Spectral Tuning in Sensory Rhodopsin I from Salinibacter ruber

Abstract

Organisms utilize light as energy sources and as signals. Rhodopsins, which have seven transmembrane ␣-helices with retinal covalently linked to a conserved Lys residue, are found in various organisms as distant in evolution as bacteria, archaea, and eukarya. One of the most notable properties of rhodopsin molecules is the large variation in their absorption spectrum. Sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII) function as photosensors and have similar properties (retinal composition, photocycle, structure, and function) except for their max (SRI, ϳ560 nm; SRII, ϳ500 nm). An expression system utilizing Escherichia coli and the high protein stability of a newly found SRI-like protein, SrSRI, enables studies of mutant proteins. To determine the residue contributing to the spectral shift from SRI to SRII, we constructed various SRI mutants, in which individual residues were substituted with the corresponding residues of SRII. Three such mutants of SrSRI showed a large spectral blue-shift (>14 nm) without a large alteration of their retinal composition. Two of them, A136Y and A200T, are newly discovered color tuning residues. In the triple mutant, the max was 525 nm. The inverse mutation of SRII (F134H/Y139A/ T204A) generated a spectral-shifted SRII toward longer wavelengths, although the effect is smaller than in the case of SRI, which is probably due to the lack of anion binding in the SRII mutant. Thus, half of the spectral shift from SRI to SRII could be explained by only those three residues taking into account the effect of Cl ؊ binding.

A Blue-shifted Light-driven Proton Pump for Neural Silencing

“…Similar to the spectral shift of the A223T mutant of HwBR (437 cm Ϫ1 ), it has already been reported that an Ala to Thr or Thr to Ala mutation at position 223 causes a spectral blue shift in HsBR and SRI or a red shift in SRII, respectively, with similar shift values (⌬ ϭ 356 -545 cm Ϫ1 ) (15,17,34), confirming that this residue is one of the important color-tuning residues in microbial rhodopsins. In the case of the residues Asp-123 and Ser-149, their contributions to the color tuning have also been reported in mutants of HsBR, SRI, and SRII (15,17,19). For Met-126, however, the shift value caused by the M118A mutation in HsBR was 3284 cm Ϫ1 (19), which is much larger than those of both HwBR (300 cm Ϫ1 ), AR3 (541 cm Ϫ1 ), and SRII (160 cm Ϫ1 ) (15).…”

“…In those studies that aimed to determine the key residue(s) contributing to the color change of SRII, several SRII mutants were analyzed in which each residue was replaced by its corresponding residue in BR. In addition to this, we also performed similar experiments for SRI (17). From the results, we have found the key residues (factors) contributing to the color change from orange (ϳ500 nm) to purple (ϳ560 nm), as well as several unique factors, as the interaction between the Dand E-helices for SRII (16) or the chloride ion binding around the ␤-ionone ring for SRI (18), respectively.…”

Directed Evolution of Gloeobacter violaceus Rhodopsin Spectral Properties

“…By replacing single amino acid residues in Salinibacter ruber sensory rhodopsin I (SrSRI, λ max = 557 nm) with the corresponding residues from Natronomonas pharaonis sensory rhodopsin II (NpSRII, λ max = 498 nm, ~30% amino acid identity to SrSRI), Sudo et al identified three blue tuning mutations, each of which shifted λ max by 14-24 nm [27]. The SrSRI variant with all three mutations had a λ max of 525 nm (32 nm blue shift), accounting for more than half the spectral shift between SrSRI and NpSRII [27].…”

“…By replacing single amino acid residues in Salinibacter ruber sensory rhodopsin I (SrSRI, λ max = 557 nm) with the corresponding residues from Natronomonas pharaonis sensory rhodopsin II (NpSRII, λ max = 498 nm, ~30% amino acid identity to SrSRI), Sudo et al identified three blue tuning mutations, each of which shifted λ max by 14-24 nm [27]. The SrSRI variant with all three mutations had a λ max of 525 nm (32 nm blue shift), accounting for more than half the spectral shift between SrSRI and NpSRII [27]. In ChRs, recombination of variants with different spectral preferences, kinetics, and ion permeability has been used to generate mutants with different combinations of these properties [28], including a highly active red-shifted mutant called ReaChR [29].…”

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

“…In general, the absorption maximum is related to the wavelength of light in environments where the organisms are living. We have previously identified the residues responsible for the color-tuning among various microbial rhodopsins, such as BR, HwBR, AR3, and sensory rhodopsin I (SRI), which show the color purple, and such as sensory rhodopsin II (SRII) and middle rhodopsin, which show the color orange (41)(42)(43)(44). On the basis of these results, we speculate that Ser-129 (in helix-D) as well as Phe-159 and Tyr-164 (both in helix-E) (Fig.…”