Keiichi Kojima | PubFacts

Publications by authors named "Keiichi Kojima"

44 Publications

Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization.

Sci Rep 2021 Jul 20;11(1):14765. Epub 2021 Jul 20.

Division of Pharmaceutical Sciences, Okayama University, Okayama, 700-8530, Japan.

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 Escherichia coli 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 Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pK = 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 OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.
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http://dx.doi.org/10.1038/s41598-021-94181-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8292405PMC
July 2021

An optogenetic assay method for electrogenic transporters using Escherichia coli co-expressing light-driven proton pump.

Protein Sci 2021 Jul 3. Epub 2021 Jul 3.

Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan.

In organisms, nutrients and wastes move across the cellular membrane, in which membrane-embedded transporters facilitate and inhibit the movement. Despite the physiological significances, the currently used assay methods for transporter activities require tedious preparation and analytical processes. In this study, we report the isotope-free and label-free measurement system for the transport activities of electrogenic transporters. In the system, two molecules, a light-driven inward proton pump rhodopsin, xenorhodopsin (XeR), and a representative of an electrogenic transporter, an oxalate transporter (OxlT), were co-expressed in Escherichia coli cells. The light illumination of the cells co-expressing XeR and OxlT showed an increase in the pH of the bulk solution and that the extent of the pH change is significantly enhanced by adding the oxalate, suggesting the light-induced inward proton transport by XeR coupled to the negative electrogenic transport by OxlT. Such a pH increase was dependent on the oxalate concentration, but not on the XeR expression level. Of note, pH increase was not observed for the nonfunctional mutants of OxlT, R272A, and K355Q, supporting the validity of the system. Thus, we successfully developed an optogenetic assay method for electrogenic transporters using E. coli co-expressing light-driven proton pump.
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http://dx.doi.org/10.1002/pro.4154DOI Listing
July 2021

Bacterium Lacking a Known Gene for Retinal Biosynthesis Constructs Functional Rhodopsins.

Microbes Environ 2020 ;35(4)

Atmosphere and Ocean Research Institute (AORI), The University of Tokyo.

Microbial rhodopsins, comprising a protein moiety (rhodopsin apoprotein) bound to the light-absorbing chromophore retinal, function as ion pumps, ion channels, or light sensors. However, recent genomic and metagenomic surveys showed that some rhodopsin-possessing prokaryotes lack the known genes for retinal biosynthesis. Since rhodopsin apoproteins cannot absorb light energy, rhodopsins produced by prokaryotic strains lacking genes for retinal biosynthesis are hypothesized to be non-functional in cells. In the present study, we investigated whether Aurantimicrobium minutum KNC, which is widely distributed in terrestrial environments and lacks any previously identified retinal biosynthesis genes, possesses functional rhodopsin. We initially measured ion transport activity in cultured cells. A light-induced pH change in a cell suspension of rhodopsin-possessing bacteria was detected in the absence of exogenous retinal. Furthermore, spectroscopic analyses of the cell lysate and HPLC-MS/MS analyses revealed that this strain contained an endogenous retinal. These results confirmed that A. minutum KNC possesses functional rhodopsin and, hence, produces retinal via an unknown biosynthetic pathway. These results suggest that rhodopsin-possessing prokaryotes lacking known retinal biosynthesis genes also have functional rhodopsins.
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http://dx.doi.org/10.1264/jsme2.ME20085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734400PMC
May 2021

Lokiarchaeota archaeon schizorhodopsin-2 (LaSzR2) is an inward proton pump displaying a characteristic feature of acid-induced spectral blue-shift.

Sci Rep 2020 11 30;10(1):20857. Epub 2020 Nov 30.

Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, 700-8530, Japan.

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 = 12.3) is stabilised by the deprotonated counterion (presumably Asp184, pK = 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.
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http://dx.doi.org/10.1038/s41598-020-77936-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7704677PMC
November 2020

Mechanism of absorption wavelength shifts in anion channelrhodopsin-1 mutants.

Biochim Biophys Acta Bioenerg 2021 02 26;1862(2):148349. Epub 2020 Nov 26.

