Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The primary proton transfer reactions of thermophilic rhodopsin, which was first discovered in an extreme thermophile, Thermus thermophilus JL-18, were investigated using time-resolved Fourier transform infrared spectroscopy at various temperatures ranging from 298 to 343 K (25 to 70 °C) and proton transport activity analysis. The analyses were performed using counterion (D95E, D95N, D229E, and D229N) and proton donor mutants (E106D and E106Q) as well. First, the initial proton transfer from the protonated retinal Schiff base (PRSB) to D95 was identified. The temperature dependency showed that the proton transfer reaction in the intermediate states dramatically changed above 318 K (45 °C). In addition, the proton transfer reaction correlated well with the structural change from turn to β-strand in the protein moiety, suggesting that this step may be regulated by the rigidity of the loop region. We also elucidated that the proton transfer reaction from proton donor E106 to the retinal Schiff base occurred synchronously with the primary proton transfer from the PRSB to D95. Surprisingly, we discovered that the direction of proton transfer was regulated by the secondary counterion, D229. Comparative analysis of Gloeobacter rhodopsin from the mesophile, Gloeobacter violaceus, highlighted that the primary proton transfer reactions in thermophilic rhodopsin were optimized at high temperatures partly due to the specific turn to β-strand structural change. This was not observed in Gloeobacter rhodopsin and other related proteins such as bacteriorhodopsin.

DOI： 10.1016/j.bbabio.2023.148980

PubMed

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

An oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source, thereby reducing the risk of kidney stone formation in host animals. The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations, occluded and outward-facing states. The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate. The occluded pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the pocket are completely blocked by extensive interdomain interactions, which can be opened solely by a flip of a single side chain neighbouring the substrate. This study shows the structural basis underlying metabolic interactions enabling favourable symbiosis.

DOI： 10.1038/s41467-023-36883-5

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Other Link： https://www.nature.com/articles/s41467-023-36883-5

Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Pharmaceutical Society of Japan  

DOI： 10.1248/cpb.c22-00774

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acsomega.2c06980

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Microbial Ecology  

Diatoms are a major phytoplankton group responsible for approximately 20% of carbon fixation on Earth. They perform photosynthesis using light-harvesting chlo-rophylls located in plastids, an organelle obtained through eukaryote-eukaryote endosymbiosis. Microbial rhodopsin, a photoreceptor distinct from chlo-rophyll-based photosystems, was recently identified in some diatoms. However, the physiological function of diatom rhodopsin remains unclear. Heterologous expression techniques were herein used to investigate the protein function and subcellular localization of diatom rhodopsin. We demonstrated that diatom rhodopsin acts as a light-driven proton pump and localizes primarily to the outermost membrane of four membrane-bound complex plastids. Using model simulations, we also examined the effects of pH changes inside the plastid due to rhodopsin-mediated proton transport on photosynthesis. The results obtained suggested the involvement of rhodopsin-mediated local pH changes in a photosynthetic CO2-concentrating mechanism in rhodopsin-possessing diatoms.

DOI： 10.1264/jsme2.me23015

PubMed

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Publishing type：Research paper (scientific journal)  

DOI： 10.2142/biophysico.bppb-v20.s021

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Publishing type：Research paper (scientific journal)  

DOI： 10.2142/biophysico.bppb-v20.s001

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Language：English   Publishing type：Research paper (scientific journal)  

Membrane transport proteins can be divided into two types: those that bind substrates in a resting state and those that do not. In this study, we demonstrate that these types can be converted by mutations through a study of two cyanobacterial anion-pumping rhodopsins, Mastigocladopsis repens halorhodopsin (MrHR) and Synechocystis halorhodopsin (SyHR). Anion pump rhodopsins, including MrHR and SyHR, initially bind substrate anions to the protein center and transport them upon illumination. MrHR transports only smaller halide ions, Cl- and Br-, but SyHR also transports SO42-, despite the close sequence similarity to MrHR. We sought a determinant that could confer SO42- pumping ability on MrHR and found that the removal of a negative charge at the anion entrance is a prerequisite for SO42- transport by MrHR. Consistently, the reverse mutation in SyHR significantly weakened SO42- pump activity. Notably, the MrHR and SyHR mutants did not show SO42- induced absorption spectral shifts or changes in the photoreactions, suggesting no bindings of SO42- in their initial states or the bindings to the sites far from the protein centers. In other words, unlike wild-type SyHR, these mutants take up SO42- into their centers after illumination and release it before the ends of the photoreactions.

DOI： 10.1038/s41598-022-20784-6

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/jacs.1c12608

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Language：English   Publishing type：Research paper (scientific journal)  

We have developed a methodology for identifying further thermostabilizing mutations for an intrinsically thermostable membrane protein. The methodology comprises the following steps: (1) identifying thermostabilizing single mutations (TSSMs) for residues in the transmembrane region using our physics-based method; (2) identifying TSSMs for residues in the extracellular and intracellular regions, which are in aqueous environment, using an empirical force field FoldX; and (3) combining the TSSMs identified in steps (1) and (2) to construct multiple mutations. The methodology is illustrated for thermophilic rhodopsin whose apparent midpoint temperature of thermal denaturation Tm is ∼91.8 °C. The TSSMs previously identified in step (1) were F90K, F90R, and Y91I with ΔTm ∼5.6, ∼5.5, and ∼2.9 °C, respectively, and those in step (2) were V79K, T114D, A115P, and A116E with ΔTm ∼2.7, ∼4.2, ∼2.6, and ∼2.3 °C, respectively (ΔTm denotes the increase in Tm). In this study, we construct triple and quadruple mutants, F90K+Y91I+T114D and F90K+Y91I+V79K+T114D. The values of ΔTm for these multiple mutants are ∼11.4 and ∼13.5 °C, respectively. Tm of the quadruple mutant (∼105.3 °C) establishes a new record in a class of outward proton pumping rhodopsins. It is higher than Tm of Rubrobacter xylanophilus rhodopsin (∼100.8 °C) that was the most thermostable in the class before this study.

DOI： 10.1021/acs.jpcb.1c08684

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cold Spring Harbor Laboratory  

Abstract

Diatoms are a major phytoplankton group responsible for about 20% of Earth’s primary production. They carry out photosynthesis inside the plastid, an organelle obtained through eukaryote-eukaryote endosymbiosis. Recently, microbial rhodopsin, a photoreceptor distinct from chlorophyll-based photosystems, has been identified in certain diatoms. However, the physiological function of diatom rhodopsin is not well understood. Here we show that the diatom rhodopsin acts as a light-driven proton pump and localizes to the outermost membrane of the four membrane-bound complex plastids. Heterologous expression techniques were used to investigate the protein function and subcellular localization of diatom rhodopsin. Using model simulations, we further evaluated the physiological role of the acidic pool in the plastid produced by proton-transporting rhodopsin. Our results propose that the rhodopsin-derived acidic pool may be involved in a photosynthetic CO₂-concentrating mechanism and assist CO₂ fixation in diatom cells.

DOI： 10.1101/2022.01.18.476826

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：eLife Sciences Publications, Ltd  

Anion channelrhodopsin from <italic>Guillardia theta</italic> (<italic>Gt</italic>ACR1) has Asp234 (3.2 Å) and Glu68 (5.3 Å) near the protonated Schiff base. Here, we investigate mutant <italic>Gt</italic>ACR1s (e.g., E68Q/D234N) expressed in HEK293 cells. The influence of the acidic residues on the absorption wavelengths was also analyzed using a quantum mechanical/molecular mechanical approach. The calculated protonation pattern indicates that Asp234 is deprotonated and Glu68 is protonated in the original crystal structures. The D234E mutation and the E68Q/D234N mutation shorten and lengthen the measured and calculated absorption wavelengths, respectively, which suggests that Asp234 is deprotonated in the wild-type <italic>Gt</italic>ACR1. Molecular dynamics simulations show that upon mutation of deprotonated Asp234 to asparagine, deprotonated Glu68 reorients toward the Schiff base and the calculated absorption wavelength remains unchanged. The formation of the proton transfer pathway via Asp234 toward Glu68 and the disconnection of the anion conducting channel are likely a basis of the gating mechanism.

DOI： 10.7554/elife.72264

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Other Link： https://cdn.elifesciences.org/articles/72264/elife-72264-v1.xml

Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

Rhodopsins act as photoreceptors with their chromophore retinal (vitamin-A aldehyde) and they regulate light-dependent biological functions. Archaerhodopsin-3 (AR3) is an outward proton pump that has been widely utilized as a tool for optogenetics, a method for controlling cellular activity by light. To characterize the retinal binding cavity of AR3, we synthesized a dimethyl phenylated retinal derivative, (2E,4E,6E,8E)-9-(2,6-Dimethylphenyl)-3,7-dimethylnona-2,4,6,8-tetraenal (DMP-retinal). QM/MM calculations suggested that DMP-retinal can be incorporated into the opsin of AR3 (archaeopsin-3, AO3). Thus, we introduced DMP-retinal into AO3 to obtain the non-natural holoprotein (AO3-DMP) and compared some molecular properties with those of AO3 with the natural A1-retinal (AO3-A1) or AR3. Light-induced pH change measurements revealed that AO3-DMP maintained slow outward proton pumping. Noteworthy, AO3-DMP had several significant changes in its molecular properties compared with AO3-A1 as follows; 1) spectroscopic measurements revealed that the absorption maximum was shifted from 556 to 508 nm and QM/MM calculations showed that the blue-shift was due to the significant increase in the HOMO-LUMO energy gap of the chromophore with the contribution of some residues around the chromophore, 2) time-resolved spectroscopic measurements revealed the photocycling rate was significantly decreased, and 3) kinetical spectroscopic measurements revealed the sensitivity of the chromophore binding Schiff base to attack by hydroxylamine was significantly increased. The QM/MM calculations show that a cavity space is present at the aromatic ring moiety in the AO3-DMP structure whereas it is absent at the corresponding <italic>β</italic>-ionone ring moiety in the AO3-A1 structure. We discuss these alterations of the difference in interaction between the natural A1-retinal and the DMP-retinal with binding cavity residues.

DOI： 10.3389/fmolb.2021.794948

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>Microbial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an <italic>Escherichia coli</italic> expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate <italic>Oxyrrhis marina</italic> (<italic>O. marina</italic> rhodopsin-2, <italic>Om</italic>R2) that can be expressed in <italic>E. coli</italic> cells. <italic>E. coli</italic> cells harboring the <italic>Om</italic>R2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified <italic>Om</italic>R2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-<italic>trans</italic> retinal chromophore, (2) the possession of the deprotonated counterion (p<italic>K</italic>_a = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of <italic>Om</italic>R2 expressed in <italic>E. coli</italic> cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

DOI： 10.1038/s41598-021-94181-w

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Other Link： http://www.nature.com/articles/s41598-021-94181-w

Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/pro.4154

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/pro.4154

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/prot.26015

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/prot.26015

Publishing type：Research paper (scientific journal)   Publisher：Biophysical Society of Japan  

DOI： 10.2142/biophysico.bppb-v18.019

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>
Microbial rhodopsin is a photoreceptor protein found in various bacteria and archaea, and it is considered to be a light-utilization device unique to heterotrophs. Recent studies have shown that several cyanobacterial genomes also include genes that encode rhodopsins, indicating that these auxiliary light-utilizing proteins may have evolved within photoautotroph lineages. To explore this possibility, we performed a large-scale genomic survey to clarify the distribution of rhodopsin and its phylogeny. Our surveys revealed a novel rhodopsin clade, cyanorhodopsin (CyR), that is unique to cyanobacteria. Genomic analysis revealed that rhodopsin genes show a habitat-biased distribution in cyanobacterial taxa, and that the CyR clade is composed exclusively of non-marine cyanobacterial strains. Functional analysis using a heterologous expression system revealed that CyRs function as light-driven outward H⁺ pumps. Examination of the photochemical properties and crystal structure (2.65 Å resolution) of a representative CyR protein, N2098R from <italic>Calothrix</italic> sp. NIES-2098, revealed that the structure of the protein is very similar to that of other rhodopsins such as bacteriorhodopsin, but that its retinal configuration and spectroscopic characteristics (absorption maximum and photocycle) are distinct from those of bacteriorhodopsin. These results suggest that the CyR clade proteins evolved together with chlorophyll-based photosynthesis systems and may have been optimized for the cyanobacterial environment.

DOI： 10.1038/s41598-020-73606-y

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Other Link： http://www.nature.com/articles/s41598-020-73606-y

Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

The photoreactive protein rhodopsin is widespread in microorganisms and has a variety of photobiological functions. Recently, a novel phylogenetically distinctive group named ‘schizorhodopsin (SzR)’ has been identified as an inward proton pump. We performed functional and spectroscopic studies on an uncharacterised schizorhodopsin from the phylum Lokiarchaeota archaeon. The protein, LaSzR2, having an all-trans-retinal chromophore, showed inward proton pump activity with an absorption maximum at 549 nm. The pH titration experiments revealed that the protonated Schiff base of the retinal chromophore (Lys188, pK_a = 12.3) is stabilised by the deprotonated counterion (presumably Asp184, pK_a = 3.7). The flash-photolysis experiments revealed the presence of two photointermediates, K and M. A proton was released and uptaken from bulk solution upon the formation and decay of the M intermediate. During the M-decay, the Schiff base was reprotonated by the proton from a proton donating residue (presumably Asp172). These properties were compared with other inward (SzRs and xenorhodopsins, XeRs) and outward proton pumps. Notably, LaSzR2 showed acid-induced spectral ‘blue-shift’ due to the protonation of the counterion, whereas outward proton pumps showed opposite shifts (red-shifts). Thus, we can distinguish between inward and outward proton pumps by the direction of the acid-induced spectral shift.

DOI： 10.1038/s41598-020-77936-9

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Other Link： https://www.nature.com/articles/s41598-020-77936-9

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.bbabio.2020.148349

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The membrane-embedded protein rhodopsin is widely produced in organisms as a photoreceptor showing a variety of light-dependent biological functions. To investigate its molecular features, rhodopsin is often extracted from cellular membrane lipids by a suitable detergent as "micelles." The extracted protein is purified by column chromatography and then is often reconstituted into "liposomes" by removal of the detergent. The styrene-maleic acid ("SMA") copolymer spontaneously forms nanostructures containing lipids without detergent. In this study, we applied SMA to characterize two microbial rhodopsins, a thermally stable rhodopsin, Rubrobacter xylanophilus rhodopsin (RxR), and an unstable one, Halobacterium salinarum sensory rhodopsin I (HsSRI), and evaluated their physicochemical properties in SMA lipid particles compared with rhodopsins in micelles and in liposomes. Those two rhodopsins were produced in Escherichia coli cells and were successfully extracted from the membrane by the addition of SMA (5 w/v %) without losing their visible color. Analysis by dynamic light scattering revealed that RxR in SMA lipid particles (RxR-SMA) formed a discoidal structure with a diameter of 54 nm, which was 10 times smaller than RxR in phosphatidylcholine liposomes. The small particle size of RxR-SMA allowed us to obtain scattering-less visible spectra with a high signal-to-noise ratio similar to RxR in detergent micelles composed of n-dodecyl-β-D-maltoside. High-speed atomic force microscopy revealed that a single particle contained an average of 4.1 trimers of RxR (12.3 monomers). In addition, RxR-SMA showed a fast cyclic photoreaction (k = 13 s-1) comparable with RxR in phosphatidylcholine liposomes (17 s-1) but not to RxR in detergent micelles composed of n-dodecyl-β-D-maltoside (0.59 s-1). By taking advantage of SMA, we determined the dissociation constant (Kd) of chloride for HsSRI as 34 mM. From these results, we conclude that SMA is a useful molecule forming a membrane-mimicking assembly for microbial rhodopsins having the advantages of detergents and liposomes.

DOI： 10.1016/j.bpj.2020.09.026

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/1348-0421.12829

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/1348-0421.12829

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acs.jpcb.0c06560

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acs.jpclett.0c01406

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Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

DOI： 10.1021/acs.jpcb.0c02254

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Language：English   Publishing type：Research paper (scientific journal)  

We develop a new methodology best suited to the identification of thermostabilizing mutations for an intrinsically stable membrane protein. The recently discovered thermophilic rhodopsin, whose apparent midpoint temperature of thermal denaturation Tm is measured to be ∼91.8 °C, is chosen as a paradigmatic target. In the methodology, we first regard the residues whose side chains are missing in the crystal structure of the wild type (WT) as the "residues with disordered side chains," which make no significant contributions to the stability, unlike the other essential residues. We then undertake mutating each of the residues with disordered side chains to another residue except Ala and Pro, and the resultant mutant structure is constructed by modifying only the local structure around the mutated residue. This construction is based on the postulation that the structure formed by the other essential residues, which is nearly optimized in such a highly stable protein, should not be modified. The stability changes arising from the mutations are then evaluated using our physics-based free-energy function (FEF). We choose the mutations for which the FEF is much lower than for the WT and test them by experiments. We successfully find three mutants that are significantly more stable than the WT. A double mutant whose Tm reaches ∼100 °C is also discovered.

