Engineering a carotenoid-binding site in Dokdonia sp. PRO95 Na+-translocating rhodopsin by a single amino acid substitution
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Light-driven H+, Cl− and Na+ rhodopsin pumps all use a covalently bound retinal molecule to capture light energy. Some H+-pumping rhodopsins (xanthorhodopsins; XRs) additionally contain a carotenoid antenna for light absorption. Comparison of the available primary and tertiary structures of rhodopsins pinpointed a single Thr residue (Thr216) that presumably prevents carotenoid binding to Na+-pumping rhodopsins (NaRs). We replaced this residue in Dokdonia sp. PRO95 NaR with Gly, which is found in the corresponding position in XRs, and produced a variant rhodopsin in a ketocarotenoid-synthesising Escherichia coli strain. Unlike wild-type NaR, the isolated variant protein contained the tightly bound carotenoids canthaxanthin and echinenone. These carotenoids were visible in the absorption, circular dichroism and fluorescence excitation spectra of the Thr216Gly-substituted NaR, which indicates their function as a light-harvesting antenna. The amino acid substitution and the bound carotenoids did not affect the NaR photocycle. Our findings suggest that the antenna function was recently lost during NaR evolution but can be easily restored by site-directed mutagenesis.
KeywordsRhodopsin Na+ pump Carotenoid antenna Canthaxanthin Echinenone Xanthorhodopsin
Na+-translocating rhodopsin of Krokinobacter eikastus
Na+-translocating rhodopsin of Dokdonia sp. PRO95
Root mean square deviation
Thr216Gly variant of NaR
This work was supported by the Russian Science Foundation Research Project 14-14-00128. We are indebted to Prof. T. Friedrich for providing us the pACCAR25ΔcrtXZcrtO plasmid and for helpful discussions.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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