7 Although this conformational change may represent the formation of functionally active state, the reaction kinetics of the change have not been elucidated. 19 A study using the HPLC analysis and the Fourier transform infrared (FTIR) spectroscopy revealed a large change in the backbone of HeR in the last O intermediate having 13- cis-retinal chromophore. 7,12,18 In addition, the putative function of HeR transporting amphiphilic molecules was suggested based on the absence of HeRs in diderm (Gram-negative) bacteria. Because the presence of the long-lived photo-intermediate without ion transport is a common feature of sensory and enzyme rhodopsins, 15–18 HeR is thought to be involved in unidentified signal transduction or cellular metabolic process. The turnover rate of HeRs is relatively longer (0.6–11 s) compared with most abundant H +-pumping type 1 rhodopsins. The K and O intermediates exhibit red-shifted visible absorption spectra compared to the dark state, while the absorption spectrum of the M intermediate, in which the Schiff-base linkage of the retinal chromophore is deprotonated, is highly blue-shifted. HeRs bind an all- trans-retinal chromophore in the dark, and it isomerized to the 13- cis form upon light illumination leading to a photocyclic reaction involving several photo-intermediates: K, L, M, and O. (b) The extracellular hydrogen bonding network of TaHeR connecting the retinal Schiff base (RSB) region and ICL1. 11 The area shown in b is indicated by an orange rectangle. 1 (a) The X-ray crystallographic structure of TaHeR dimer (PDB ID: 6IS6). 7,11 HeR 48C12 and HeR from Thermoplasmatales archaeon SG8-52-1 ( TaHeR) were revealed to form dimers by X-ray crystallography in which protomers are bridged to each other by their long extracellular loops between TM1 and 2 ( Fig. 7 Although the biological function of most HeRs is not known except for a few members, 8–10 they exhibit a characteristic inverted orientation relative to the canonical microbial and animal rhodopsins with the N- and C-termini facing the intracellular and extracellular sides, respectively. In 2018, the third class of rhodopsin, heliorhodopsin (HeR), was discovered by the functional metagenome analysis. Microbial rhodopsins exhibit a wide variety of functions, light-driven ion pumps, light-gated ion channels, light-dependent enzymes, and so on, 4,5 whereas most animal rhodopsins activate heterotrimeric G proteins in a light-dependent manner as a sub-group of the G protein-coupled receptor (GPCR) superfamily. 1–3 Both microbial and animal rhodopsins consist of seven transmembrane helical architecture (TM1–7), in which a retinylidene Schiff base (retinal chromophore) is covalently bound to a lysine residue in TM7. Two distinctive types of rhodopsins, microbial (type 1) and animal (type 2) rhodopsins, are known. Introduction Rhodopsin is a ubiquitous family of photoreceptive membrane proteins. It is suggested that the structure of hydrophilic residues in the ICL1 helix is changed during this process. These results are interpreted in terms of different sensitivities of TG and CD methods, that is, D is sensitive to the structure of the solvent-exposed surface and selectively observes the conformational change in the ICL1 region. It was found that this replacement partially suppresses the D-change, although the CD-change is almost the same as that of the wild type. To examine the contribution of a characteristic helix in the intracellular loop 1 (ICL1 helix), Tyr93 on the ICL1 helix was replaced by Gly (Y93G), and the reaction of this mutant was also investigated. Light-induced unfolding of helical structure is detected by the CD method. Although two time constants (202 μs and 2.6 ms) are observed for the conversion from the M to O by the absorption detection, D decreases only at the first step (202 μs). Subsequently, D decreases upon the O formation. The TG method reveals that the diffusion coefficient ( D) does not change until the O formation suggesting no significant conformation change at the surface of the protein during the early steps of the reaction. In this study, the kinetics of conformational changes of HeR from Thermoplasmatales archaeon SG8-52-1 ( TaHeR) were studied by the transient grating (TG) and circular dichroism (CD) methods. HeRs bind all- trans-retinal as a chromophore in the dark, and its isomerization to the 13- cis form by light illumination leads to a photocyclic reaction involving several photo-intermediates: K, L, M, and O. They exist as a dimer and exhibit a characteristic inverted topology. Heliorhodopsins (HeRs) are a new category of rhodopsins.
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