DOI： 10.1021/acs.jcim.0c00063

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Language：Japanese   Publisher：(公社)日本薬学会  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Rubrobacter xylanophilus rhodopsin (RxR) is a phylogenetically distinct and thermally stable seven-transmembrane protein that functions as a light-driven proton (H+) pump with the chromophore retinal. To characterize its vectorial proton transport mechanism, mutational and theoretical investigations were performed for carboxylates in the transmembrane region of RxR and the sequential proton transport steps were revealed as follows: (i) a proton of the retinylidene Schiff base (Lys209) is transferred to the counterion Asp74 upon formation of the blue-shifted M-intermediate in collaboration with Asp205, and simultaneously, a respective proton is released from the proton releasing group (Glu187/Glu197) to the extracellular side, (ii) a proton of Asp85 is transferred to the Schiff base during M-decay, (iii) a proton is taken up from the intracellular side to Asp85 during decay of the red-shifted O-intermediate. This ion transport mechanism of RxR provides valuable information to understand other ion transporters since carboxylates are generally essential for their functions.

DOI： 10.1038/s41598-019-57122-2

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Authorship：Lead author,　Corresponding author   Publisher：Biophysical Society of Japan  

DOI： 10.2142/biophys.60.209

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Microbial Ecology  

DOI： 10.1264/jsme2.me20085

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Language：Japanese   Publisher：GEOCHEMICAL SOCIETY OF JAPAN  

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DOI： 10.14862/geochemproc.67.0_186

CiNii Article

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Publishing type：Research paper (scientific journal)  

DOI： 10.1021/acs.jpcb.0c02254

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1038/s41598-019-44308-x

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Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

DOI： 10.1021/acs.jpcb.8b09660

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biophysical Society  

Pharanois phoborhodopsin (ppR) from Natronomonas pharaonis is a transmembrane photoreceptor protein involved in negative phototaxis. Structural changes in ppR triggered by photoisomerization of the retinal chromophore are transmitted to its cognate transducer protein (pHtrII) through a cyclic photoreaction pathway involving several photointermediates. This pathway is called the photocycle. It is important to understand the detailed configurational changes of retinal during the photocycle. We previously observed one of the photointermediates (M-intermediates) by in situ photoirradiation solid-state NMR experiments. In this study, we further observed the 13C cross-polarization magic-angle-spinning NMR signals of late photointermediates such as O- and N′-intermediates by illumination with green light (520 nm). Under blue-light (365 nm) irradiation of the M-intermediates, 13C cross-polarization magic-angle-spinning NMR signals of 14- and 20-13C-labeled retinal in the O-intermediate appeared at 115.4 and 16.4 ppm and were assigned to the 13-trans, 15-syn configuration. The signals caused by the N′-intermediate appeared at 115.4 and 23.9 ppm and were assigned to the 13-cis configuration, and they were in an equilibrium state with the O-intermediate during thermal decay of the M-intermediates at −60°C. Thus, photoirradiation NMR studies revealed the photoreaction pathways from the M- to O-intermediates and the equilibrium state between the N′- and O-intermediate. Further, we evaluated the detailed retinal configurations in the O- and N′-intermediates by performing a density functional theory chemical shift calculation. The results showed that the N′-intermediate has a 63° twisted retinal state due to the 13-cis configuration. The retinal configurations of the O- and N′-intermediates were determined to be 13-trans, 15-syn, and 13-cis, respectively, based on the chemical shift values of [20-13C] and [14-13C] retinal obtained by photoirradiation solid-state NMR and density functional theory calculation.

DOI： 10.1016/j.bpj.2018.05.030

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acs.jpcb.8b04894

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society  

Rhodopsin is widely distributed in organisms as a membrane-embedded photoreceptor protein, consisting of the apoprotein opsin and vitamin-A aldehyde retinal, A1-retinal and A2-retinal being the natural chromophores. Modifications of opsin (e.g., by mutations) have provided insight into the molecular mechanism of the light-induced functions of rhodopsins as well as providing tools in chemical biology to control cellular activity by light. Instead of the apoprotein opsin, in this study, we focused on the retinal chromophore and synthesized three vinylene derivatives of A2-retinal. One of them, C(14)-vinylene A2-retinal (14V-A2), was successfully incorporated into the opsin of a light-driven proton pump archaerhodopsin-3 (AR3). Electrophysiological experiments revealed that the opsin of AR3 (archaeopsin3, AO3) with 14V-A2 functions as a light-gated proton channel. The engineered proton channel showed characteristic photochemical properties, which are significantly different from those of AR3. Thus, we successfully produced a proton channel by replacing the chromophore of AR3.

DOI： 10.1021/acs.jpclett.8b00879

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Language：English   Publishing type：Research paper (scientific journal)  

Light-driven ion-pumping rhodopsins are widely distributed among bacteria, archaea, and eukaryotes in the euphotic zone of the aquatic environment. H+-pumping rhodopsin (proteorhodopsin: PR), Na+-pumping rhodopsin (NaR), and Cl--pumping rhodopsin (ClR) have been found in marine bacteria, which suggests that these genes evolved independently in the ocean. Putative microbial rhodopsin genes were identified in the genome sequences of marine Cytophagia. In the present study, one of these genes was heterologously expressed in Escherichia coli cells and the rhodopsin protein named Rubricoccus marinus halorhodopsin (RmHR) was identified as a light-driven inward Cl- pump. Spectroscopic assays showed that the estimated dissociation constant (Kd,int.) of this rhodopsin was similar to that of haloarchaeal halorhodopsin (HR), while the Cl--transporting photoreaction mechanism of this rhodopsin was similar to that of HR, but different to that of the already-known marine bacterial ClR. This amino acid sequence similarity also suggested that this rhodopsin is similar to haloarchaeal HR and cyanobacterial HRs (e.g., SyHR and MrHR). Additionally, a phylogenetic analysis revealed that retinal biosynthesis pathway genes (blh and crtY) belong to a phylogenetic lineage of haloarchaea, indicating that these marine Cytophagia acquired rhodopsin-related genes from haloarchaea by lateral gene transfer. Based on these results, we concluded that inward Cl--pumping rhodopsin is present in genera of the class Cytophagia and may have the same evolutionary origins as haloarchaeal HR.

DOI： 10.1264/jsme2.ME17197

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

A new group of microbial rhodopsins named xenorhodopsins (XeR), which are closely related to the cyanobacterial Anabaena sensory rhodopsin, show a light-driven "inward" proton transport activity, as reported for one representative of this group from Parvularcula oceani (PoXeR). In this study, we functionally and spectroscopically characterized a new member of the XeR clade from a marine bacterium Rubricoccus marinus SG-29T (RmXeR). Escherichia coli cells expressing recombinant RmXeR showed a light-induced alkalization of the cell suspension, which was strongly impaired by a protonophore, suggesting that RmXeR is a light-driven "inward" proton pump as is PoXeR. The spectroscopic properties of purified RmXeR were investigated and compared with those of PoXeR and a light-driven "outward" proton pump, bacteriorhodopsin (BR) from the archaeon Halobacterium salinarum. Action spectroscopy revealed that RmXeR with all-trans retinal is responsible for the light-driven inward proton transport activity, but not with 13-cis retinal. From pH titration experiments and mutational analysis, we estimated the pKa values for the protonated Schiff base of the retinal chromophore and its counterion as 11.1 ± 0.07 and 2.1 ± 0.07, respectively. Of note, the direction of both the retinal composition change upon light-dark adaptation and the acid-induced spectral shift was opposite that of BR, which is presumably related to the opposite directions of ion transport (from outside to inside for RmXeR and from inside to outside for BR). Flash photolysis experiments revealed the appearances of three intermediates (L, M and O) during the photocycle. The proton uptake and release were coincident with the formation and decay of the M intermediate, respectively. Together with associated findings from other microbial rhodopsins, we propose a putative model for the inward proton transport mechanism of RmXeR.

DOI： 10.1039/c7cp05033j

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biophysical Society of Japan  

DOI： 10.2142/biophysico.15.0_179

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Publishing type：Research paper (scientific journal)  

DOI： 10.1021/acs.jpcb.8b09660

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We present a novel design of a few-cycle noncollinear optical parametric amplifier (NOPA) pumped by the second harmonic of a Ti: sapphire laser. A quasi-transform-limited sub-6 fs pulse width was realized by static dispersion compensation with commercially available chirped mirrors. The performance of the NOPA was tested by performing transient absorption spectroscopy on sensory rhodopsin II, and we observe short-lived oscillatory components that are associated with the vibrational coherence from the isomerizing molecule in the excited electronic state. (C) 2017 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.cplett.2017.04.001

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Rhodopsins are seven-transmembrane proteins that function as photoreceptors for a variety of biological processes. Their characteristic visible colors make rhodopsins a good model for membrane-embedded proteins. In this study, by utilizing their color changes, we performed comparative studies on the stability of five microbial rhodopsins using the same instruments, procedures and media. As denaturants, we employed four physicochemical stimuli: (i) thermal perturbation, (ii) the water-soluble reagent hydroxylamine, (iii) the detergent sodium dodecyl sulfate, and (iv) the organic solvent ethanol. On the basis of the results, models for stabilization mechanisms in rhodopsins against each stimulus is proposed. (C) 2017 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.cplett.2017.05.055

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Rhodopsins are proteins that contain seven transmembrane domains with a chromophore retinal and that function as photoreceptors for light-energy conversion and light-signal transduction in a wide variety of organisms. Here we characterized a phylogenetically distinctive new rhodopsin from the thermophilic eubacterium Rubrobacter xylanophilus DSM 9941(T) that was isolated from thermally polluted water. Although R. xylanophilus rhodopsin (RxR) is from Actinobacteria, it is located between eukaryotic and archaeal rhodopsins in the phylogenetic tree. Escherichia coli cells expressing RxR showed a light-induced decrease in environmental pH and inhibition by a protonophore, indicating that it works as a light-driven outward proton pump. We characterized purified RxR spectroscopically, and showed that it has an absorption maximum at 541 nm and binds nearly 100% all-trans retinal. The pK(a) values for the protonated retinal Schiff base and its counterion were estimated to be 10.7 and 1.3, respectively. Time-resolved flash-photolysis experiments revealed the formation of a red-shifted intermediate. Of note, RxR showed an extremely high thermal stability in comparison with other proton pumping rhodopsins such as thermophilic rhodopsin TR (by 16-times) and bacteriorhodopsin from Halobacterium salinarum (HsBR, by 4-times).

DOI： 10.1038/srep44427

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

In organisms, ion transporters play essential roles in the generation and dissipation of ion gradients across cell membranes. Microbial rhodopsins selectively transport cognate ions using solar energy, in which the substrate ions identified to date have been confined to monovalent ions such as H+, Na+ and Cl-. Here we report a novel rhodopsin from the cyanobacterium Synechocystis sp. PCC 7509, which inwardly transports a polyatomic divalent sulfate ion, SO42-, with changes of its spectroscopic properties in both unphotolyzed and photolyzed states. Upon illumination, cells expressing the novel rhodopsin, named Synechocystis halorhodopsin (SyHR), showed alkalization of the medium only in the presence of Cl- or SO42-. That alkalization signal was enhanced by addition of a protonophore, indicating an inward transport of and SO42- with a subsequent secondary inward H+ movement across the membrane. The anion binding to SyHR was suggested by absorption spectral shifts from 542 to 536 nm for Cl- and from 542 to 556 nm for SO42-, and the affinities of Cl- and SO42- were estimated as 0.112 and 5.81 rriM, respectively. We then performed time-resolved spectroscopic measurements ranging from femtosecond to millisecond time domains to elucidate the structure and structural changes of SyHR during the photoreaction. Based on the results, we propose a photocycle model for SyHR in the absence or presence of substrate ions with the timing of their uptake and release. Thus, we demonstrate SyHR as the first light-driven polyatomic divalent anion (SO42-) transporter and report its spectroscopic characteristics.

DOI： 10.1021/jacs.6b12139

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Several new retinal-based photoreceptor proteins that act as light-driven electrogenic halide ion pumps have recently been discovered. Some of them, called "NTQ' rhodopsins, contain a conserved Asn-Thr-Gln motif in the third or C-helix. In this study, we investigated the photochemical characteristics of an NTQ rhodopsin, Nonlabens marinus rhodopsin 3 (NM-R3), which was discovered in the N. marinus S1-08(T) strain, using static and time-resolved spectroscopic techniques. We demonstrate that NM-R3 binds a Cl-in the-vicinity of the retinal chromophore accompanied by a spectral blueshift from 568 nm in the absence of Cl-to 534 nm in the presence of Cl-. From the Cl- concentration dependence, we estimated the affinity (dissociation constant, K-d) for Cl- in the original state as 24 mM, which is ca. 10 times weaker than that of archaeal halorhodopsins but ca. 3 times stronger than that of a marine bacterial Cl-pumping rhodopsin (C1R). NM-R3 showed no dark-light adaptation of the retinal chromophore and predominantly possessed an all-trans-retinal, which is responsible for the light-driven Cl-pump function. Flash-photolysis experiments suggest that NM-R3 passes through five or six photochemically distinct intermediates (K, L(N), O-1, O-2, and NM-R3'). From these results, we assume that the Cl-is released and taken up during the L(N)-O-1 transition from a transiently formed cytoplasmic (CP) binding site and the O-2-NM R3' or the NM-R3'-original NM-R3 transitions from the extracellular (EC) side, respectively. We propose a mechanism for the Cl-transport by NM-R3 based on our results and its recently reported crystal structure.

DOI： 10.1021/acs.jpcb.6b11101

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Anion channelrhodopsin-2 (ACR2) was recently identified from the cryptophyte algae Guillardia theta and has become a focus of interest in part because of its novel light-gated anion channel activity and its extremely high neural silencing activity. In this study, we tried to express ACR2 in Escherichia coli cells as a recombinant protein. The E. coli cells expressing ACR2 showed an increase in pH upon blue-light illumination in the presence of monovalent anions and the protonophore carbonyl cyanide mchlorophenylhydrazone (CCCP), indicating an inward anion channel activity. Then, taking advantage of the E. coli expression system, we performed alanine-scanning mutagenesis on conserved basic amino acid residues. One of them, R84A, showed strong signals compared with the wild-type, indicating an inhibitory role of R84 on Cl-transportation. The signal was strongly enhanced in R84E, whereas R84K was less effective than the wild-type (i.e., R84). These results suggest that the positive charge at position 84 is critical for the inhibition. Thus we succeeded in functional expression of ACR2 in E. coli and found the inhibitory role of R84 during the anion transportation.

DOI： 10.1038/srep41879

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/acs.langmuir.7b00463

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

DOI： 10.1093/jmcb/mjw027

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Primary photochemical events in the unusually thermostable proton pumping rhodopsin of Thermus thermophilus bacterium (TR) are reported for the first time. Internal conversion in this protein is shown to be significantly faster than in bacteriorhodopsin (BR), making it the most rapidly isomerizing microbial proton pump known. Internal conversion (IC) dynamics of TR and BR were recorded from room temperature to the verge of thermal denaturation at 70 degrees C and found to be totally independent of temperature in this range. This included the well documented multiexponential nature of IC in BR, suggesting that assignment of this to ground state structural inhomogeneity needs revision. TR photodynamics were also compared with that of the phylogenetically more similar proton pump Gloeobacter rhodopsin (GR). Despite this similarity GR has poor thermal stability, and the excited state decays significantly more slowly and exhibits very prominent stretched exponential behavior. Coherent torsional wave-packets induced by impulsive photoexcitation of TR and GR show marked resemblance to each other in frequency and amplitude and differ strikingly from similar signatures in pump-probe data of BR and other microbial retinal proteins. Possible correlations between IC rates and thermal stability and the promise of using torsional coherence signatures for understanding chromophore protein binding in microbial retinal proteins are discussed.

DOI： 10.1021/jacs.6b05002

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Thermophilic rhodopsin (TR) is a photoreceptor protein with an extremely high thermal stability and the first characterized light-driven electrogenic proton pump derived from the extreme thermophile Thermus thermophilus JL-18. In this study, we confirmed its high thermal stability compared with other microbial rhodopsins and also report the potential availability of TR for optogenetics as a light-induced neural silencer. The x-ray crystal structure of TR revealed that its overall structure is quite similar to that of xanthorhodopsin, including the presence of a putative binding site for a carotenoid antenna; but several distinct structural characteristics of TR, including a decreased surface charge and a larger number of hydrophobic residues and aromatic-aromatic interactions, were also clarified. Based on the crystal structure, the structural changes of TR upon thermal stimulation were investigated by molecular dynamics simulations. The simulations revealed the presence of a thermally induced structural substate in which an increase of hydrophobic interactions in the extracellular domain, the movement of extracellular domains, the formation of a hydrogen bond, and the tilting of transmembrane helices were observed. From the computational and mutational analysis, we propose that an extracellular LPGG motif between helices F and G plays an important role in the thermal stability, acting as a "thermal sensor." These findings will be valuable for understanding retinal proteins with regard to high protein stability and high optogenetic performance.

