Thermal and Spectroscopic Characterization of a Proton Pumping Rhodopsin From an Extreme Thermophile

J Biol Chem. 2013 Jul 26;288(30):21581-92. doi: 10.1074/jbc.M113.479394. Epub 2013 Jun 5.

Abstract

So far retinylidene proteins (∼rhodopsin) have not been discovered in thermophilic organisms. In this study we investigated and characterized a microbial rhodopsin derived from the extreme thermophilic bacterium Thermus thermophilus, which lives in a hot spring at around 75 °C. The gene for the retinylidene protein, named thermophilic rhodopsin (TR), was chemically synthesized with codon optimization. The codon optimized TR protein was functionally expressed in the cell membranes of Escherichia coli cells and showed active proton transport upon photoillumination. Spectroscopic measurements revealed that the purified TR bound only all-trans-retinal as a chromophore and showed an absorption maximum at 530 nm. In addition, TR exhibited both photocycle kinetics and pH-dependent absorption changes, which are characteristic of rhodopsins. Of note, time-dependent thermal denaturation experiments revealed that TR maintained its absorption even at 75 °C, and the denaturation rate constant of TR was much lower than those of other proton pumping rhodopsins such as archaerhodopsin-3 (200 ×), Haloquadratum walsbyi bacteriorhodopsin (by 10-times), and Gloeobacter rhodopsin (100 ×). Thus, these results suggest that microbial rhodopsins are also distributed among thermophilic organisms and have high stability. TR should allow the investigation of the molecular mechanisms of ion transport and protein folding.

Keywords: 7-Helix Receptor; Ion Pump; Membrane Proteins; Membrane Transport; Photobiology; Photoreceptors; Protein Denaturation; Retinal; Rhodopsin; Thermal Stability.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Hot Temperature*
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Sequence Data
  • Phylogeny
  • Protein Stability
  • Proton Pumps / genetics
  • Proton Pumps / metabolism*
  • Rhodopsins, Microbial / classification
  • Rhodopsins, Microbial / genetics
  • Rhodopsins, Microbial / metabolism*
  • Sequence Homology, Amino Acid
  • Spectrophotometry / methods*
  • Thermus thermophilus / genetics
  • Thermus thermophilus / metabolism*
  • Time Factors

Substances

  • Proton Pumps
  • Rhodopsins, Microbial