掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/jacs.3c02833

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Light-driven inward proton pump rhodopsin RmXeR was embedded in pH-sensitive liposomes. Substance release from the proteoliposomes was observed following light illumination both in vitro and in cells, indicating the successful production of light-induced disruptive liposomes (LiDL). Thus, LiDL is a photoswitchable carrier utilized for intracellular substance delivery.

DOI： 10.1039/d3cc02056h

PubMed

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

Abstract

Microbial rhodopsins, a family of photoreceptive membrane proteins containing the chromophore retinal, show a variety of light-dependent molecular functions. Channelrhodopsins work as light-gated ion channels and are widely utilized for optogenetics, which is a method for controlling neural activities by light. Since two cation channelrhodopsins were identified from the chlorophyte alga Chlamydomonas reinhardtii, recent advances in genomic research have revealed a wide variety of channelrhodopsins including anion channelrhodopsins (ACRs), describing their highly diversified molecular properties (e.g., spectral sensitivity, kinetics and ion selectivity). Here, we report two channelrhodopsin-like rhodopsins from the Colpodellida alga Vitrella brassicaformis, which are phylogenetically distinct from the known channelrhodopsins. Spectroscopic and electrophysiological analyses indicated that these rhodopsins are green- and blue-sensitive pigments (λ_max =  ~ 550 and ~ 440 nm) that exhibit light-dependent ion channeling activities. Detailed electrophysiological analysis revealed that one of them works as a monovalent anion (Cl⁻, Br⁻ and NO₃⁻) channel and we named it V. brassicaformis anion channelrhodopsin-2, VbACR2. Importantly, the absorption maximum of VbACR2 (~ 440 nm) is blue-shifted among the known ACRs. Thus, we identified the new blue-shifted ACR, which leads to the expansion of the molecular diversity of ACRs.

DOI： 10.1038/s41598-023-34125-8

PubMed

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その他リンク： https://www.nature.com/articles/s41598-023-34125-8

担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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掲載種別：研究論文（学術雑誌）   出版者・発行元：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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その他リンク： https://www.nature.com/articles/s41467-023-36883-5

担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Pharmaceutical Society of Japan  

DOI： 10.1248/cpb.c22-00774

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/acsomega.2c06980

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

PubMed

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/jacs.1c12608

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

PubMed

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担当区分：責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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その他リンク： https://cdn.elifesciences.org/articles/72264/elife-72264-v1.xml

担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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その他リンク： http://www.nature.com/articles/s41598-021-94181-w

担当区分：責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1002/pro.4154

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/pro.4154

担当区分：最終著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Biophysical Society of Japan  

DOI： 10.2142/biophysico.bppb-v18.019

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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その他リンク： https://www.nature.com/articles/s41598-020-77936-9

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bbabio.2020.148349

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.bpj.2020.09.026

PubMed

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

Abstract

It is known that a hyperthermostable protein tolerable at temperatures over 100°C can be designed from a soluble globular protein by introducing mutations. To expand the applicability of this technology to membrane proteins, here we report a further thermo‐stabilization of the thermophilic rhodopsin from Thermus thermophilus JL‐18 as a model membrane protein. Ten single mutations in the extramembrane regions were designed based on a computational prediction of folding free‐energy differences upon mutation. Experimental characterizations using the UV‐visible spectroscopy and the differential scanning calorimetry revealed that four of ten mutations were thermo‐stabilizing: V79K, T114D, A115P, and A116E. The mutation‐structure relationship of the TR constructs was analyzed using molecular dynamics simulations at 300 K and at 1800 K that aimed simulating structures in the native and in the random‐coil states, respectively. The native‐state simulation exhibited an ion‐pair formation of the stabilizing V79K mutant as it was designed, and suggested a mutation‐induced structural change of the most stabilizing T114D mutant. On the other hand, the random‐coil‐state simulation revealed a higher structural fluctuation of the destabilizing mutant S8D when compared to the wild type, suggesting that the higher entropy in the random‐coil state deteriorated the thermal stability. The present thermo‐stabilization design in the extramembrane regions based on the free‐energy calculation and the subsequent evaluation by the molecular dynamics may be useful to improve the production of membrane proteins for structural studies.