DOI： 10.1074/jbc.M116.719815

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Photoactive retinal proteins are widely distributed throughout the domains of the microbial world (i.e., bacteria, archaea, and eukarya). Here we describe three retinal proteins belonging to a phylogenetic clade with a unique DTG motif. Light-induced decrease in the environmental pH and its inhibition by carbonyl cyanide m-chlorophenylhydrazone revealed that these retinal proteins function as light-driven outward electrogenic proton pumps. We further characterized one of these proteins, Pantoea vagans rhodopsin (PvR), spectroscopically. Visible spectroscopy and high-performance liquid chromatography revealed that PvR has an absorption maximum at 538 nm with the retinal chromophore predominantly in the all-trans form (&gt;90%) under both dark and light conditions. We estimated the pK(a) values of the protonated Schiff base of the retinal chromophore and its counterion as approximately 13.5 and 2.1, respectively, by using pH titration experiments, and the photochemical reaction cycle of PvR was measured by time-resolved flash-photolysis in the millisecond timeframe. We observed a blue-shifted and a red-shifted intermediate, which we assigned as M-like and O-like intermediates, respectively. Decay of the M-like intermediate was highly sensitive to environmental pH, suggesting that proton uptake is coupled to decay of the M-like intermediate. From these results, we propose a putative model for the photoreaction of PvR.

DOI： 10.1111/php.12585

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

A novel planar architecture has been developed for the study of photodetectors utilizing the transient photocurrent response induced by a metal/insulator/semi-conductor/metal (MISM) structured device, where the insulator is an ionic liquid (IL-MISM). Using vanadyl 2,3-naphthalocyanine, which absorbs in the communications-relevant near-infrared wavelength region (lambda(max,film) approximate to 850 nm) in conjunction with C-60 as a bulk heterojunction, the high capacitance of the formed electric double layers at the ionic liquid interfaces yields high charge Separation efficiency within the semiconductor layer, and the minimal potential drop in the bulk ionic liquid allows the electrodes to be offset by distances of over 7 mm. Furthermore) the decrease in operational speed with increased electrode separation is beneficial for a dear modeling of the waveform of the photocurrent signal, free from the influence of measurement circuitry. Despite the use of a molecular semiconductor as the active layer in conjunction with a liquid insulating layer, devices with a stability of several days could be achieved, and the operational stability of such devices was shown to be dependent solely on the solubility of the active layer in the ionic liquid, even under atmospheric conditions. Furthermore) the greatly simplified device construction process, which does not rely on transparent electrode materials or direct electrode deposition, provides a highly reproducible platform for the study of the electronic processes within IL-MISM detectors that is largely free from architectural constraints.

DOI： 10.1021/la504972q

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Microbial opsins with a bound chromophore function as photosensitive ion transporters and have been employed in optogenetics for the optical control of neuronal activity. Molecular engineering has been utilized to create colour variants for the functional augmentation of optogenetics tools, but was limited by the complexity of the protein-chromophore interactions. Here we report the development of blue-shifted colour variants by rational design at atomic resolution, achieved through accurate hybrid molecular simulations, electrophysiology and X-ray crystallography. The molecular simulation models and the crystal structure reveal the precisely designed conformational changes of the chromophore induced by combinatory mutations that shrink its p-conjugated system which, together with electrostatic tuning, produce large blue shifts of the absorption spectra by maximally 100 nm, while maintaining photosensitive ion transport activities. The design principle we elaborate is applicable to other microbial opsins, and clarifies the underlying molecular mechanism of the blue-shifted action spectra of microbial opsins recently isolated from natural sources.

DOI： 10.1038/ncomms8177

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

There are two types of membrane-embedded ion transport machineries in nature. The ion pumps generate electrochemical potential by energy-coupled active ion transportation, while the ion channels produce action potential by stimulus-dependent passive ion transportation. About 80% of the amino acid residues of the light-driven proton pump archaerhodopsin-3 (AR3) and the light-gated cation channel channelrhodopsin (ChR) differ although they share the close similarity in architecture. Therefore, the question arises: How can these proteins function differently? The absorption maxima of ion pumps are red-shifted about 30100 nm compared with ChRs, implying a structural difference in the retinal binding cavity. To modify the cavity, a blue-shifted AR3 named AR3-T was produced by replacing three residues located around the retinal (i.e., M128A, G132V, and A225T). AR3-T showed an inward H+ flux across the membrane, raising the possibility that it works as an inward H+ pump or an H+ channel. Electrophysiological experiments showed that the reverse membrane potential was nearly zero, indicating light-gated ion channeling activity of AR3-T. Spectroscopic characterization of AR3-T revealed similar photochemical properties to some of ChRs, including an all-trans retinal configuration, a strong hydrogen bond between the protonated retinal Schiff base and its counterion, and a slow photocycle. From these results, we concluded that the functional determinant in the H+ transporters is localized at the center of the membrane-spanning domain, but not in the cytoplasmic and extracellular domains.

DOI： 10.1021/ja511788f

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

A novel platform for transient photodetector component screening has been developed whereby an optical fiber tip serves as the counter electrode when placed in a variety of dielectric media, connected to a photoresponsive working electrode. The soft processing conditions allow for ubiquitous photodetection for organic and biological systems.

DOI： 10.1039/c5cc06237c

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Channelrhodopsins have become a focus of interest because of their ability to control neural activity by light, used in a technology called optogenetics. The channelrhodopsin in the eukaryote Chlamydomonas reinhardtii (CrChR-1) is a light-gated cation channel responsible for motility changes upon photo-illumination and a member of the membrane-embedded retinal protein family. Recent crystal structure analysis revealed that CrChR-1 has unique extended modules both at its N- and C-termini compared to other microbial retinal proteins. This study reports the first successful expression of a ChR-1 variant in Escherichia coli as a holoprotein: the ChR-1 variant lacking both the N- and C-termini (CrChR-1_82-308). However, compared to ChR-1 having the extended modules (CrChR-1_1-357), truncation of the termini greatly altered the absorption maximum and photochemical properties, including the pK(a) values of its charged residues around the chromophore, the reaction rates in the photocycle and the photo-induced ion channeling activity. The results of some experiments regarding ion transport activity suggest that CrChR-1_82-308 has a proton channeling activity even in the dark. On the basis of these results, we discuss the structural and functional roles of the N- and C-terminal extended modules in CrChR-1.

DOI： 10.1039/c5pp00213c

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Assembly is one of the keys to understand biological molecules, and it takes place in spatial and temporal domains upon stimulation. Microbial rhodopsin (also called retinal protein) is a membrane-embedded protein that has a retinal chromophore within seven-transmembrane alpha-helices and shows homo-, di-, tri-, penta-, and hexameric assemblies. Those assemblies are closely related to critical physiological properties such as stabilizing the protein structure and regulating their photoreaction dynamics. Here we investigated the assembly and disassembly of thermophilic rhodopsin (TR), which is a novel proton-pumping rhodopsin derived from a thermophile living at 75 degrees C. TR was characterized using size-exclusion chromatography and circular dichroism spectroscopy, and formed a trimer at 25 degrees C, but irreversibly dissociated into monomers upon thermal stimulation. The transition temperature was estimated to be 68 degrees C. The irreversible nature made it possible to investigate the photochemical properties of both the trimer and the monomer independently. Compared with the trimer, the absorption maximum of the monomer is blue-shifted by 6 nm without any changes in the retinal composition, pKa value for the counterion or the sequence of the proton movement. The photocycling rate of the monomeric TR was similar to that of the trimeric TR. A similar trimer-monomer transition upon thermal stimulation was observed for another eubacterial rhodopsin GR but not for the archaeal rhodopsins AR3 and HwBR, suggesting that the transition is conserved in bacterial rhodopsins. Thus, the thermal stimulation of TR induces the irreversible disassembly of the trimer.

DOI： 10.1021/jp507374q

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

SRI (sensory rhodopsin I) can discriminate multiple colors for the attractant and repellent phototaxis. Studies aimed at revealing the color-dependent mechanism show that SRI is a challenging system not only in photobiology but also in photochemistry. During the photoreaction of SRI, an M-intermediate (attractant) transforms into a P-intermediate (repellent) by absorbing blue light. Consequently, SRI then cycles back to the G-state. The photoreactions were monitored with the C-13 NMR signals of [20-C-13]retnal-SrSRI using in situ photo-irradiation solid-state NMR spectroscopy. The M-intermediate was trapped at -40 degrees C by illumination at 520 nm. It was transformed into the P-intermediate by subsequent illumination at 365 nm. These results reveal that the G-state could be directly transformed to the P-intermediate by illumination at 365 nm. Thus, the stationary trapped M- and P-intermediates are responsible for positive and negative phototaxis, respectively.

DOI： 10.1002/anie.201309258

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Language：English   Publisher：ELSEVIER SCIENCE BV  

Retinal proteins (similar to rhodopsins) are photochemically reactive membrane-embedded proteins, with seven transmembrane alpha-helices which bind the chromophore retinal (vitamin A aldehyde). They are widely distributed through all three biological kingdoms, eukarya, bacteria and archaea, indicating the biological significance of the retinal proteins. Light absorption by the retinal proteins triggers a photoisomerization of the chromophore, leading to the biological function, light-energy conversion or light-signal transduction. This article reviews molecular and evolutionary aspects of the light-signal transduction by microbial sensory receptors and their related proteins. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks. (C) 2013 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbabio.2013.05.005

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Light absorption by the photoreceptor microbial rhodopsin triggers trans cis isomerization of the retinal chromophore surrounded by seven transmembrane alpha-helices. Sensory rhodopsin I (SRI) is a dual functional photosensory rhodopsin both for positive and negative phototaxis in microbes. By making use of the highly stable SRI protein from Salinibacter ruber (SrSRI), the early steps in the photocycle were studied by time-resolved spectroscopic techniques. All of the temporal behaviors of the S-n &lt;- S-1 absorption, ground-state bleaching, K intermediate absorption, and stimulated emission were observed in the femto- to picosecond time region by absorption spectroscopy. The primary process exhibited four dynamics similar to other microbial rhodopsins. The first dynamics (tau(1) similar to 54 fs) corresponds to the population branching process from the Franck-Condon region to the reactive (S-1(r)) and nonreactive (S-1(nr)) S-1 states. The second dynamics (tau(2) = 0.64 ps) is the isomerization process of the Sir state to generate the ground-state 13-cis form, and the third dynamics (tau(3) = 1.8 ps) corresponds to the internal conversion of the S-1(nr) state. The fourth component (tau(3)' = 2.5 ps) is assignable to the J-decay (K-formation). This reaction scheme was further supported by the results of fluorescence spectroscopy. To investigate the protein response(s), the spectral changes of the tryptophan bands were monitored by ultraviolet resonance Raman spectroscopy. The intensity change following the K formation in the chromophore structure (tau similar to 17 ps) was significantly small in SrSRI as compared with other microbial rhodopsins. We also analyzed the effect(s) of Cl- binding on the ultrafast dynamics of SrSRI. Compared with a chloride pump Halorhodopsin, Cl- binding to SrSRI was less effective for the excited-state dynamics, whereas the binding altered the structural changes of tryptophan following the K-formation, which was the characteristic feature for SrSRI. On the basis of these results, a primary photoreaction scheme of SrSRI together with the role of chloride binding is proposed.

DOI： 10.1021/jp4112662

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S180_1

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S233_5

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S235_4

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S234_6

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Language：English   Publishing type：Research paper (scientific journal)  

Background: Rhodopsin is distributed among various organisms. Results: A proton pumping rhodopsin named TR was characterized. Conclusion: TR showed high stability. Significance: TR should be a useful protein for research on retinylidene proteins. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M113.479394

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Language：English   Publishing type：Research paper (scientific journal)  

Ion-transporting rhodopsins are widely utilized as optogenetic tools both for light-induced neural activation and silencing. The most studied representative is Bacteriorhodopsin (BR), which absorbs green/red light (∼570 nm) and functions as a proton pump. Upon photoexcitation, BR induces a hyperpolarization across the membrane, which, if incorporated into a nerve cell, results in its neural silencing. In this study, we show that several residues around the retinal chromophore, which are completely conserved among BR homologs from the archaea, are involved in the spectral tuning in a BR homolog (HwBR) and that the combination mutation causes a large spectral blue shift (λmax = 498 nm) while preserving the robust pumping activity. Quantum mechanics/molecular mechanics calculations revealed that, compared with the wild type, the β-ionone ring of the chromophore in the mutant is rotated ∼130° because of the lack of steric hindrance between the methyl groups of the retinal and the mutated residues, resulting in the breakage of the π conjugation system on the polyene chain of the retinal. By the same mutations, similar spectral blue shifts are also observed in another BR homolog, archearhodopsin-3 (also called Arch). The color variant of archearhodopsin-3 could be successfully expressed in the neural cells of Caenorhabditis elegans, and illumination with blue light (500 nm) led to the effective locomotory paralysis of the worms. Thus, we successfully produced a blue-shifted proton pump for neural silencing. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M113.475533

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society  

Rhodopsin contains retinal as the chromophore within seven transmembrane helices. Recently, we found a unique rhodopsin (middle rhodopsin, MR), which is evolutionarily located between the well-studied bacteriorhodopsin and sensory rhodopsin II, and which accommodates three retinal isomers in its ground state (the all-trans, the 13-cis, and, uniquely, the 11-cis isomers). In this study, we investigated structural changes of both the protein moiety and the retinal chromophore during photocycles of MR by time-resolved Fourier-transform infrared spectroscopy. Three photointermediates with decay time constants of 95 μs, 0.9 ms, and &gt
∼10 ms were identified by the global exponential fitting analysis. The first and third intermediates were attributed to the all-trans photocycle, in accordance with recently published results, whereas the second intermediate was likely one that was spectroscopically silent in the visible region and that was formed between the first and third states or resulted from the activation of the 13-cis isomer. By comparing light-induced difference spectra with various isotope labels in either the retinal or the protein moiety, we concluded that a β-sheet structure in the hydrophilic part was significantly altered during the all-trans photocycle of MR, which may involve an active state of the protein. This feature is characteristic of MR among microbial (type-1) rhodopsins. © 2013 American Chemical Society.

DOI： 10.1021/jp308765t

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Other Link： http://orcid.org/0000-0001-5284-8773

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier B.V.  

Rhodopsins are photoactive molecules functioning as photo-energy or photo-signal converters with the chromophore retinal. Recently we characterized a unique microbial rhodopsin (middle rhodopsin, MR) which can also bind 11-cis retinal besides all-trans and 13-cis retinal at a particular ratio. In this study, we investigated the structural characteristics around the retinal binding cavity in MR. The results suggest that the space of the retinal binding site of MR is less restricted to the retinal chromophore and the presence of the 11-cis conformer is regulated by the residues located around the retinal. Furthermore, although the triple mutant of MR has identical residues with the well-studied microbial rhodopsin bacteriorhodopsin (BR) within 5 Å from the retinal, the absorption maximum and retinal composition of MR did not reach those of BR, indicating that some long-range effect(s) (&gt
5 Å) is also important for the maintenance of the chemical properties of MR. © 2012 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.chemphys.2012.11.020

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Flagellar motors embedded in bacterial membranes are molecular machines powered by specific ion flows. Each motor is composed of a stator and a rotor and the interactions of those components are believed to generate the torque. Na+ influx through the PomA/PomB stator complex of Vibrio alginolyticus is coupled to torque generation and is speculated to trigger structural changes in the cytoplasmic domain of PomA that interacts with a rotor protein in the C-ring, FliG, to drive the rotation. In this study, we tried to overproduce the cytoplasmic loop of PomA (PomA-Loop), but it was insoluble. Thus, we made a fusion protein with a small soluble tag (GB1) which allowed us to express and characterize the recombinant protein. The structure of the PomA-Loop seems to be very elongated or has a loose tertiary structure. When the PomA-Loop protein was produced in E. coli, a slight dominant effect was observed on motility. We conclude that the cytoplasmic loop alone retains a certain function. © 2013 THE BIOPHYSICAL SOCIETY OF JAPAN.

