Engineering a Carotenoid-Binding Site in Dokdonia Sp. PRO95 Na +-translocating Rhodopsin by a Single Amino Acid Substitution

Photosynth Res. 2018 May;136(2):161-169. doi: 10.1007/s11120-017-0453-0. Epub 2017 Oct 5.

Abstract

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.

Keywords: Canthaxanthin; Carotenoid antenna; Echinenone; Na+ pump; Rhodopsin; Xanthorhodopsin.

MeSH terms

  • Amino Acid Substitution
  • Binding Sites
  • Canthaxanthin / metabolism
  • Carotenoids / metabolism*
  • Circular Dichroism
  • Evolution, Molecular
  • Flavobacteriaceae / metabolism*
  • Glycine
  • Molecular Dynamics Simulation
  • Mutagenesis, Site-Directed
  • Protein Conformation
  • Protein Engineering
  • Rhodopsins, Microbial / chemistry
  • Rhodopsins, Microbial / genetics*
  • Rhodopsins, Microbial / metabolism*
  • Sodium / metabolism
  • Spectrometry, Fluorescence

Substances

  • Rhodopsins, Microbial
  • Carotenoids
  • Canthaxanthin
  • Sodium
  • echinenone
  • Glycine