DOI： 10.1002/prot.26015

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/prot.26015

掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

Abstract

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 Calothrix 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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その他リンク： https://www.nature.com/articles/s41598-020-73606-y

掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

Abstract

Vibrio vulnificus is a foodborne pathogen causing septicemia with high mortality rate. In this study, we explored how Escherichia coli, one of the commensal bacteria in the human gastrointestinal tract, can interact with V. vulnificus. Our study results show that the amount of biofilm produced by V. vulnificus was reduced in the presence of E. coli ATCC 35218, although the growth of V. vulnificus L‐180 remained unaffected. We also detected an antibiofilm effect of E. coli culture supernatant against V. vulnificus, which could not be reduced even after heat treatment. These findings indicate that E. coli and its culture supernatant may be suitable to prevent biofilm formation by V. vulnificus. By contrast, live cells of V. vulnificus could reduce the amount of preformed E. coli biofilm, but its culture supernatant could not. This suggests that the cell‐associated factors contribute toward reduction in E. coli biofilm. Therefore, we speculate that ingestion of an infectious dose of V. vulnificus might induce dislodging of the commensal bacteria from the intestinal epithelia and thus can colonize to initiate the infection.

DOI： 10.1111/1348-0421.12829

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/1348-0421.12829

担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/acs.jpcb.0c06560

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/acs.jpclett.0c01406

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/acs.jcim.0c00063

PubMed

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/acs.jpcb.9b10700

PubMed

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41598-019-57122-2

PubMed

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Japanese Society of Microbial Ecology  

DOI： 10.1264/jsme2.me20085

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Proceedings of the National Academy of Sciences  

Giant viruses are remarkable for their large genomes, often rivaling those of small bacteria, and for having genes thought exclusive to cellular life. Most isolated to date infect nonmarine protists, leaving their strategies and prevalence in marine environments largely unknown. Using eukaryotic single-cell metagenomics in the Pacific, we discovered a Mimiviridae lineage of giant viruses, which infects choanoflagellates, widespread protistan predators related to metazoans. The ChoanoVirus genomes are the largest yet from pelagic ecosystems, with 442 of 862 predicted proteins lacking known homologs. They are enriched in enzymes for modifying organic compounds, including degradation of chitin, an abundant polysaccharide in oceans, and they encode 3 divergent type-1 rhodopsins (VirR) with distinct evolutionary histories from those that capture sunlight in cellular organisms. One (VirR _DTS ) is similar to the only other putative rhodopsin from a virus (PgV) with a known host (a marine alga). Unlike the algal virus, ChoanoViruses encode the entire pigment biosynthesis pathway and cleavage enzyme for producing the required chromophore, retinal. We demonstrate that the rhodopsin shared by ChoanoViruses and PgV binds retinal and pumps protons. Moreover, our 1.65-Å resolved VirR _DTS crystal structure and mutational analyses exposed differences from previously characterized type-1 rhodopsins, all of which come from cellular organisms. Multiple VirR types are present in metagenomes from across surface oceans, where they are correlated with and nearly as abundant as a canonical marker gene from Mimiviridae . Our findings indicate that light-dependent energy transfer systems are likely common components of giant viruses of photosynthetic and phagotrophic unicellular marine eukaryotes.