DOI： 10.2142/biophysics.9.21

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.S199_6

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DOI： 10.2142/biophys.53.S146_3

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.S146_6

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.S176_6

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DOI： 10.2142/biophys.53.S146_2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：44  

In nature, organisms are subjected to a variety of environmental stimuli to which they respond and adapt. They can show avoidance or attractive behaviors away from or toward such stimuli in order to survive in the various environments in which they live. One such stimuli is light, to which, for example, the receptor sensory rhodopsin I (SRI) has been found to respond by regulating both negative and positive phototaxis in, e.g., the archaeon Halobacterium salinarum. Interestingly, to date, all organisms having SRI-like proteins live in highly halophilic environments, suggesting that salt significantly influences the properties of SRIs. Taking advantage of the discovery of the highly stable SRI homologue from Salinibacter ruber (SrSRI), which maintains its color even in the absence of salt, the importance of the chloride ion for the color tuning and for the slow M-decay, which is thought to be essential for the phototaxis function of SRIs, has been reported previously [Suzuki, D., et al. (2009) J. Mol. Biol.392, 48-62]. Here the effects of the anion binding on the structure and structural changes of SRI during its photocycle are investigated by means of Fourier transform infrared (FTIR) spectroscopy and electrochemical experiments. Our results reveal that, among other things, the structural change and proton movement of a characteristic amino acid residue, Asp102 in SrSRI, is suppressed by the binding of an anion in its vicinity, both in the K- and M-intermediate. The presence of this anion also effects the extent of chromophore distrotion, and tentative results indicate an influence on the number and/or properties of internal water molecules. In addition, a photoinduced proton transfer could only be observed in the absence of the bound anion. Possible proton movement pathways, including the residues Asp102 and the putative Cl binding site His131, are discussed. In conclusion, the results show that the anion binding to SRI is not only important for the color tuning, and for controlling the photocycle kinetics, but also induces some structural changes which facilitate the observed properties. © 2012 American Chemical Society.

DOI： 10.1021/bi3009592

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Rhodopsin molecules are photochemically reactive membrane-embedded proteins, with seven transmembrane α-helices, which bind the chromophore retinal (vitamin A aldehyde). They are roughly divided into two groups according to their basic functions: (i) ion transporters such as proton pumps, chloride pumps, and cation channels
and (ii) photo-sensors such as sensory rhodopsin from microbes and visual pigments from animals. Anabaena sensory rhodopsin (ASR), found in 2003 in the cyanobacterium Anabaena PCC7120, is categorized as a microbial sensory rhodopsin. To investigate the function of ASR in vivo, ASR and the promoter sequence of the pigment protein phycocyanin were co-introduced into Escherichia coli cells with the reporter gene crp. The result clearly showed that ASR functions as a repressor of the CRP protein expression and that this is fully inhibited by the light activation of ASR, suggesting that ASR would directly regulate the transcription of crp. The repression is also clearly inhibited by the truncation of the C-terminal region of ASR, or mutations on the C-terminal Arg residues, indicating the functional importance of the C-terminal region. Thus, our results demonstrate a novel function of rhodopsin molecules and raise the possibility that the membrane-spanning protein ASR could work as a transcriptional factor. In the future, the ASR activity could be utilized as a tool for arbitrary protein expression in vivo regulated by visible light. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M112.390864

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Background: Optogenetic techniques using light-driven ion channels or ion pumps for controlling excitable cells have greatly facilitated the investigation of nervous systems in vivo. A model organism, C. elegans, with its small transparent body and well-characterized neural circuits, is especially suitable for optogenetic analyses. Methodology/Principal Findings: We describe the application of archaerhodopsin-3 (Arch), a recently reported optical neuronal silencer, to C. elegans. Arch::GFP expressed either in all neurons or body wall muscles of the entire body by means of transgenes were localized, at least partially, to the cell membrane without adverse effects, and caused locomotory paralysis of worms when illuminated by green light (550 nm). Pan-neuronal expression of Arch endowed worms with quick and sustained responsiveness to such light. Worms reliably responded to repeated periods of illumination and non-illumination, and remained paralyzed under continuous illumination for 30 seconds. Worms expressing Arch in different subsets of motor neurons exhibited distinct defects in the locomotory behavior under green light: selective silencing of A-type motor neurons affected backward movement while silencing of B-type motor neurons affected forward movement more severely. Our experiments using a heat-shock-mediated induction system also indicate that Arch becomes fully functional only 12 hours after induction and remains functional for more than 24 hour. Conclusions/Sgnificance: Arch can be used for silencing neurons and muscles, and may be a useful alternative to currently widely used halorhodopsin (NpHR) in optogenetic studies of C. elegans. © 2012 Okazaki et al.

DOI： 10.1371/journal.pone.0035370

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society  

Photoactive proteins with cognate chromophores are widespread in organisms, and function as lightenergy converters or receptors for light-signal transduction. Rhodopsins, which have retinal (vitamin A aldehyde) as their chromophore within their seven transmembrane α-helices, are classified into two groups, microbial (type-1) and animal (type-2) rhodopsins. In general, light absorption by type-1 or type-2 rhodopsins triggers a trans-cis or cis-trans isomerization of the retinal, respectively, initiating their photochemical reactions. Recently, we found a new microbial rhodopsin (middle rhodopsin, MR), binding three types of retinal isomers in its original state: all-trans, 13-cis, and 11-cis. Here, we identified the absolute absorption spectra of MR by a combination of high performance liquid chromatography (HPLC) and UV-vis spectroscopy under varying light conditions. The absorption maxima of MR with all-trans, 13-cis, or 11-cis retinal are located at 485, 479, and 495 nm, respectively. Their photocycles were analyzed by time-resolved laser spectroscopy using various laser wavelengths. In conclusion, we propose that the photocycles of MR are MR(trans) → MRK:lifetime = 93 μs → MRM:lifetime = 12 ms → MR, MR(13-cis) → MR O-like:lifetime = 5.1 ms → MR, and MR(11-cis) → MR K-like:lifetime = 8.2 μs → MR, respectively. Thus, we demonstrate that a single photoactive protein drives three independent photochemical reactions. © 2012 American Chemical Society.

DOI： 10.1021/jp302357m

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.52.S30_5

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Language：Japanese   Publisher：The Pharmaceutical Society of Japan  

Light is one of the most important energy sources and signals providing critical information to biological systems. The photoreceptor rhodopsin, which possesses retinal chromophore (vitamin A aldehyde) surrounded by seven transmembrane alpha-helices, is widely dispersed in prokaryotes and in eukaryotes. Although rhodopsin molecules work as distinctly different photoreceptors, they can be divided according to their two basic functions such as light-energy conversion and light-signal transduction. Thus rhodopsin molecules have great potential for controlling cellular activity by light. Indeed, a light-energy converter channel rhodopsin is used to control neural activity. From 2001, we have been working on various microbial sensory rhodopsins functioning as light-signal converters. In this review, we will introduce rhodopsin molecules from microbes, and will describe artificial and light-dependent protein expression system in Escherichia coli using Anabeana sensory rhodopsin (ASR). The newly developed tools would be widely useful for life scientists.<br>

DOI： 10.1248/yakushi.132.407

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Other Link： https://jlc.jst.go.jp/DN/JALC/10000140750?from=CiNii

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Organisms sense and respond to environmental stimuli through membrane-embedded receptors and transducers. Sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII) are the photoreceptors for the positive and negative phototaxis in microorganisms, respectively. They form signaling complexes in the membrane with their cognate transducer proteins, HtrI and HtrII, and these SRI-HtrI and SRII-HtrII complexes transmit a light signal through their cytoplasmic sensory signaling system, inducing opposite effects (i.e., the inactivation or activation of the kinase CheA). Here we found, by using Fourier transformed infrared spectroscopy, that a conserved residue, Asp102 in Salinibacter SRI (SrSRI), which is located close to the β-ionone ring of the retinal chromophore, is deprotonated upon formation of the active M-intermediate. Furthermore, the D102E mutant of SrSRI affects the structure and/or structural changes of Cys130. This mutant shows a large spectral shift and is comparably unstable, especially in the absence of Cl-. These phenomena have not been observed in the wild-type, or the N105Q and N105D mutants of Natronomonas pharaonis SRII (NpSRII), indicating differences in the structure and structural changes between SrSRI and NpSRII around the β-ionone ring. These differences could also be supported by the measurements of the reactivity with the water-soluble reagent azide. On the basis of these results, we discuss the structure and structural changes around the retinal chromophore in SrSRI. © 2011 American Chemical Society.

DOI： 10.1021/bi200284s

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Sensory rhodopsin II (SRII) is a negative phototaxis receptor containing retinal as its chromophore, which mediates the avoidance of blue light. The signal transduction is initiated by the photoisomerization of the retinal chromophore, resulting in conformational changes of the protein which are transmitted to a transducer protein. To gain insight into the SRII sensing mechanism, we employed time-resolved ultraviolet resonance Raman spectroscopy monitoring changes in the protein structure in the picosecond time range following photoisomerization. We used a 450 nm pump pulse to initiate the SRII photocycle and two kinds of probe pulses with wavelengths of 225 and 238 nm to detect spectral changes in the tryptophan and tyrosine bands, respectively. The observed spectral changes of the Raman bands are most likely due to tryptophan and tyrosine residues located in the vicinity of the retinal chromophore, i.e., Trp76, Trp171, Tyr51, or Tyr174. The 225 nm UVRR spectra exhibited bleaching of the intensity for all the tryptophan bands within the instrumental response time, followed by a partial recovery with a time constant of 30 ps and no further changes up to 1 ns. In the 238 nm UVRR spectra, a fast recovering component was observed in addition to the 30 ps time constant component. A comparison between the spectra of the WT and Y174F mutant of SRII indicates that Tyr174 changes its structure and/or environment upon chromophore photoisomerization. These data represent the first real-time observation of the structural change of Tyr174, of which functional importance was pointed out previously. © 2011 American Chemical Society.

DOI： 10.1021/bi101817y

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Organisms utilize light as energy sources and as signals. Rhodopsins, which have seven transmembrane α-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 λmax (SRI, ∼560 nm
SRII, ∼500 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 (&gt
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 λmax 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. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M110.187948

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Salinibacter sensory rhodopsin I (SrSRI) is a microbial rhodopsin discovered from the eubacterium Salinibacter ruber. It is thought to be a photoreceptor engaging the signal transductions for both positive and negative phototaxis. To elucidate the photoreactions of SrSRI in the presence and absence of chloride ions, we measured the refractive index change after the photoexcitation by the transient grating method. As a result, two spectrally silent processes were identified after the formation of M intermediate, and we named the spectrally identical intermediates M-1, M-2, and M-3. The enthalpy changes (Delta H) were estimated as Delta H = 136, 99, and 63 kJ/mol for K, M-1, and M-2 intermediates, respectively. The Delta H values were significantly decreased (36-55 kJ/mol) by the removal of chloride ions, suggesting their importance for structural changes of SrSRI. Volume expansions of SrSRI were observed on the spectrally silent steps (44 and 11 mL/mol). They may be related to the signaling process because blue-shifted intermediates of sensory rhodopsins are thought to be active state(s) for phototaxis.

DOI： 10.1021/jp2000706

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Rhodopsins possess retinal chromophore surrounded by seven transmembrane alpha-helices, are widespread in prokaryotes and in eukaryotes, and can be utilized as optogenetic tools. Although rhodopsins work as distinctly different photo-receptors in various organisms, they can be roughly divided according to their two basic functions, light-energy conversion and light-signal transduction. In microbes, light-driven proton transporters functioning as light-energy converters have been modified by evolution to produce sensory receptors that relay signals to transducer proteins to control motility. In this study, we cloned and characterized two newly identified microbial rhodopsins from Haloquadratum walsbyi. One of them has photochemical properties and a proton pumping activity similar to the well known proton pump bacteriorhodopsin (BR). The other, named middle rhodopsin (MR), is evolutionarily transitional between BR and the phototactic sensory rhodopsin II (SRII), having an SRII-like absorption maximum, a BR-like photocycle, and a unique retinal composition. The wild-type MR does not have a light-induced proton pumping activity. On the other hand, a mutant MR with two key hydrogen-bonding residues located at the interaction surface with the transducer protein HtrII shows robust phototaxis responses similar to SRII, indicating that MR is potentially capable of the signaling. These results demonstrate that color tuning and insertion of the critical threonine residue occurred early in the evolution of sensory rhodopsins. MR may be a missing link in the evolution from type 1 rhodopsins (microorganisms) to type 2 rhodopsins (animals), because it is the first microbial rhodopsin known to have 11-cis-retinal similar to type 2 rhodopsins.

DOI： 10.1074/jbc.M110.190058

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.51.S159_1

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DOI： 10.2142/biophys.51.S102_2

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DOI： 10.2142/biophys.51.S13_6

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DOI： 10.2142/biophys.51.S159_4

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DOI： 10.1016/j.bpj.2011.10.022

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DOI： 10.2142/biophys.50.160

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Sensory rhodopsin I (SRI) functions as a dual receptor regulating both negative and positive phototaxis. It transmits light signals through changes in protein-protein interactions with its transducer protein, HtrI. The phototaxis function of Halobacterium salinarum SRI (HsSRI) has been well characterized using genetic and molecular techniques, whereas that of Salinibacter ruber SRI (SrSRI) has not. SrSRI has the advantage of high protein stability compared with HsSRI and, therefore, provided new information about structural changes and Cl- binding of SRI. However, nothing is known about the functional role of SrSRI in phototaxis behavior. In this study, we expressed a SRI homologue from the archaeon Haloarcula vallismortis (HvSRI) as a recombinant protein which uses all-trans-retinal as a chromophore. Functionally important residues of HsSRI are completely conserved in HvSRI (unlike in SrSRI), and HvSRI is extremely stable in buffers without Cl-. Taking advantage of the high stability, we characterized the photochemical properties of HvSRI under acidic and basic conditions and observed the effects of Cl- on the protein under both conditions. Fourier transform infrared results revealed that the structural changes in HvSRI were quite similar to those in HsSRI and SrSRI. Thus, HvSRI can become a useful protein model for improving our understanding of the molecular mechanism of the dual photosensing by SRI. © 2010 American Chemical Society.

DOI： 10.1021/bi901824a

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DOI： 10.1016/j.jmb.2009.10.046

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Language：English   Publisher：Hindawi Publishing Corporation  

Negative phototaxis in Natronomonas pharaonis is initiated by transient interaction changes between photoreceptor and transducer. pharaonis phoborhodopsin (ppR
also called pharaonis sensory rhodopsin II, psR-II) and the cognate transducer protein, pHtrII, form a tight 2:2 complex in the unphotolyzed state, and the interaction is somehow altered during the photocycle of ppR. We have studied the signal transduction mechanism in the ppR/pHtrII system by means of low-temperature Fourier-transform infrared (FTIR) spectroscopy. In the paper, spectral comparison in the absence and presence of pHtrII provided fruitful information in atomic details, where vibrational bands were identified by the use of isotope-labeling and site-directed mutagenesis. From these studies, we established the two pathways of light-signal conversion from the receptor to the transducer
(i) from Lys205 (retinal) of ppR to Asn74 of pHtrII through Thr204 and Tyr199, and (ii) from Lys205 of ppR to the cytoplasmic loop region of pHtrII that links Gly83. Copyright © 2010 Hideki Kandori et al.

DOI： 10.1155/2010/424760

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DOI： 10.2142/biophys.50.S130_6

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DOI： 10.2142/biophys.50.S65_3

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DOI： 10.2142/biophys.50.S67_3

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Bacterial flagellar motors are molecular machines powered by the electrochemical potential gradient of specific ions across the membrane. The PomA-PomB stator complex of Vibrio alginolyticus couples Na+ influx to torque generation in this supramolecular motor, but little is known about how Na+ associates with the PomA-PomB complex in the energy conversion process. Here, by means of attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, we directly observed binding of Na+ to carboxylates in the PomA-PomB complex, including the functionally essential residue Asp24. The Na+ affinity of Asp24 is estimated to be similar to 85 mM, close to the apparent K-m value from the swimming motility of the cells (78 mM). At least two other carboxylates are shown to be capable of interacting with Na+, but with somewhat lower affinities. We conclude that Asp24 and at least two other carboxylates constitute Na+ interaction sites in the PomA-PomB complex. This work reveals features of the Na+ pathway in the PomA-PomB Na+ channel by using vibrational spectroscopy.

DOI： 10.1021/bi901517n

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Sensory rhodopsin I (SRI) exists in the cell membranes of microorganisms such as the archaeon Halobacterium salinarum and is a photosensor responsible for positive and negative phototaxis. SRI forms a signaling complex with its cognate transducer protein, HtrI, in the membrane. That complex transmits light signals to the flagellar motor through changes in protein-protein interactions with the kinase CheA and the adaptor protein CheW, which controls the direction of the rotation of the flagellar motor. Recently, we cloned and characterized Salinibacter sensory rhodopsin I (SrSRI), which is the first SRI-like protein identified in eubacteria [Kitajima-Ihara, T., et al. (2008) J. Biol. Chem. 283, 23533-23541]. Here we cloned and expressed SrSRI with its full-length transducer protein, SrHtrI, as a fusion construct. We succeeded in producing the complex in Escherichia coli as a recombinant protein with high quality having all-trans-retinal as a chromophore for SRI, although the expression level was low (0.10 mg/L of culture). In addition, we report here the photochemical properties of the SrSRI-SrHtrI complex using time-resolved laser flash spectroscopy and other spectroscopic techniques and compare them to SrSRI without SrHtrI. ©2009 American Chemical Society.