Department of Applied Chemistry, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654, Japan; Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan. Electronic address:

Using a quantum mechanical/molecular mechanical approach, we show the mechanisms of how the protein environment of Guillardia theta anion channelrhodopsin-1 (GtACR1) can shift the absorption wavelength. The calculated absorption wavelengths for GtACR1 mutants, M105A, C133A, and C237A are in agreement with experimentally measured wavelengths. Among 192 mutant structures investigated, mutations at Thr101, Cys133, Pro208, and Cys237 are likely to increase the absorption wavelength. In particular, T101A GtACR1 was expressed in HEK293T cells. The measured absorption wavelength is 10 nm higher than that of wild type, consistent with the calculated wavelength. (i) Removal of a polar residue from the Schiff base moiety, (ii) addition of a polar or acidic residue to the β-ionone ring moiety, and (iii) addition of a bulky residue to increase the planarity of the β-ionone and Schiff base moieties are the basis of increasing the absorption wavelength.
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http://dx.doi.org/10.1016/j.bbabio.2020.148349DOI Listing
February 2021

Applicability of Styrene-Maleic Acid Copolymer for Two Microbial Rhodopsins, RxR and HsSRI.

Biophys J 2020 11 29;119(9):1760-1770. Epub 2020 Sep 29.

Dentistry and Pharmaceutical Sciences, Graduate School of Medicine, Okayama University, Okayama, Japan. Electronic address:

The membrane-embedded protein rhodopsin is widely produced in organisms as a photoreceptor showing a variety of light-dependent biological functions. To investigate its molecular features, rhodopsin is often extracted from cellular membrane lipids by a suitable detergent as "micelles." The extracted protein is purified by column chromatography and then is often reconstituted into "liposomes" by removal of the detergent. The styrene-maleic acid ("SMA") copolymer spontaneously forms nanostructures containing lipids without detergent. In this study, we applied SMA to characterize two microbial rhodopsins, a thermally stable rhodopsin, Rubrobacter xylanophilus rhodopsin (RxR), and an unstable one, Halobacterium salinarum sensory rhodopsin I (HsSRI), and evaluated their physicochemical properties in SMA lipid particles compared with rhodopsins in micelles and in liposomes. Those two rhodopsins were produced in Escherichia coli cells and were successfully extracted from the membrane by the addition of SMA (5 w/v %) without losing their visible color. Analysis by dynamic light scattering revealed that RxR in SMA lipid particles (RxR-SMA) formed a discoidal structure with a diameter of 54 nm, which was 10 times smaller than RxR in phosphatidylcholine liposomes. The small particle size of RxR-SMA allowed us to obtain scattering-less visible spectra with a high signal-to-noise ratio similar to RxR in detergent micelles composed of n-dodecyl-β-D-maltoside. High-speed atomic force microscopy revealed that a single particle contained an average of 4.1 trimers of RxR (12.3 monomers). In addition, RxR-SMA showed a fast cyclic photoreaction (k = 13 s) comparable with RxR in phosphatidylcholine liposomes (17 s) but not to RxR in detergent micelles composed of n-dodecyl-β-D-maltoside (0.59 s). By taking advantage of SMA, we determined the dissociation constant (K) of chloride for HsSRI as 34 mM. From these results, we conclude that SMA is a useful molecule forming a membrane-mimicking assembly for microbial rhodopsins having the advantages of detergents and liposomes.
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http://dx.doi.org/10.1016/j.bpj.2020.09.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7677245PMC
November 2020

A unique clade of light-driven proton-pumping rhodopsins evolved in the cyanobacterial lineage.

Sci Rep 2020 10 7;10(1):16752. Epub 2020 Oct 7.

Atmosphere and Ocean Research Institute, The University of Tokyo, Chiba, 277-8564, Japan.

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.
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http://dx.doi.org/10.1038/s41598-020-73606-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541481PMC
October 2020

Comparative Studies of the Fluorescence Properties of Microbial Rhodopsins: Spontaneous Emission Versus Photointermediate Fluorescence.

J Phys Chem B 2020 08 18;124(34):7361-7367. Epub 2020 Aug 18.

Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan.