DOI： 10.1073/pnas.1907517116

PubMed

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その他リンク： https://pnas.org/doi/pdf/10.1073/pnas.1907517116

掲載種別：研究論文（学術雑誌）   出版者・発行元：Informa UK Limited  

DOI： 10.1080/15421406.2019.1648052

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掲載種別：研究論文（学術雑誌）   出版者・発行元：eLife Sciences Publications, Ltd  

Sleep/wakefulness cycle is regulated by coordinated interactions between sleep- and wakefulness-regulating neural circuitry. However, the detailed mechanism is far from understood. Here, we found that glutamic acid decarboxylase 67-positive GABAergic neurons in the ventral tegmental area (VTA_Gad67+) are a key regulator of non-rapid eye movement (NREM) sleep in mice. VTA_Gad67+ project to multiple brain areas implicated in sleep/wakefulness regulation such as the lateral hypothalamus (LH). Chemogenetic activation of VTA_Gad67+ promoted NREM sleep with higher delta power whereas optogenetic inhibition of these induced prompt arousal from NREM sleep, even under highly somnolescent conditions, but not from REM sleep. VTA_Gad67+ showed the highest activity in NREM sleep and the lowest activity in REM sleep. Moreover, VTA_Gad67+ directly innervated and inhibited wake-promoting orexin/hypocretin neurons by releasing GABA. As such, optogenetic activation of VTA_Gad67+ terminals in the LH promoted NREM sleep. Taken together, we revealed that VTA_Gad67+ play an important role in the regulation of NREM sleep.

DOI： 10.7554/elife.44928

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その他リンク： https://cdn.elifesciences.org/articles/44928/elife-44928-v2.xml

担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/acs.biochem.9b00257

PubMed

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41598-019-44308-x

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/acs.jpcb.8b06795

Scopus

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.bpj.2018.05.030

Scopus

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/acs.jpcb.8b04894

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/acs.jpclett.8b00879

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Japanese Society of Microbial Ecology  

DOI： 10.1264/jsme2.me17197

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Royal Society of Chemistry (RSC)  

<p>On the basis of functional and spectroscopic characterization, we propose a model for the inward proton transport inRmXeR, a newly discovered microbial rhodopsin.</p>

DOI： 10.1039/c7cp05033j

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Biophysical Society of Japan  

DOI： 10.2142/biophysico.15.0_179

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.cplett.2017.04.001

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.cplett.2017.05.055

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/acs.langmuir.7b00463

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/jacs.6b12139

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

Abstract

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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その他リンク： https://www.nature.com/articles/srep44427

担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/acs.jpcb.6b11101

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

Abstract

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 m-chlorophenylhydrazone (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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その他リンク： https://www.nature.com/articles/srep41879

掲載種別：研究論文（学術雑誌）  

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/jacs.6b05002

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Oxford University Press (OUP)  

DOI： 10.1093/jmcb/mjw027

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1074/jbc.m116.719815

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担当区分：筆頭著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

Abstract

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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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

DOI： 10.1039/c5pp00213c

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その他リンク： https://link.springer.com/article/10.1039/c5pp00213c/fulltext.html

掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

Abstract

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 π-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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その他リンク： https://www.nature.com/articles/ncomms8177

掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/la504972q

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/ja511788f

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Royal Society of Chemistry (RSC)  

<p>Using an optical fiber tip as a sampling probe allows for efficient photoresponse screening of any photoactive film.</p>

DOI： 10.1039/c5cc06237c

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/jp507374q

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担当区分：責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

Abstract

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 ¹³C NMR signals of [20‐¹³C]retnal‐SrSRI using in situ photo‐irradiation solid‐state NMR spectroscopy. The M‐intermediate was trapped at −40 °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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担当区分：筆頭著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/jp4112662

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担当区分：筆頭著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1074/jbc.m113.475533

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1074/jbc.m113.479394

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.chemphys.2012.11.020

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/jp308765t

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Biophysical Society of Japan  

DOI： 10.2142/biophysics.9.21

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/bi3009592

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1074/jbc.m112.390864

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Public Library of Science (PLoS)  

DOI： 10.1371/journal.pone.0035370

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/jp302357m

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.bpj.2011.10.022

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/bi200284s

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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担当区分：筆頭著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1074/jbc.m110.187948

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/jp2000706

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担当区分：筆頭著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1074/jbc.m110.190058

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担当区分：筆頭著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Biophysical Society of Japan  

DOI： 10.2142/biophysics.7.51

CiNii Article

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/bi901824a

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/bi901517n

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担当区分：筆頭著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/bi901338d

