Salinibacter Sensory Rhodopsin

Abstract

Halobacterium salinarum sensory rhodopsin I (HsSRI), a dual receptor regulating both negative and positive phototaxis in haloarchaea, transmits light signals through changes in protein-protein interactions with its transducer, halobacterial transducer protein I (HtrI). Haloarchaea also have another sensor pigment, sensory rhodopsin II (SRII), which functions as a receptor regulating negative phototaxis. Compared with HsSRI, the signal relay mechanism of SRII is well characterized because SRII from Natronomonus pharaonis (NpSRII) is much more stable than HsSRI and HsSRII, especially in dilute salt solutions and is much more resistant to detergents. Two genes encoding SRI homologs were identified from the genome sequence of the eubacterium Salinibacter ruber. Those sequences are distantly related to HsSRI (ϳ40% identity) and contain most of the amino acid residues identified as necessary for its function. To determine whether those genes encode functional protein(s), we cloned and expressed them in Escherichia coli. One of them (SrSRI) was expressed well as a recombinant protein having alltrans retinal as a chromophore. UV-Vis, low-temperature UVVis, pH-titration, and flash photolysis experiments revealed that the photochemical properties of SrSRI are similar to those of HsSRI. In addition to the expression system, the high stability of SrSRI makes it possible to prepare large amounts of protein and enables studies of mutant proteins that will allow new approaches to investigate the photosignaling process of SRI-HtrI.

Color‐Discriminating Retinal Configurations of Sensory Rhodopsin I by Photo‐Irradiation Solid‐State NMR Spectroscopy

“…These configurations of retinal in the M-intermediate of SrSRI could be clearly determined by comparing with those of ppR [10] and BR [11][12][13] (Supporting Information, Table S1). Furthermore, the absorption maximum at 390 nm [8] indicated a deprotonated SB. Similar results were obtained by irradiation with 520 nm LED light (Figure 1 A) and 595 nm LED light (Figure 1 B).…”

“…[8] In the dark state, SrSRI has an all-trans retinal as a chromophore, has an absorption maximum at 557 nm, and has a slower photocycle than the light-driven ion pumping rhodopsin (BR and HR), indicating similarities with HsSRI. [8,9] SrSRI is much more stable than HsSRI, especially both in dilute salt solutions and in detergent micelles. These characteristics of SrSRI will allow new approaches to investigate the photo-signaling process of SRI.…”

“…Because the absorption maximum of the Pintermediate is located at higher wavelength (ca. 525 nm), [8] it is likely that the P-intermediate has a protonated retinal Schiff base. Additionally, as the chemical shift value of the Pintermediate is significantly different from that of the Mintermediate, a rather large conformational change was observed, which may cause large changes in protein interactions, acting as a switch between positive and negative phototaxis functions.…”

“…Subsequently the P-intermediate was trapped stationary by illumination with a second UV light (365 nm) in a repellent state. The configuration of the retinal in the M-intermediate was determined to be 13-cis, 15-anti with deprotonation of SB (absorption maximum at 390 nm) [8] and in the P-intermediate to be 13-cis with protonation of the SB (absorption maximum at 525 nm). [8] In Figure 4, we tentatively assigned the configuration of the P-intermediate to be 13-cis, 15-anti, although 15-anti or 15-syn was not determined in this experiment.…”

“…The configuration of the retinal in the M-intermediate was determined to be 13-cis, 15-anti with deprotonation of SB (absorption maximum at 390 nm) [8] and in the P-intermediate to be 13-cis with protonation of the SB (absorption maximum at 525 nm). [8] In Figure 4, we tentatively assigned the configuration of the P-intermediate to be 13-cis, 15-anti, although 15-anti or 15-syn was not determined in this experiment. Interestingly, transformation from the M-to the P-intermediate occurred without retinal isomerization of C13=C14 bond from 13-cis to all-trans.…”