DOI： 10.1021/bi901338d

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Microbial organisms utilize light not only as energy sources but also as signals by which rhodopsins (containing retinal as a chromophore) work as photoreceptors. Sensory rhodopsin I (SRI) is a dual photoreceptor that regulates both negative and positive phototaxis in microbial organisms, such as the archaeon Halobacterium salinarum and the eubacterium Salinibacter ruber. These organisms live in highly halophilic environments, suggesting the possibility of the effects of salts on the function of SRI. However, such effects remain unclear because SRI proteins from H. salinarum (HsSRI) are unstable in dilute salt solutions. Recently, we characterized a new SRI protein (SrSRI) that is stable even in the absence of salts, thus allowing us to investigate the effects of salts on the photochemical properties of SRI. In this study, we report that the absorption maximum of SrSRI is shifted from 542 to 556 nm in a Cl--dependent manner with a Km of 307 ± 56 mM, showing that Cl--binding sites exist in SRI. The bathochromic shift was caused not only by NaCl but also by other salts (NaI, NaBr, and NaNO3), implying that I-, Br-, and NO3- can also bind to SrSRI. In addition, the photochemical properties during the photocycle are also affected by chloride ion binding. Mutagenesis studies strongly suggested that a conserved residue, His131, is involved in the Cl--binding site. In light of these results, we discuss the effects of the Cl- binding to SRI and the roles of Cl- binding in its function. © 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2009.06.050

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Torque generation in the Salmonella flagellar motor is coupled to translocation of H+ ions through the protonconducting channel of the Mot protein stator complex. The Mot complex is believed to be anchored to the peptidoglycan (PG) layer by the putative peptidoglycan-binding (PGB) domain of MotB. Proton translocation is activated only when the stator is installed into the motor. We report the crystal structure of a C-terminal periplasmic fragment of MotB (MotBc) that contains the PGB domain and includes the entire periplasmic region essential for motility. Structural and functional analyses indicate that the PGB domains must dimerize in order to form the proton-conducting channel. Drastic conformational changes in the N-terminal portion of MotBc are required both for PG binding and the proton channel activation. © 2009 Blackwell Publishing Ltd.

DOI： 10.1111/j.1365-2958.2009.06802.x

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Flagellar motor proteins, MotA/B and PomA/B, are essential for the motility of Escherichia coli and Vibrio alginolyticus, respectively. Those complexes work as a H+ and a Na+ channel, respectively and play important roles in torque generation as the stators of the flagellar motors. Although Asp32 of MotB and Asp24 of PomB are believed to function as ion binding site(s), the ion flux pathway from the periplasm to the cytoplasm is still unclear. Conserved residues, Ala39 of MotB and Cys31 of PomB, are located on the same sides as Asp32 of MotB and Asp24 of PomB, respectively, in a helical wheel diagram. In this study, a series of mutations were introduced into the Ala39 residue of MotB and the Cys31 residue of PomB. The motility of mutant cells were markedly decreased as the volume of the side chain increased. The loss of function due to the MotB(A39V) and PomB(L28A/C31A) mutations was suppressed by mutations of MotA(M206S) and PomA(L183F), respectively, and the increase in the volume caused by the MotB(A39V) mutation was close to the decrease in the volume caused by the MotA(M206S) mutation. These results demonstrate that Ala39 of MotB and Cys31 of PomB form part of the ion flux pathway and pore with Met206 of MotA and Leu183 of PomA in the MotA/B and PomA/B stator units, respectively. © 2009.

DOI： 10.2142/biophysics.5.45

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DOI： 10.2142/biophys.49.S57_2

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DOI： 10.2142/biophys.49.S94_2

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Sensory rhodopsin I (SRI) is one of the most interesting photosensory receptors in nature because of its ability to mediate opposite signals depending on light color by photochromic one-photon and two-photon reactions. Recently, we characterized SRI from eubacterium Salinibacter ruber (SrSRI). This protein allows more detailed information about the structure and structural changes of SRI during its action to be obtained. In this paper, Fourier transform infrared (FTIR) spectroscopy is applied to SrSRI, and the spectral changes upon formation of the K and M intermediates are compared with those of other archaeal rhodopsins, SRI from Halobacterium salinarum (AsSRI), sensory rhodopsin II (SRII), bacteriorhodopsin (BR), and halorhodopsin (HR). Spectral comparison of the hydrogen out-of-plane (HOOP) vibrations of the retinal chromophore in the K intermediates shows that extended choromophore distortion takes place in SrSRI and HsSRI, as well as in SRII, whereas the distortion is localized in the Schiff base region in BR and HR. It appears that sensor and pump functions are distinguishable from the spectral feature of HOOP modes. The HOOP band at 864 cm-1 in SRII, important for negative phototaxis, is absent in SrSRI, suggesting differences in signal transfer mechanism between SRI and SRII. The strongly hydrogen-bound water molecule, important for proton pumps, is observed at 2172 cm-1 in SrSRI, as well as in BR and SRII. The formation of the M intermediate accompanies the appearance of peaks at 1753 (+) and 1743 (-) cm-1, which can be interpreted as the protonation signal of the counterion (Asp72) and the proton release signal from an unidentified carboxylic acid, respectively. The structure and structural changes of SrSRI are discussed on the basis of the present infrared spectral comparisons with other rhodopsins. © 2008 American Chemical Society.

DOI： 10.1021/bi801358b

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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 (∼40% 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 all-trans 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. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M802990200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biophysical Society  

Pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II) is a seven transmembrane helical retinal protein. ppR forms a signaling complex with pharaonis Halobacterial transducer II (pHtrII) in the membrane that transmits a light signal to the sensory system in the cytoplasm. The M-state during the photocycle of ppR (λmax = 386 nm) is one of the active (signaling) intermediates. However, progress in characterizing the M-state at physiological temperature has been slow because its lifetime is very short (decay half-time ∼1 s). In this study, we identify a highly stable photoproduct that can be trapped at room temperature in buffer solution containing n-octyl-β-D-glucoside, with a decay half-time and an absorption maximum of ∼2 h and 386 nm, respectively. HPLC analysis revealed that this stable photoproduct contains 13-cis-retinal as a chromophore. Previously, we reported that water-soluble hydroxylamine reacts selectively with the M-state, and we found that this stable photoproduct also reacts selectively with that reagent. These results suggest that the physical properties of the stable photoproduct (named the M-like state) are very similar with the M-state during the photocycle. By utilizing the high stability of the M-like state, we analyzed interactions of the M-like state and directly estimated the pKa value of the Schiff base in the M-like state. These results suggest that the dissociation constant of the ppRM-like/pHtrII complex greatly increases (to 5 μM) as the pKa value greatly decreases (from 12 to 1.5). The proton transfer reaction of ppR from the cytoplasmic to the extracellular side is proposed to be caused by this change in pKa. © 2008 by the Biophysical Society.

DOI： 10.1529/biophysj.107.125294

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：WILEY-VCH  

Pharaonis halorhodopsin (pHR) functions as a light-driven inward chloride ion pump in Natoronomonas pharaonis, while pharaonis phoborhodopsin (ppR
also called pharaonis sensory rhodopsin II, pSRII), is a light sensor for negative phototaxis. ppR forms a 2:2 complex with its cognate transducer protein (pHtrII) through intramembranous hydrogen bonds: Tyr199ppR-Asn74 pHtrII and Thr189ppR-Glu43pHtrII, Ser62 pHtrII. It was reported that a pHR mutant (P240T / F250Y), which possesses the hydrogen-bonding sites, impairs its pumping activity upon complexation with pHtrII. In this study, effect of the complexation with pHtrII on the structural changes upon formation of the K, L1 and L 2 intermediates of pHR was investigated by use of Fourier-transform infrared spectroscopy. The vibrational changes of Tyr250pHR and Asn74pHtrII were detected for the L1 and L2 intermediates, supporting that Tyr250pHR forms a hydrogen bond with Asn74pHtrII as similarly to Tyr199ppR. The conformational changes of the retinal chromophore were never affected by complexation with pHtrII, but amide-I vibrations were clearly different in the absence and presence of pHtrII. The molecular environment around Asp156pHR in helix D is also slightly affected. These additional structural changes are probably related to blocking of translocation of a chloride ion from the extracellular to the cytoplasmic side during the photocycle.

DOI： 10.1111/j.1751-1097.2008.00317.x

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Sensory rhodopsin II (SRII, also called pharaonis phoborhodopsin, ppR) is responsible for negative phototaxis in Natronomonas pharaonis. Photoisomerization of the retinal chromophore from all-trans to 13-cis initiates conformational changes in the protein, leading to activation of the cognate transducer protein (HtrII). We previously observed enhancement of the C 14-D stretching vibration of the retinal chromophore at 2244 cm -1 upon formation of the K state and interpreted that a steric constraint occurs at the C14D group in SRIIK. Here, we identify the counterpart of the C14D group as Thr204, because the C14-D stretching signal disappeared in T204A, T204S, and T204C mutants as well as a C14-HOOP (hydrogen out-of-plane) vibration at 864 cm-1. Although the K state of the wild-type bacteriorhodopsin (BR), a light-driven proton pump, possesses neither 2244 nor 864 cm-1 bands, both signals appeared for the K state of a triple mutant of BR that functions as a light sensor (P200T/V210Y/A215T). We found a positive correlation between these vibrational amplitudes of the C14 atom at 77 K and the physiological phototaxis response. These observations strongly suggest that the steric constraint between the C14 group of retinal and Thr204 of the protein is a prerequisite for light-signal transduction by SRII. © 2008 American Chemical Society.

DOI： 10.1021/bi8003507

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Sensory rhodopsin I (SRI) functions in both positive and negative phototaxis in complex with halobacterial transducer protein I (HtrI). Orange light activation of SRI results in deprotonation of the retinylidene chromophore of SRI to produce the S373 photocycle intermediate, the signaling state for positive phototaxis. In this study, we observed pH dependence on structural coupling between the two molecules upon the formation of the S 373 intermediate by means of Fourier transform infrared spectroscopy. At alkaline pH, where Asp76 (one of the counterions of the protonated retinylidene Schiff base) is deprotonated, HtrI-dependent alteration of the light-induced difference spectra is limited to reduction of amide I bands at 1661 (+)/ 1647 (-) cm-1, and perturbation of one of the protonated carboxylic acid bands occurs at 1734 (-) cm-1 (which appears to become ionized only when complexed with HtrI). However, at acidic pH, HtrI-complexed SRI exhibits not only light-induced reduction of the amide I changes but a wider range of spectral alterations including the appearance of several new amide I bands, perturbation of the chromophore-related vibrational modes, and other additional changes characteristic of tyrosine, glutamate, and aspartate residues. Since such pH dependence of structural changes was not observed in the complex of the D76N mutant of SRI, which behaves much like HtrI-complexed SRI in acidic conditions, we conclude that extensive orange light-induced conformational coupling between SRI and HtrI occurs only when Asp76 is neutralized. © 2008 American Chemical Society.

DOI： 10.1021/bi702050c

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pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a receptor for negative phototaxis in Natronomonas pharaonis. The X-ray crystallographic structure of ppR is very similar to those of the ion-pumping rhodopsins, bacteriorhodopsin (BR) and halorhodopsin (hR). However, the decay processes of the photocycle intermediates such as M and O are much slower than those of BR and hR, which is advantageous for the sensor function of ppR. Iwamoto et al. previously found that, in a quadruple mutant (P182S/P183E/V194T/T204C
denoted as SETC) of ppR, the decay of the O intermediate was accelerated by ×100 times (t1/2 ∼6.6 ms vs 690 ms for the wild type of ppR), being almost equal to that of BR (Iwamoto, M., et al. (2005) Biophys. J. 88, 1215-1223). The mutated residues are located on the extracellular surface (Pro182, Pro183, and Val194) and near the Schiff base (Thr204). The present Fourier-transform infrared (FTIR) spectroscopy of SETC revealed that protein structural changes in the K and M states were similar to those of the wild type. In contrast, the ppRo minus ppR infrared difference spectra of SETC are clearly different from those of the wild type in amide-I (1680-1640 cm-1) and S-H stretching (2580-2520 cm -1) vibrations. The 1673 (+) and 1656 (-) cm-1 bands newly appear for SETC in the frequency region typical for the amide-I vibration of the αII- and αI-helices, respectively. The intensities of the 1673 (+) cm-1 band of various mutants were well correlated with their O-decay half-times. Since the αII-helix possesses a considerably distorted structure, the result implies that distortion of the helix is required for fast O-decay. In addition, the characteristic changes in the S-H stretching vibration of Cys204 were different between SETC and T204C, suggesting that structural change near the Schiff base was induced by mutations of the extracellular surface. We conclude that the lifetime of the O intermediate in ppR is regulated by the distorted α-helix and strengthened hydrogen bond of Cys204. © 2008 American Chemical Society.

DOI： 10.1021/bi701885k

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Language：English   Publisher：Trivandrum : Research Trends  

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S118_5

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DOI： 10.2142/biophys.48.S10_3

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DOI： 10.2142/biophys.48.S49_2

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DOI： 10.2142/biophys.48.S63_6

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DOI： 10.2142/biophys.48.S116_6

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Halobacterial pharaonis phoborhodopsin [ppR, also called Natronomonas pharaonis sensory rhodopsin II (NpSRII)] is a phototaxis protein which transmits a light signal to the cytoplasm through its transducer protein (pHtrII). pHtrII, a two-transmembrane protein that interacts with ppR, belongs to the group of methyl-accepting chemotaxis proteins (MCPs). Several mutation studies have indicated that the linker region connecting the transmembrane and methylation regions is necessary for signal transduction. However, the three-dimensional (3D) structure of an MCP linker region has yet to be reported, and hence, details concerning the signal transduction mechanism remain unknown. Here the structure of the pHtrII linker region was investigated biochemically and biophysically. Following limited proteolysis, only one trypsin resistant fragment in the pHtrII linker region was identified. This fragment forms a homodimer with a Kd value of 115 μM. The 3D structure of this fragment was determined by solution NMR, and only one α-helix was found between two HAMP domains of the linker region. This α-helix was significantly stabilized within transmembrane protein pHtrII as revealed by CW-EPR. The presence of Af1503 HAMP domain-like structures in the linker region was supported by CD, NMR, and ELDOR data. The α-helix determined here presumably works as a mechanical joint between two HAMP domains in the linker region to transfer the photoactivated conformational change downstream. © 2007 American Chemical Society.

DOI： 10.1021/bi701837n

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Biochemistry and Molecular Biology Inc. (ASBMB)  

Bacteriorhodopsin (BR) and sensory rhodopsin II (SRII) function as a light-driven proton pump and a receptor for negative phototaxis in haloarchaeal membranes, respectively. SRII transmits light signals through changes in protein-protein interaction with its transducer HtrII. Recently, we converted BR by three mutations into a form capable of transmitting photosignals to HtrII to mediate phototaxis responses. The BR triple mutant (BR-T) provides an opportunity to identify structural changes necessary to activate HtrII by comparing light-induced infrared spectral changes of BR, BR-T, and SRII. The hydrogen out-of-plane (HOOP) vibrations of the BR-T were very similar to those of SRII, indicating that they are distributed more extensively along the retinal chromophore than in BR, as in SRII. On the other hand, the bands of the protein moiety in BR-T are similar to those of BR, indicating that they are not specific to photosensing. The alteration of the O-H stretching vibration of Thr-204 in SRII, which we had previously shown to be essential for signal relay to HtrII, occurs also in BR-T. In addition, 1670(+)/1664(-) cm-1 bands attributable to a distorted α-helix were observed in BR-T in a HtrII-dependent manner, as is seen in SRII. Thus, we identified similarities and dissimilarities of BR-T to BR and SRII. The results suggest signaling function of the structural changes of the HOOP vibrations, the O-H stretching vibration of the Thr-215 residue, and a distorted α-helix for the signal generation. We also succeeded in measurements of L minus initial state spectra of BR-T, which are the first FTIR spectra of L intermediates among sensory rhodopsins. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M701271200

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：2  

An alkali-halophilic archaeum, Natronomonas pharaonis, contains two rhodopsins that are halorhodopsin (phR), a light-driven inward Cl- pump and phoborhodopsin (ppR), the receptor of negative phototaxis functioning by forming a signaling complex with a transducer, pHtrII (Sudo Y. et al., J. Mol. Biol. 357 [2006] 1274). Previously, we reported that the phR double mutant, P240T/F250YphR, can bind with pHtrII. This mutant itself can transport Cl-, while the net transport was stopped upon formation of the complex. The flash-photolysis data were analyzed by a scheme in which phR→P1→P2→P3→P 4→phR. The P3 of the wild-type and the double mutant contained two components, X- and O-intermediates. After the complex formation, however, the P3 of the double mutant lacked the X-intermediate. These observations imply that the X-intermediate (probably the N-intermediate) is the state having Cl- in the cytoplasmic binding site and that the complex undergoes an extracellular Cl- circulation because of the inhibition of formation of the X-intermediate. © 2007 American Society for Photobiology.

