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.
Highlights
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
JL-18 strain contains a gene encoding a microbial rhodopsin composed of 260 amino acid residues (Fig. 2) (NCBI accession ID YP_006059019)
The amino acid sequence of thermophilic rhodopsin (TR) is relatively related to green proteorhodopsin (31% identity, 72% similarity) and more closely to xanthorhodopsin (54% identity, 83% similarity) (Fig. 1) and contains characteristic amino acids found in PR-like proton pumps, such as Asp-95, Asp-229, Glu-106, and His-61, which correspond to Asp-97, Asp-227, Glu-108, and His-75 in PR, respectively
Summary
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 ؋) These results suggest that microbial rhodopsins are distributed among thermophilic organisms and have high stability. In addition to the sensory rhodopsins responsible for photo-signal transduction, a significant number of proton-pumping rhodopsins have been identified from the genes of prokaryotes (archaea and bacteria) and eukaryotes (fungi and algae) (Fig. 1) [7]. In addition to the expression system, the high stability of TR should allow the use of various methods to investigate the molecular mechanisms both of the ion transport and the protein folding
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