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.jmb.2009.06.050

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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担当区分：筆頭著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Biophysical Society of Japan  

DOI： 10.2142/biophysics.5.45

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/bi801358b

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1074/jbc.m802990200

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担当区分：筆頭著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1529/biophysj.107.125294

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記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）   出版者・発行元：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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その他リンク： http://orcid.org/0000-0001-5284-8773

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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担当区分：筆頭著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/bi701885k

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）  

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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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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その他リンク： http://orcid.org/0000-0001-5284-8773

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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その他リンク： http://orcid.org/0000-0001-5284-8773

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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その他リンク： http://orcid.org/0000-0001-5284-8773

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）  

DOI： 10.1073/pnas.0607467103

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Biophysical Society  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Biophysical Society  

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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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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その他リンク： http://orcid.org/0000-0001-5284-8773

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society ({ACS})  

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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その他リンク： http://orcid.org/0000-0001-5284-8773

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Biophysical Society  

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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その他リンク： http://orcid.org/0000-0001-5284-8773

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Biophysical Society  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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担当区分：最終著者,　責任著者   掲載種別：記事・総説・解説・論説等（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.xpro.2021.101046

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担当区分：筆頭著者,　責任著者   記述言語：日本語   掲載種別：記事・総説・解説・論説等（学術雑誌）  

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担当区分：筆頭著者,　責任著者   記述言語：日本語   掲載種別：記事・総説・解説・論説等（商業誌、新聞、ウェブメディア）  

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掲載種別：記事・総説・解説・論説等（学術雑誌）   出版者・発行元：Pharmaceutical Society of Japan  

DOI： 10.1248/bpb.b21-00544

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担当区分：筆頭著者,　責任著者   掲載種別：記事・総説・解説・論説等（商業誌、新聞、ウェブメディア）  

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出版者・発行元：Biophysical Society of Japan  

DOI： 10.2142/biophys.61.049

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記述言語：日本語   出版者・発行元：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.60.047

CiNii Article

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担当区分：筆頭著者,　責任著者  

DOI： 10.2142/biophys.60.209

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出版者・発行元：Springer Science and Business Media {LLC}  

DOI： 10.1007/s12551-017-0335-x

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記述言語：日本語  

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担当区分：筆頭著者   記述言語：日本語   掲載種別：書評論文，書評，文献紹介等  

DOI： 10.1248/yakushi.15-00229-3

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DOI： 10.1248/yakushi.15-00229-3

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記述言語：英語   出版者・発行元：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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記述言語：英語   出版者・発行元：一般社団法人 日本生物物理学会  

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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記述言語：日本語   出版者・発行元：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.55.092

CiNii Article

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担当区分：最終著者,　責任著者   掲載種別：書評論文，書評，文献紹介等   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.bbabio.2013.05.005

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DOI： 10.1002/9780470015902.a0022838

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担当区分：筆頭著者,　責任著者  

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担当区分：筆頭著者  

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記述言語：日本語   出版者・発行元：公益社団法人 日本薬学会  

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

CiNii Article

CiNii Books

J-GLOBAL

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その他リンク： https://jlc.jst.go.jp/DN/JALC/10000140750?from=CiNii

記述言語：日本語   出版者・発行元：[極限環境生物学会]学会事務局  

CiNii Article

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記述言語：日本語   出版者・発行元：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.160

CiNii Article

CiNii Books

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記述言語：英語   掲載種別：書評論文，書評，文献紹介等  

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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PubMed

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記述言語：英語   掲載種別：書評論文，書評，文献紹介等   出版者・発行元：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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PubMed

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記述言語：英語   掲載種別：記事・総説・解説・論説等（学術雑誌）   出版者・発行元：Trivandrum : Research Trends  

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PubMed

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記述言語：日本語   出版者・発行元：(株)メディカルドゥ  

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担当区分：最終著者   記述言語：英語   掲載種別：書評論文，書評，文献紹介等   出版者・発行元：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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