 97, 819–828 (2013). https://doi.org/10.1007/s00253-012-4452-y
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DOI: https://doi.org/10.1007/s00253-012-4452-y