DOI： 10.1562/2006-06-09-RA-916

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We have recorded 13C solid state NMR spectra of [3- 13C]Ala-labeled pharaonis phoborhodopsin (ppR) and its mutants, A149S and A149V, complexed with the cognate transducer pharaonis halobacterial transducer II protein (pHtrII) (1-159), to gain insight into a possible role of their cytoplasmic surface structure including the C-terminal α-helix and E-F loop for stabilization of the 2:2 complex, by both cross-polarization magic angle spinning (CP-MAS) and dipolar decoupled (DD)-MAS NMR techniques. We found that 13C CP-MAS NMR spectra of [3-13C]Ala-ppR, A149S and A149V complexed with the transducer pHtrII are very similar, reflecting their conformation and dynamics changes caused by mutual interactions through the transmembrane α-helical surfaces. In contrast, their DD-MAS NMR spectral features are quite different between [3-13C]Ala- A149S and A149V in the complexes with pHtrII: 13C DD-MAS NMR spectrum of [3- 13C]Ala-A149S complex is rather similar to that of the uncomplexed form, while the corresponding spectral feature of A149V complex is similar to that of ppR complex in the C-terminal tip region. This is because more flexible surface structure detected by the DD-MAS NMR spectra are more directly influenced by the dynamics changes than the CP-MAS NMR. It turned out, therefore, that an altered surface structure of A149S resulted in destabilized complex as viewed from the 13C NMR spectrum of the surface areas, probably because of modified conformation at the corner of the helix E in addition to the change of hydropathy. It is, therefore, concluded that the surface structure of ppR including the C-terminal α-helix and the E-F loops is directly involved in the stabilization of the complex through conformational stability of the helix E. © 2007 American Society for Photobiology.

DOI： 10.1562/2006-06-20-RA-940

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S262_2

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DOI： 10.2142/biophys.47.S261_2

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DOI： 10.2142/biophys.47.S161_4

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DOI： 10.2142/biophys.47.S16_4

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DOI： 10.2142/biophys.47.S261_4

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DOI： 10.2142/biophys.47.S262_1

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DOI： 10.2142/biophys.47.S160_3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Biochemistry and Molecular Biology Inc. (ASBMB)  

Sensory rhodopsin II (SRII), a receptor for negative phototaxis in haloarchaea, transmits light signals through changes in protein-protein interaction with its transducer HtrII. Light-induced structural changes throughout the SRII-HtrII interface, which spans the periplasmic region, membrane-embedded domains, and cytoplasmic domains near the membrane, have been identified by several studies. Here we demonstrate by site-specific mutagenesis and analysis of phototaxis behavior that two residues in SRII near the membrane-embedded interface (Tyr174 on helix F and Thr204 on helix G) are essential for signaling by the SRII-HtrII complex. These residues, which are the first in SRII shown to be required for phototaxis function, provide biological significance to the previous observation that the hydrogen bond between them is strengthened upon the formation of the earliest SRII photointermediate (SRIIK) only when SRII is complexed with HtrII. Here we report frequency changes of the S-H stretch of a cysteine substituted for SRII Thr204 in the signaling state intermediates of the SRII photocycle, as well as an influence of HtrII on the hydrogen bond strength, supporting a direct role of the hydrogen bond in SRII-HtrII signal relay chemistry. Our results suggest that the light signal is transmitted to HtrII from the energized interhelical hydrogen bond between Thr204 and Tyr174, which is located at both the retinal chromophore pocket and in helices F and G that form the membrane-embedded interaction surface to the signal-bearing second transmembrane helix of HtrII. The results argue for a critical process in signal relay occurring at this membrane interfacial region of the complex. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M605907200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

pharaonis phoborhodopsin (ppR
also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor protein for negative phototaxis in Natronomonas pharaonis. Photoisomerization of the retinal chromophore from all-trans to 13-cis initiates conformational changes of the protein leading to activation of the cognate transducer protein (pHtrII). Elucidation of the initial photoreaction, formation of the K intermediate of ppR, is important for understanding the mechanism of storage of photon energy. We have reported the K minus ppR Fourier transform infrared (FTIR) spectra, including several vibrational bands of the retinal, the protein, and internal water molecules. It is interesting that more vibrational bands were observed in the hydrogen-out-of-plane (HOOP) region than for the light-driven proton pump, bacteriorhodopsin. This result implied that the steric constraints on the retinal chromophore in the binding pocket of ppR are distributed more widely upon formation of the initial intermediate. In this study, we assigned the HOOP and hydrogen-in-plane vibrations by means of low-temperature FTIR spectroscopy applied to ppR reconstituted with retinal deuterated at C7, C8, C10-C12, C14, and C15. As a result, the 966 (+)/ 971 (-) and 958 (+)/961 (-) cm-1 bands were assigned to the C7=C8 and C11=C12 Au HOOP modes, respectively, suggesting that the structural changes spread to the middle part of the retinal. The positive bands at 1001, 994, 987, and 979 cm-1 were assigned to the C15-HOOP vibrations of the K intermediate, whose frequencies are similar to those of the KL intermediate of bacteriorhodopsin trapped at 135 K. Another positive band at 864 cm-1 was assigned to the C14-HOOP vibration. Relatively many positive bands of hydrogen-in-plane vibrations supported the wide distribution of structural changes of the retinal as well. These results imply that the light energy was stored mainly in the distortions around the Schiff base region while some part of the energy was transferred to the distal part of the retinal. © 2006 American Chemical Society.

DOI： 10.1021/bi0610597

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

Pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a receptor for negative phototaxis in Natronomonas pharaonis. In membranes, it forms a 2:2 complex with its transducer protein pHtrII, and the association is weakened by 2 orders of magnitude in the M intermediate (ppR M). Such a change is believed to correspond to the transfer of the light signal to pHtrII. A previous Fourier transform infrared (FTIR) study observed hydrogen-bonding alteration of Asn74 in pHtrII in the M state, suggesting a light-signaling pathway from the receptor to the transducer [Furutani, Y., Kamada, K., Sudo, Y., Shimono, K., Kamo, N., and Kandori, H. (2005) Biochemistry 44, 2909-2915]. In this paper, we measure temperature dependence of the ppRM minus ppR spectra in the absence and presence of pHtrII at 250-293 K. Significant temperature dependence was observed for the amide-I vibrations of helices only for the ppR/pHtrII complex, where the amplitude of amide-I vibrations was reduced at room temperature. 13C-Labeling of ppR or pHtrII revealed that such spectral changes of helices originate from ppR and not pHtrII. The hydrogen-bonding alteration of Asn74 in pHtrII was temperature-independent, implying that the observed helical structural perturbation in ppR takes place in different region. On the other hand, temperature-dependent structural changes of helices were diminished for the complex of ppR with the G83C and G83F mutants of pHtrII. Gly83 is believed to connect the transmembrane helix and cytosolic linker region in a flexible kink near the membrane surface of pHtrII, and its replacement by Cys or Phe abolishes the photosensory function. The present study provides direct experimental evidence that Gly83 plays an important structural role in the activation processes of the ppR/pHtrII complex. A molecular mechanism of protein structural changes in the ppR/pHtrII complex is discussed on the basis of the present FTIR results. © 2006 American Chemical Society.

DOI： 10.1021/bi060047i

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Four rhodopsins, bacteriorhodopsin (bR), halorhodopsin (hR), sensory rhodopsin (sR) and phoborhodopsin (pR) exist in archaeal membranes. bR and hR work as a light-driven ion pump. sR and pR work as a photo-sensor of phototaxis, and form signaling complexes in membranes with their respective cognate transducer proteins HtrI (with sR) and HtrII (with pR), through which light signals are transmitted to the cytoplasm. What is the determining factor(s) of the specific binding to form the complex? Binding of the wild-type or mutated rhodopsins with HtrII was measured by isothermal titration calorimetric analysis (ITC). bR and hR could not bind with HtrII. On the other hand, sR could bind to HtrII, although the dissociation constant (KD) was about 100 times larger than that of pR. An X-ray crystallographic structure of the pR/HtrII complex revealed formation of two specific hydrogen bonds whose pairs are Tyr199pR/Asn74HtrII and Thr189pR/Glu43 HtrII/Ser62HtrII. To investigate the importance of these hydrogen bonds, the KD value for the binding of various mutants of bR, hR, sR and pR with HtrII was estimated by ITC. The KD value of T189VpR/Y199FpR, double mutant/HtrII complex, was about 100-fold larger than that of the wild-type pR, whose KD value was 0.16 μM. On the other hand, bR and hR double mutants, P200T bR/V210YbR and P240ThR/F250YhR, were able to bind with HtrII. The KD value of these complexes was estimated to be 60.1(±10.7) μM for bR and to be 29.1(±6.1) μM for hR, while the wild-type bR and hR did not bind with HtrII. We concluded that these two specific hydrogen bonds play important roles in the binding between the rhodopsins and transducer protein. © 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2006.01.061

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DOI： 10.1073/pnas.0607467103

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

In visual and archaeal rhodopsins, light energy is stored in the chromophore-protein interaction after retinal photoisomerization. This paper reports a novel method to monitor the steric constraint after retinal isomerization by use of enhanced C-D stretching vibrations. In the difference FTIR spectra between an archaeal light-sensor pharaonis phoborhodopsin (ppR) and the primary K intermediate at 77 K, no peaks were observed in the 2160-2330 cm-1 region for deuterated retinals at position 7, 8, 10, 11, 12, and 15, whereas a strong peak appeared at 2244 cm-1 for the K intermediate of ppR possessing a C14-D-labeled retinal. The 2244-cm-1 band is assigned as the C14-D stretching vibration, and enhanced absorption in the K state probably originates from the local steric constraint at the C14-D position (also possible electrostatic field effects) after the C13=C14 double bond rotation. Copyright © 2005 American Chemical Society.

DOI： 10.1021/ja056203a

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S99_2

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pHtrII, a pharaonis halobacterial transducer protein, possesses two transmembrane helices and forms a signaling complex with pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, NpSRII) within the halobacterial membrane. This complex transmits a light signal to the sensory system located in the cytoplasm. It has been suggested that the linker region connecting the transmembrane region and the methylation region of pHtrII is important for binding to ppR and subsequent photosignal transduction. In this study, we present evidence to suggest that the linker region itself interacts directly with ppR in addition to the interaction in the membrane region. An in vitro pull-down assay revealed that the linker region bound to ppR, and its dissociation constant (KD) was estimated to be approximately 10 μM using isothermal titration calorimetry (ITC). Solution NMR analyses showed that ppR interacted with the linker region of pHtrII (pHtrIIG83-Q149) and resulted in the broadening of many peaks, indicating structural changes within this region. These results suggest that the pHtrII linker region interacts directly with ppR. There was no demonstrable interaction between the C-terminal region of ppR (ppRGly224-His247) and either the linker region (pHtrIIG83-Q149) or the transmembrane region (pHtrII M1-E114) of pHtrII. On the basis of the NMR, CD, and photochemical data, we discuss the structural changes and role of the linker region of pHtrII in relation to photosignal transduction. © 2005 American Chemical Society.

DOI： 10.1021/bi047573z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a receptor for negative phototaxis in Natronobacterium pharaonis. It forms a 2:2 complex with its transducer protein, pHtrII, in membranes, and the association is weakened by 2 orders of magnitude in the M intermediate. Such change is believed to correspond to the transfer of the light signal to pHtrII. In this paper, we applied Fourier transform infrared (FTIR) spectroscopy to the active M intermediate in the absence and presence of pHtrII. The obtained difference FTIR spectra were surprisingly similar, notwithstanding the presence of pHtrII. This result strongly suggests that the transducer activation in the ppR-pHtrII system does not induce secondary structure alterations of the pHtrII itself. On the other hand, we found that the hydrogen bond of the OH group of Thr204 is altered in the primary K intermediate, but restored in the M intermediate. The hydrogen bond of Asn74 in pHtrII is strengthened in M, presumably because of the change in interaction with Tyr199 of ppR. These facts provided a light signaling pathway from Lys205 (retinal) of the receptor to Asn74 of the transducer through Thr204 and Tyr199. Transducer activation is likely to involve a relaxation of Thr204 in the receptor and hydrogen bonding alteration of Asn74 in the transducer, during which the helices of the transducer perform rigid-body motion without changing their secondary structures. © 2005 American Chemical Society.

DOI： 10.1021/bi047893i

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biophysical Society  

Pharaonis phoborhodopsin (ppR), also called pharaonis sensory rhodopsin II, NpSRII, is a photoreceptor of negative phototaxis in Natronomonas (Natronobacterium) pharaonis. The photocycle rate of ppR is slow compared to that of bacteriorhodopsin, despite the similarity in their x-ray structures. The decreased rate of the photocycle of ppR is a result of the longer lifetime of later photo-intermediates such as M- (ppRM) and O-intermediates (ppRO). In this study, mutants were prepared in which mutated residues were located on the extracellular surface (P182, P183, and V194) and near the Schiff base (T204) including single, triple (P182S/P183E/V194T), and quadruple mutants. The decay of ppRO of the triple mutant was accelerated ∼20-times from 690 ms for the wild-type to 36 ms. Additional mutation resulting in a triple mutant at the 204th position such as T204C or T204S further decreased the decay half-time to 6.6 or 8 ms, almost equal to that of bacteriorhodopsin. The decay half-times of the ppRO of mutants (11 species) and those of the wild-type were well-correlated with the pK a value of Asp-75 in the dark for the respective mutants as spectroscopically estimated, although there are some exceptions. The implications of these observations are discussed in detail. © 2005 by the Biophysical Society.

DOI： 10.1529/biophysj.104.045583

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DOI： 10.2142/biophys.45.S98_3

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DOI： 10.2142/biophys.45.S187_4

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DOI： 10.2142/biophys.45.S187_2

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DOI： 10.2142/biophys.45.S187_3

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DOI： 10.2142/biophys.45.S7_4

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pharaonis phoborhodopsin (ppR
also called pharaonis sensory rhodopsin II, NpSRII) is a receptor for negative phototaxis in Natronomonas (Natronobacterium) pharaonis. In membranes, it forms a 2:2 complex with its transducer protein, pHtrII, which transmits light signals into the cytoplasmic space through protein-protein interactions. We previously found that a specific deprotonated carboxyl of ppR or pHtrII strengthens their binding [Sudo, Y., et al. (2002) Biophys. J. 83, 427-432]. In this study we aim to identify this carboxyl group. Since the D75N mutant has only one photointermediate (ppRo-like) whose existence spans the millisecond time range, the analysis of its decay rate is simple. We prepared various D75N mutants such as D75N/D214N, D75N/K157Q/R162Q/R164Q (D75N/3Gln), D75N/D193N, and D75N/D193E, among which only D75N/D193N did not show pH dependence with regard to the ppRo-like decay rate and KD value for binding, implying that the carboxyl group in question is from Asp-193. The pKa of this group decreased to below 2 when a complex was formed. Therefore, we conclude that Asp-193ppR is connected to the distant transducer-ppR binding surface via hydrogen bonds, thereby modulating its pKa. In addition, we discuss the importance of Arg-162 ppR with respect to the binding activity.

DOI： 10.1021/bi048803c

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Pharaonis phoborhodopsin (ppR) is a photosensor of negative phototaxis in Natronomonas (Natronobacterium) pharaonis, an alkalophilic halophile. This protein has seven transmembrane helices into which a chromophore, all-trans retinal, binds to a specific lysine residue (located in helix G) via a protonated Schiff base. Various mutants were engineered to have a single cysteine in the F-helix. In the presence of a bulky fluorescent SH-reagent, MIANS, (2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid, illumination decreased the photoreactivity or flash-yield (absorbance deflection immediately after the flash) of the L163C ppR mutant (in which Leu-163 was replaced with Cys) without changing the photocycling rate. The fluorescence of the isolated protein increased with increasing illumination. These observations suggest that during photocycling, the space around Cys-163 in the F-helix might open, permitting reaction with the relatively large molecule. This reaction occurred only at the M-state and not at the O-state. The implications are discussed. © The Royal Society of Chemistry and Owner Societies 2004.

DOI： 10.1039/b315454h

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pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a photo-receptor for negative phototaxis in Natronobacterium pharaonis. During the photoreaction cycle (photocycle), ppR exhibits intraprotein proton movements, resulting in proton pumping from the cytoplasmic to the extracellular side, although it is weak. In this study, light-induced proton uptake and release of ppR reconstituted with phospholipid were analyzed using a SnO2 electrode. The reconstituted ppR exhibited properties in proton uptake and release that are different from those of dodecyl maltoside solubilized samples. It showed fast proton release before the decay of ppR M (M-photointermediate) followed by proton uptake, which was similar to that of bacteriorhodopsin (BR), a light-driven proton pump. Mutant analysis assigned Asp193 to one (major) of the members of the proton-releasing group (PRG). Fast proton release was observed only when the pH was approximately 5-8 in the presence of Cl-. When Cl- was replaced with SO 42-, the reconstituted ppR did not exhibit fast proton release at any pH, suggesting Cl- binding around PRG. PRG in BR consists of Glu204 (Asp193 in ppR) and Glu194 (Pro183 in ppR). Replacement of Pro183 by Glu/Asp, a negatively charged residue, led to Cl --independent fast proton release. The transducer binding affected the properties of PRG in ppR in the ground state and in the ppRM state, suggesting that interaction with the transducer extends to the extracellular surface of ppR. Differences and similarities in the molecular mechanism of the proton movement between ppR and BR are discussed.

DOI： 10.1021/bi035960n

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Pharaonis phoborhodopsin (ppR, or pharaonis sensory rhodopsin II, NpsRII) is a sensor for the negative phototaxis of Natronomonas (Natronobacterium) pharaonis. Arginine 72 of ppR corresponds to Arg-82 of bacteriorhodopsin, which is a highly conserved residue among microbial rhodopsins. Using various Arg-72 ppR mutants, we obtained the following results: 1), Arg-72ppR together possibly with Asp-193 influenced the pKa of the counterion of the protonated Schiff base. 2), The M-rise became approximately four times faster than the wild-type. 3), Illumination causes proton uptake and release, and the pH profiles of the sequence of these two proton movements were different between R72A mutant and the wild-type
it is inferred that Arg-72 connects the proton transfer events occurring at both the Schiff base and an extracellular proton-releasing residue (Asp-193). 4), The M-decays of Arg-72 mutants were faster (∼8-27 folds at pH 8 depending on mutants) than the wild-type, implying that the guanidinium prevents the proton transfer from the extracellular space to the deprotonated Schiff base. 5), The proton-pumping activities were decreased for mutants having increased M-decay rates, but the extent of the decrease was smaller than expected. The role of Arg-72 of ppR on the photochemistry was discussed.

DOI： 10.1016/S0006-3495(04)74359-3

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We have recorded 13C NMR spectra of the [3-13C]Ala, [1-13C]Val-labeled pharaonis transducer pHtrll(1-159) in the presence and absence of phoborhodopsin (ppR or sensory rhodopsin II) in egg phosphatidylcholine or dimyristoylphosphatidylcholine bilayers by means of site-directed (amino acid specific) solid-state NMR. Two kinds of 13C NMR signals of [3-13C]Ala-pHtrll complexed with ppR were clearly seen with dipolar decoupled magic angle spinning (DD-MAS) NMR. One of these resonances was at the peak position of the low-field α-helical peaks (αII-helix) and is identified with cytoplasmic α-helices protruding from the bilayers
the other was the high-field α-helical peak (αI-helix) and is identified with the transmembrane α-helices. The first peaks, however, were almost completely suppressed by cross-polarization magic angle spinning (CP-MAS) regardless of the presence or absence of ppR or by DD-MAS NMR in the absence of ppR. This is caused by an increased fluctuation frequency of the cytoplasmic α-helix from 105 Hz in the uncomplexed states to &gt
106 Hz in the complexed states, leading to the appearance of peaks that were suppressed because of the interference of the fluctuation frequency with the frequency of proton decoupling (105 Hz), as viewed from the 13C NMR spectra of [3-13C]Ala-labeled pHtrll. Consistent with this view, the 13C DD-MAS NMR signals of the cytoplasmic α-helices of the complexed [3-13C]Ala-pHtrll in the dimyristoylphosphatidylcholine (DMPC) bilayer were partially suppressed at 0°C due to a decreased fluctuation frequency at the low temperature. In contrast, examination of the 13C CP-MAS spectra of [1-13C]Val-labeled complexed pHtrll showed that the 13C NMR signals of the transmembrane α-helix were substantially suppressed. These spectral changes are again interpreted in terms of the increased fluctuation frequency of the transmembrane α-helices from 103 Hz of the uncomplexed states to 10 4 Hz of the complexed states. These findings substantiate the view that the transducers alone are in an aggregated or clustered state but the ppR-pHtrll complex is not aggregated. We show that 13C NMR is a very useful tool for achieving a better understanding of membrane proteins which will serve to clarify the molecular mechanism of signal transduction in this system.

DOI： 10.1016/S0006-3495(04)74361-1

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DOI： 10.2142/biophys.44.S95_1

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DOI： 10.2142/biophys.44.S94_4

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DOI： 10.2142/biophys.44.S59_1

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DOI： 10.2142/biophys.44.S94_2

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DOI： 10.2142/biophys.44.S94_3

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DOI： 10.2142/biophys.44.S94_1

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DOI： 10.2142/biophys.44.S95_2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a receptor for negative phototaxis in Natronobacterium pharaonis. It forms a 2:2 complex with its transducer protein, pHtrII, in membranes and transmits light signals through the change in the protein-protein interaction. We previously found that the ppRK minus ppR spectrum in D20 possesses vibrational bands of ppR at 3479 (-)/3369 (+) cm-1 only in the presence of pHtrII [Furutani, Y., Sudo, Y., Kamo, N., and Kandori, H. (2003) Biochemistry 42, 4837-4842]. A D/H-unexchangeable X-H group appears to form a stronger hydrogen bond upon retinal photoisomerization in the ppR-pHtrII complex. This article aims to identify the group by use of various mutant proteins. According to the crystal structure, Tyr-199 of ppR forms a hydrogen bond with Asn-74 of pHtrII in the complex. Nevertheless, the 3479 (-)/3369 (+) cm-1 bands were preserved in the Y199F mutant, excluding the possibility that the bands are O-H stretches of Tyr-199. On the other hand, Thr-204 and Tyr-174 form a hydrogen bond between the retinal chromophore pocket and the binding surface of the ppR-pHtrII complex. These FTIR measurements revealed that the bands at 3479 (-)/3369 (+) cm-1 disappeared in the T204A mutant, while being shifted to 3498 (-) and 3474 (+) cm-1 in the T204S mutant. They appear at 3430 (-)/3402 (+) cm-1 in the Y174F mutant. From these results, we concluded that the bands at 3479 (-)/3369 (+) cm-1 originate from the O-H stretch of Thr-204. A stronger hydrogen bond as shown by a large spectral downshift (110 cm-1) suggests that the specific hydrogen bonding alteration of Thr-204 takes place upon retinal photoisomerization, which does not occur in the absence of the transducer protein. Thr-204 has been known as an important residue for color tuning and photocycle kinetics in ppR. The results presented here point to an additional important role of Thr-204 in ppR for the interaction with pHtrII. Specific interaction in the complex that involves Thr-204 presumably affects the decay kinetics and binding affinity in the M intermediate.

DOI： 10.1021/bi035678g

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Pharaonis phoborhodopsin (ppR, also called Natronobacterium pharaonis sensory rhodopsin II) and its transducer protein, pharaonis halobacterial transducer of ppR (pHtrII), form a signaling complex, and light signals are transmitted from the sensor to the transducer by the protein-protein interaction. A truncated pHtrII(1-159) consisting of intramembrane helices (expressing amino acid residues from the first to the 159th position) and ppR form the complex in a solution containing 0.1% n-dodecyl-β-D-maltoside. At 75-85°C, the time-dependent color loss of ppR was caused by denaturation. We found that pHtrII(1-159) retarded the denaturation rate of ppR. This increase in the thermal stability was used as a probe for the binding ability in the dark. Tyr199 of ppR and Asn74 of pHtrII(1-114) were proposed as amino acid residues interacting with each other through hydrogen bonding. Then, ppR and pHtrII(1-159) mutants at these positions were prepared to examine the effect on the binding in the dark. The wild-type and Y199F mutant can bind pHtrII(1-159), suggesting that the hydrogen bonding between these specific amino acid residues may not be the only cause of the binding, but the hydrophobic interaction via phenyl ring of ppR may contribute dominantly.

DOI： 10.1562/0031-8655(2003)078<0511:IONPPS>2.0.CO;2

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DOI： 10.2142/biophys.43.S183_3

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Natronobacterium pharaonis phoborhodopsin (ppR
also called N. pharaonis sensory rhodopsin II, NpsRII) is a photophobic sensor in N. pharaonis, and has a shorter absorption maximum (λmax 500 nm) than those of other archaeal retinal proteins (λmax, 560-590 nm) such as bacteriorhodopsin (bR). We constructed chimeric proteins between bR and ppR to investigate the long range interactions effecting the color regulation among archaeal retinal proteins. The λmax of B-DEFG/P-ABC was 545 nm, similar to that of bR expressed in Escherichia coli (λmax, 550 nm). B-DEFG/P-ABC means a chimera composed of helices D, E, F, and G of bR and helices A, B, and C of ppR. This indicates that the major factor(s) determining the difference in λmax between bR and ppR exist in helices DEFG. To specify the more minute regions for the color determination between bR and ppR, we constructed 15 chimeric proteins containing helices D, E, F, and G of bR. According to the absorption spectra of the various chimeric proteins, the interaction between helices D and E as well as the effect of the hydroxyl group around protonated Schiff base on helix G (Thr-204 for ppR and Ala-215 for bR) are the main factors for spectral tuning between bR and ppR.

DOI： 10.1074/jbc.M301200200

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pharaonis phoborhodopsin (ppR
also called pharaonis sensory rhodopsin II, psRII) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. ppR activates the cognate transducer protein, pHtrII, upon absorption of light. ppR and pHtrII form a tight 2:2 complex in the unphotolyzed state, and the interaction is somehow altered during the photocycle of ppR. In this paper, we studied the influence of pHtrII on the structural changes occurring upon retinal photoisomerization in ppR by means of low-temperature FTIR spectroscopy. We trapped the K intermediate at 77 K and compared the ppRK minus ppR spectra in the absence and presence of pHtrII. There are no differences in the X-D stretching vibrations (2700-1900 cm-1) caused by presence of pHtrII. This result indicates that the hydrogen-bonding network in the Schiff base region is not altered by interaction with pHtrII, which is consistent with the same absorption spectrum of ppR with or without pHtrII. In contrast, the ppRK minus ppR infrared difference spectra are clearly influenced by the presence of pHtrII in amide-I (1680-1640 cm-1) and amide-A (3350-3250 cm-1) vibrations. The identical spectra for the complex of the unlabeled ppR and 13C- or 15N-labeled pHtrII indicate that the observed structural changes for the peptide backbone originate from ppR only and are altered by retinal photoisomerization. The changes do not come from pHtrII, implying that the light signal is not transmitted to pHtrII in ppRK. In addition, we observed D2O-insensitive bands at 3479 (-)/3369 (+) cm-1 only in the presence of pHtrII, which presumably originate from an X-H stretch of an amino acid side chain inside the protein.

DOI： 10.1021/bi034317y

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We have recorded 13C nuclear magnetic resonance (NMR) spectra of [3-13C]Ala, [1-13C]Val-labeled pharaonis phoborhodopsin (ppR or sensory rhodopsin II) incorporated into egg PC (phosphatidylcholine) bilayer, by means of site-directed high-resolution solid-state NMR techniques. Seven 13C NMR signals from transmembrane α-helices were resolved for [3-13C]Ala-ppR at almost the same positions as those of bacteriorhodopsin (bR), except for the suppressed peaks in the loop regions in spite of the presence of at least three Ala residues. In contrast, 13C NMR signals from the loops were visible from [1-13C]Val-ppR but their peak positions of the transmembrane α-helices are not always the same between ppR and bR. The motional frequency of the loop regions in ppR was estimated as 105 Hz in view of the suppressed peaks from [3-13C]Ala-ppR due to interference with proton decoupling frequency. We found that conformation and dynamics of ppR were appreciably altered by complex formation with a cognate truncated transducer pHtr II (1-159). In particular, the C-terminal α-helix protruding from the membrane surface is involved in the complex formation and subsequent fluctuation frequency is reduced by one order of magnitude. © 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(03)00065-6

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In bacteriorhodopsin (bR), Arg-82bR has been proven to be a very important residue for functional role of this light-driven proton pump. The arginine residue at this position is a super-conserved residue among archaeal rhodopsins. pharaonis phoborhodopsin (ppR
or called as "pharaonis sensory rhodopsin II") has its absorption maximum at 498 nm and acts as a sensor in the membrane of Natronobacterium pharaonis, mediating the negative phototaxis from the light of wavelength shorter than 520 nm. To investigate the role of the arginine residue (Arg-72ppR) of ppR corresponding to Arg-82bR, mutants whose Arg-72ppR was replaced by alanine (R72A), lysine (R72K), glutamine (R72Q) and serine (R72S) were prepared. These mutants were unstable in low concentrations of NACl and lost their color gradually when the proteins were solubilized with 0.1% n-dodecyl-β-D-maltoside. The order of instability was R72S &gt
R72A &gt
R72K &gt
R72Q &gt
the wild type. The rates of denaturation were reduced in a solution of high concentrations of monovalent anions.

DOI： 10.1562/0031-8655(2003)077<0096:AOPPSR>2.0.CO;2

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DOI： 10.2142/biophys.43.S192_3

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DOI： 10.2142/biophys.43.S193_3

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DOI： 10.2142/biophys.43.S192_2

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DOI： 10.2142/biophys.43.S193_2

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DOI： 10.2142/biophys.43.S192_1

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DOI： 10.2142/biophys.43.S194_1

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pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II [psRII]) is a member of the archaeal rhodopsin family and acts as a repellent phototaxis receptor of Natronobacterium pharaonis. Upon illumination, ppR is excited and undergoes a linear cyclic photoreaction, namely, a photocycle that constitutes photointermediates such as M- and O-intermediates (ppRM and ppRO, respectively). Under a constant background illumination (&gt
600 nm) that irradiates ppRO, the decay rate of the flash-induced ppRO increased with an increase in the background light intensity, indicating the photoreactivity of ppRO. Azide did not influence the light-accelerated ppRO decay, but the time required for the cycle to be completed became shortened in an azide concentration-dependent manner because of acceleration of ppRM decay. Hence, the turnover rate of photocycling increased appreciably in the presence of both the background illumination and the azide. The observation reported previously (Schmies, G. et al. 2000, Biophys. J. 78:967-976) is discussed in connection with the present observations.

DOI： 10.1562/0031-8655(2002)076<0462:IATDOT>2.0.CO;2

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Pharaonis phoborhodopsin (ppR or pharaonis sensory rhodopsin II) is a receptor of the negative phototaxis of Natronobacterium pharaonis and forms a complex with its transducer pHtrII in membranes. Flash-photolyis of a D75N mutant did not yield the M-intermediate, but an O-like intermediate is observed in a ms time range. We examined the interaction between the D75N of ppR and t-Htr (truncated pHtrII). These formed a complex in the presence of 0.1% n-dodecyl-β-maltoside, and the association accelerated the decay of the O of D75N from 15 to 56 s-1. From the decay time constants under varying ratios of D75N and t-Htr, n, the molar ratio of D75N/t-Htr in the complex, and KD, the dissociation constant, were estimated. The value of n was unity and KD was estimated to 146 nM. This KD value can be considered to be the association between the photo-intermediate and t-Htr, which is deduced by the method of estimation. Previously we (Photochem. Photobiol. 74 (2001) 489) reported a KD of 15 μM for the interaction between the wild-type and t-Htr by means of the change in M-decay rates. Therefore, this value should be the KD value for the interaction between M of the wild-type and t-Htr. © 2002 Elsevier Science B. V. All rights reserved.

DOI： 10.1016/S1011-1344(02)00322-6

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pharaonis phoborhodopsin (ppR
also pharaonis sensory rhodopsin II, psRII) is a receptor of the negative phototaxis of Natronobacterium pharaonis. In halobacterial membrane, ppR forms a complex with its transducer pHtrII, and this complex transmits the light signal to the sensory system in the cytoplasm. In the present work, the truncated transducer, t-Htr, was used which interacts with ppR [Sudo et al. (2001) Photochem. Photobiol. 74, 489-494]. Two water-soluble reagents, hydroxylamine and azide, reacted both with the transducer-free ppR and with the complex ppR/t-Htr (the complex between ppR and its truncated transducer). In the dark, the bleaching rates caused by hydroxylamine were not significantly changed between transducer-free ppR and ppR/t-Htr, or that of the free ppR was a little slower. Illumination accelerated the bleach rates, which is consistent with our previous conclusion that the reaction occurs selectively at the M-intermediate, but the rate of the complex was about 7.4-fold slower than that of the transducer-free ppR. Azide accelerated the M-decay, and its reaction rate of ppR/t-Htr was about 4.6-fold slower than free ppR. These findings suggest that the transducer binding decreases the water accessibility around the chromophore at the M-intermediate. Its implication is discussed. © 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0005-2736(01)00423-0

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pharaonis phoborhodopsin (ppR; also pharaonis sensory rhodopsin II, psRII) is a receptor of the negative phototaxis of Natronobacterium pharaonis. In halobacterial membrane, ppR forms a complex with its transducer pHtrII, and this complex transmits the light signal to the sensory system in the cytoplasm. In the present work, the truncated transducer, t-Htr, was used which interacts with ppR [Sudo et al. (2001) Photochem. Photobiol. 74, 489-494]. Two water-soluble reagents, hydroxylamine and azide, reacted both with the transducer-free ppR and with the complex ppR/t-Htr (the complex between ppR and its truncated transducer). In the dark, the bleaching rates caused by hydroxylamine were not significantly changed between transducer-free ppR and ppR/t-Htr, or that of the free ppR was a little slower. Illumination accelerated the bleach rates, which is consistent with our previous conclusion that the reaction occurs selectively at the M-intermediate, but the rate of the complex was about 7.4-fold slower than that of the transducer-free ppR. Azide accelerated the M-decay, and its reaction rate of ppR/t-Htr was about 4.6-fold slower than free ppR. These findings suggest that the transducer binding decreases the water accessibility around the chromophore at the M-intermediate. Its implication is discussed.

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pharaonis Phoborhodopsin (ppR
also pharaonis sensory rhodopsin II, psRII) is a retinal protein in Natronobacterium pharaonis and is a receptor of negative phototaxis. It forms a complex with its transducer, pHtrII, in membranes and transmits light signals by protein-protein interaction. Tyr-199 is conserved completely in phoborhodopsins among a variety of archaea, but it is replaced by Val (for bacteriorhodopsin) and Phe (for sensory rhodopsin I). Previously, we (Sudo, Y., M. Iwamoto, K. Shimono, and N. Kamo, submitted for publication) showed that analysis of flash-photolysis data of a complex between D75N and the truncated pHtrII (t-Htr) give a good estimate of the dissociation constant KD in the dark. To investigate the importance of Tyr-199, KD of double mutants of D75N/Y199F or D75N/Y199V with t-Htr was estimated by flash-photolysis and was ∼10-fold larger than that of D75N, showing the significant contribution of Tyr-199 to binding. The KD of the D75N/t-Htr complex increased with decreasing pH, and the data fitted well with the Henderson-Hasselbach equation with a single pKa of 3.86 ± 0.02. This suggests that certain deprotonated carboxyls at the surface of the transducer (possibly Asp-102, Asp-104, and Asp-106) are needed for the binding.

DOI： 10.1016/S0006-3495(02)75180-1

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Pharaonis phoborhodopsin (ppR
also pharaonis sensory rhodopsin II, psRII) is a receptor of the negative phototaxis of Natronobacterium pharaonis. By spectroscopic titration of D193N and D193E mutants, the PKa of the Schiff base was evaluated. Asp193 corresponds to Glu204 of bacteriorhodopsin (bR). The pKa of the Schiff base (SBH+) of D193N was ∼10.1-10.0 (at XH+) and ∼11.4-11.6 (at X) depending on the protonation state of a certain residue (designated by X) and independent of Cl-, whereas those of the wild type and D193E were &gt
12. The pKa values of XH+ were ∼11.8-11.2 at the state of SB, 10.5 at SBH+ state in the presence of Cl-, and 9.6 at SBH+ without Cl-. These imply the presence of a long-range interaction in the extracellular channel. Asp193 was suggested to be deprotonated in the present dodecylmaltoside (DDM) solubilized wild-type ppR, which is contrary to Glu204 of bR. In the absence of salts, the irreversible denaturation of D193N (but not the wild type and D193E) occurred via a metastable state, into which the addition of Cl- reversed the intact pigment. This suggests that the negative charge at residue 193, which can be substituted by Cl-, is necessary to maintain the proper conformation in the DDM-solubilized ppR.

DOI： 10.1016/S0006-3495(02)75236-3

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.42.S155_2

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.42.S119_1

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.42.S156_1

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.42.S155_3

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.42.S154_4

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.42.S155_1

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Language：English   Publishing type：Research paper (scientific journal)  

Phoborhodopsin (pR or sensory rhodopsin II, sRII) and pharaonis phoborhodopsin (ppR or pharaonis sRII, psRII) have a unique absorption maximum (λmax) compared with three other archaeal rhodopsins: λmax of pR and ppR is approx. 500 nm and of others (e.g. bacteriorhodopsin, bR) is 560-590 nm. To determine the residue contributing to the opsin shift from ppR to bR, we constructed various ppR mutants, in which a single residue was substituted for a residue corresponding to that of bR. The residues mutated were those which differ from that of bR and locate within 5 Å from the conjugated polyene chain of the chromophore or any methyl group of the polyene chain. The shifts of λmax of all mutants were small, however. We constructed a mutant in which all residues which differ from those of bR in the retinal binding site were simultaneously substituted for those of bR, but the shift was only from 499 to 509 nm. Next, we constructed a mutant in which 10 residues located within 5 Å from the polyene as described above were simultaneously substituted. Only 44% of the opsin shift (λmax of 524 nm) from ppR to bR was obtained even when all amino acids around the chromophore were replaced by the same residues as bR. We therefore conclude that the structural factor is more important in accounting for the difference of λmax between ppR and bR rather than amino acid substitutions. The possible structural factors are discussed. © 2001 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0005-2736(01)00394-7

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Language：English   Publisher：MAIK NAUKA/INTERPERIODICA  

Phoborhodopsin (pR or sensory rhodopsin II, sRII) is a photoreceptor of the negative phototaxis of Halobacterium salinarum, and pharaonis phoborhodopsin (ppR or pharaonis sensory rhodopsin II, psRII) is a corresponding protein of Natronobacterium pharaonis. The photocycle of ppR is essentially as follows: ppR(498) --&gt; ppR(K)(similar to540) --&gt; ppR(KL)(512) --&gt; ppR(L)(488) --&gt; ppR(M)(390) --&gt; ppR(O)(560) --&gt; ppR (numbers in parenthesis denote the maximum absorbance). The photocycle is very similar to that of bacteriorhodopsin, but the rate of initial pigment recovery is about two-orders of magnitude slower. By low-temperature spectroscopy, two K-intermediates were found but the L intermediate was not detected. The lack of L indicates extraordinary stability of K at low temperature. ppR(M) is photoactive similar to M of bR. The ground state ppR contains only all-trans retinal whereas ppR(M) and ppR(O) contain 13-cis and all-trans, respectively. ppR has the ability of light-induced proton transport from the inside to the outside. Proton uptake occurs at the formation of ppRO and the release at its decay. ppR associates with its transducer and this complex transmits a signal to the cytoplasm, The proton transport ability is lost when the complex forms, but the proton uptake and release still occur, suggesting that the proton movement is non-electrogenic (release and uptake occur from the same side). The stoichiometry of the complex between ppR and the transducer is 1 : 1. ppR or pR has absorption maximum at similar to500 nm, which is blue-shifted from those of other archaeal rhodopsins. The molecular mechanism of this color regulation is not yet solved.

DOI： 10.1023/A:1013187403599

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Language：English   Publishing type：Research paper (scientific journal)  

Phoborhodopsin (pR
also called sensory rhodopsin II, sRII) is a receptor of negative phototaxis of Halobacterium salinarum, and pharaonis phoborhodopsin (ppR
also pharaonis sensory rhodopsin II, psRII) is a corresponding protein of Natronobacterium pharaonis. These receptors contain retinal as a chromophore which binds to a lysine residue via Schiff base. This Schiff base can be cleaved with hydroxylamine to loose their color (bleaching). In dark, the bleaching rate of ppR was very slow whereas illumination accelerated considerably the bleaching rate. Addition of azide accelerated the decay of the M-intermediate while its formation (decay of the L-intermediate) is not affected. The bleaching rate of ppR under illumination was decreased by addition of azide. Essentially no reactivity with hydroxylamine under illumination was observed in the case of D75N mutant which lacks the M-intermediate in its photocycle. Moreover, we provided illumination by flashes to ppR in the presence of varying concentrations of azide to measure the bleaching rate per one flash. A good correlation was obtained between the rate and the mean residence time, MRT, which was calculated from flash photolysis data of the M-decay. These findings reveal that water-soluble hydroxylamine reacts selectively with the M-intermediate and its implication was discussed. © 2001 Published by Elsevier Science B.V.

DOI： 10.1016/S0005-2736(01)00380-7

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Pharaonis phoborhodopsin (ppR) (also pharaonis sensory rhodopsin II) is a receptor of the negative phototaxis of Natronobacterium pharaonis. ppR forms a complex with its pharaonis halobacterial transducer (pHtrII), and this complex transmits the light signal to the sensory system in the cytoplasm. The expressed C-terminal-His tagged ppR and C-terminal-His tagged truncated pHtrII (t-Htr) in Escherichia coli (His means the 6x histidine tag) form a complex even in the presence of 0.1% of n-dodecyl-β-D-maltoside, and the M-decay of the complex became about twice slower than that of ppR alone. The photocycling rates under varying concentration ratios of ppR to t-Htr in the presence of detergent were measured. The data were analyzed on the following assumptions: (1) the M-decay of both ppR alone and the complex followed a single exponential decay with different time constants
and (2) the M-decay under varying concentration ratios of ppR to t-Htr, therefore, followed a biexponential decay function which combined the decay of the free ppR and that of the complex as photoreactive species. From these analyses we estimated the dissociation constant (15.2 ± 1.8 μM) and the number of binding sites (1.2 ± 0.08).

DOI： 10.1562/0031-8655(2001)074<0489:PPBTIC>2.0.CO;2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Archaeal rhodopsins possess a retinal molecule as their chromophores, and their light energy and light signal conversions are triggered by all-traps to 13-cis isomerization of the retinal chromophore. Relaxation through structural changes of the protein then leads to functional processes, proton pump in bacteriorhodopsin and transducer activation in sensory rhodopsins. In the present paper, low-temperature Fourier transform infrared spectroscopy is applied to phoborhodopsin from Natronobacterium pharaonis (ppR), a photoreceptor for the negative phototaxis of the bacteria, and infrared spectral changes before and after photoisomerization are compared with those of bacteriorhodopsin (BR) at 77 K. Spectral comparison of the C-C stretching vibrations of the retinal chromophore shows that chromophore conformation of the polyene chain is similar between ppR and BR. This fact implies that the unique chromophore-protein interaction in ppR, such as the blue-shifted absorption spectrum with vibrational fine structure, originates from both ends, the beta -ionone ring and the Schiff base regions. In fact, less planer ring structure and stronger hydrogen bond of the Schiff base were suggested for ppR. Similar frequency changes upon photoisomerization are observed for the C=N stretch of the retinal Schiff base and the stretch of the neighboring threonine side chain (Thr79 in ppR and Thr89 in BR), suggesting that photoisomerization in ppR is driven by the motion of the Schiff base like BR. Nevertheless, the structure of the K state after photoisomerization is different between ppR and BR. In BR, chromophore distortion is localized in the Schiff base region, as shown in its hydrogen out-of-plane vibrations. In contrast, more extended structural changes take place in ppR in view of chromophore distortion and protein structural changes. Such structure of the K intermediate of ppR is probably correlated with its high thermal stability. In fact, almost identical infrared spectra are obtained between 77 and 170 K in ppR. Unique chromophore-protein interaction and photoisomerization processes in ppR are discussed on the basis of the present infrared spectral comparison with BR.

DOI： 10.1021/bo0103819

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biophysical Society  

Phoborhodopsin (pR
also sensory rhodopsin II, sRII) is a retinoid protein in Halobacterium salinarum and works as a receptor of negative phototaxis. Pharaonis phoborhodopsin (ppR
also pharaonis sensory rhodopsin II, psRII) is a corresponding protein of Natronobacterium pharaonis. In bacterial membrane, ppR forms a complex with its transducer pHtrII, and this complex transmits the light signal to the sensory system in the cytoplasm. We expressed pHtrII-free ppR or ppR-pHtrII complex in H. salinarum Pho81/wr- cells. Flash-photolysis experiments showed no essential changes between pHtrII-free ppR and the complex. Using SnO2 electrode, which works as a sensitive pH electrode, and envelope membrane vesicles, we showed the photo-induced outward proton transport. This membranous proton transport was also shown using membrane vesicles from Escherichia coli in which ppR was functionally expressed. On the other hand, the proton transport was ceased when ppR formed a complex with pHtrII. Using membrane sheet, it was shown that the complex undergoes first proton uptake and then release during the photocycle, the same as pHtrII-free ppR, although the net proton transport ceases. Taking into consideration that the complex of sRII (pR) and its transducer undergoes extracellular proton circulation (J. Sasaki and J. L. Spudich, 1999, Biophys. J. 77:2145-2152), we inferred that association with pHtrII closes a cytoplasmic channel of ppR, which lead to the extracellular proton circulation.

DOI： 10.1016/S0006-3495(01)76070-5

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.41.S63_2

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.41.S63_3

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.40.S131_2

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Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：Pharmaceutical Society of Japan  

DOI： 10.1248/bpb.b21-00544

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Authorship：Lead author,　Corresponding author   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Publisher：Biophysical Society of Japan  

DOI： 10.2142/biophys.61.049

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.60.047

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Publisher：Springer Science and Business Media {LLC}  

DOI： 10.1007/s12551-017-0335-x

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Language：Japanese  

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Authorship：Lead author   Language：Japanese   Publishing type：Book review, literature introduction, etc.   Publisher：PHARMACEUTICAL SOC JAPAN  

Retinal proteins possess vitamin A aldehyde (retinal) as a chromophore within seven transmembrane a-helices. Visible light absorption of them triggers trans-cis photoisomerization of the retinal chromophore and induces structural changes in the protein moiety, resulting in a variety of biological functions such as vision, ion transportation, and photosensing. Environmental genomics revealed that retinal proteins are widely distributed through all three biological kingdoms, eukarya, bacteria, and archaea, indicating the biological significance of their light energy conversion. In addition to their biological aspect, retinal proteins have become a focus of interest in part because of applications for optogenetics. On the basis of our results and other findings, we highlight the recent progress in structural and functional studies on retinal proteins.

DOI： 10.1248/yakushi.15-00229-3

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DOI： 10.1248/yakushi.15-00229-3

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Language：English   Publisher：Springer Japan  

Retinylidene proteins (also called rhodopsins) are membrane-embedded photoreceptors that contain a vitamin A aldehyde linked to a lysine residue by a Schiff base as their light-sensing chromophore. The chromophore is surrounded by seven-transmembrane α-helices and absorbs light at different wavelengths due to differences in the electronic energy gap between its ground and excited states. The variation in the wavelength of maximal absorption (λmax: 360–620 nm) of rhodopsins arises due to interaction between the apoprotein (opsin) and the retinyl chromophore, the ‘opsin shift’. This chapter reviews the color tuning mechanisms in type-1 microbial and type-2 animal rhodopsins as revealed mainly by our experimental and theoretical studies.

DOI： 10.1007/978-4-431-55516-2_7

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Language：English   Publisher：The Biophysical Society of Japan  

One of the major topics in biophysics and physicobiology is to understand and utilize biological functions using various advanced techniques. Taking advantage of the photoreactivity of the seven-transmembrane rhodopsin protein family has been actively investigated by a variety of methods. Rhodopsins serve as models for membrane-embedded proteins, for photoactive proteins and as a fundamental tool for optogenetics, a new technology to control biological activity with light. In this review, we summarize progress of microbial rhodopsin research from the viewpoint of distribution, diversity and potential.

DOI： 10.2142/biophysico.12.0_121

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DOI： 10.20632/vso.89.2_83

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.55.092

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DOI： 10.1002/9780470015902.a0022838

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Language：Japanese   Publisher：[極限環境生物学会]学会事務局  

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Language：English   Publishing type：Book review, literature introduction, etc.  

Microorganisms show attractant and repellent responses to survive in the various environments in which they live. Those phototaxic (to light) and chemotaxic (to chemicals) responses are regulated by membrane-embedded receptors and transducers. This article reviews the following: (1) the signal relay mechanisms by two photoreceptors, Sensory Rhodopsin I (SRI) and Sensory Rhodopsin II (SRII) and their transducers (HtrI and HtrII) responsible for phototaxis in microorganisms
and (2) the signal relay mechanism of a chemoreceptor/transducer protein, Tar, responsible for chemotaxis in E. coli. Based on results mainly obtained by our group together with other findings, the possible molecular mechanisms for phototaxis and chemotaxis are discussed. © 2010 by the authors.

DOI： 10.3390/s100404010

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