Rhodopsins are seven-transmembrane photoreceptor proteins that bind to the retinal chromophore and have been utilized as a genetically encoded voltage indicator (GEVI). So far, archaerhodopsin-3 (AR3) has been successfully used as a GEVI, despite its low fluorescence intensity. We performed comparative and quantitative fluorescence analyses of 15 microbial rhodopsins to explore these highly fluorescent molecules and to clarify their fluorescence mechanism. These rhodopsins showed a wide range of fluorescence intensities in mouse hippocampal neurons. Some of them, GR, HwBR, IaNaR, MR, and NpHR, showed fluorescence intensities comparable with or higher than that of AR3, suggesting their potential for GEVIs. The fluorescence intensity in neurons correlated with that of the bright fluorescent photointermediate such as a Q-intermediate ( = 0.75), suggesting that the fluorescence in neurons originates from the fluorescence of the photointermediate. Our findings provide a crucial step for producing next-generation rhodopsin-based GEVIs.
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http://dx.doi.org/10.1021/acs.jpcb.0c06560DOI Listing
August 2020

Green-Sensitive, Long-Lived, Step-Functional Anion Channelrhodopsin-2 Variant as a High-Potential Neural Silencing Tool.

J Phys Chem Lett 2020 Aug 22;11(15):6214-6218. Epub 2020 Jul 22.

Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan.

Anion channelrhodopsin-2 (GtACR2) was identified from the alga as a light-gated anion channel, providing a powerful neural silencing tool for optogenetics. To expand its molecular properties, we produced here GtACR2 variants by strategic mutations on the four residues around the retinal chromophore (i.e., R129, G152, P204, and C233). After the screening with the expression system, we estimated spectral sensitivities and the anion channeling function by using the HEK293 expression system. Among the mutants, triple (R129M/G152S/C233A) and quadruple (R129M/G152S/P204T/C233A) mutants showed the significantly red-shifted absorption maxima (λ = 498 and 514 nm, respectively) and the long-lived channel-conducting states (the half-life times were 3.4 and 5.4 s, respectively). In addition, both mutants can be activated and inactivated by different wavelengths, representing their step-functional ability. We nicknamed the quadruple mutant "GLaS-ACR2" from its green-sensitive, long-lived, step-functional properties. The unique characteristics of GLaS-ACR2 suggest its high potential as a neural silencing tool.
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http://dx.doi.org/10.1021/acs.jpclett.0c01406DOI Listing
August 2020

Methodology for Further Thermostabilization of an Intrinsically Thermostable Membrane Protein Using Amino Acid Mutations with Its Original Function Being Retained.

J Chem Inf Model 2020 03 10;60(3):1709-1716. Epub 2020 Mar 10.

Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan.

We develop a new methodology best suited to the identification of thermostabilizing mutations for an intrinsically stable membrane protein. The recently discovered thermophilic rhodopsin, whose apparent midpoint temperature of thermal denaturation is measured to be ∼91.8 °C, is chosen as a paradigmatic target. In the methodology, we first regard the residues whose side chains are missing in the crystal structure of the wild type (WT) as the "residues with disordered side chains," which make no significant contributions to the stability, unlike the other essential residues. We then undertake mutating each of the residues with disordered side chains to another residue except Ala and Pro, and the resultant mutant structure is constructed by modifying only the local structure around the mutated residue. This construction is based on the postulation that the structure formed by the other essential residues, which is nearly optimized in such a highly stable protein, should not be modified. The stability changes arising from the mutations are then evaluated using our physics-based free-energy function (FEF). We choose the mutations for which the FEF is much lower than for the WT and test them by experiments. We successfully find three mutants that are significantly more stable than the WT. A double mutant whose reaches ∼100 °C is also discovered.
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http://dx.doi.org/10.1021/acs.jcim.0c00063DOI Listing
March 2020

How Does a Microbial Rhodopsin RxR Realize Its Exceptionally High Thermostability with the Proton-Pumping Function Being Retained?

J Phys Chem B 2020 02 3;124(6):990-1000. Epub 2020 Feb 3.

Institute of Advanced Energy , Kyoto University , Gokasho, Uji, Kyoto 611-0011 , Japan.

We often encounter a case where two proteins, whose amino-acid sequences are similar, are quite different with regard to the thermostability. As a striking example, we consider the two seven-transmembrane proteins: recently discovered rhodopsin (RxR) and long-known bacteriorhodopsin from (HsBR). They commonly function as a light-driven proton pump across the membrane. Though their sequence similarity and identity are ∼71 and ∼45%, respectively, RxR is much more thermostable than HsBR. In this study, we solve the three-dimensional structure of RxR using X-ray crystallography and find that the backbone structures of RxR and HsBR are surprisingly similar to each other: The root-mean-square deviation for the two structures calculated using the backbone C atoms of the seven helices is only 0.86 Å, which makes the large stability difference more puzzling. We calculate the thermostability measure and its energetic and entropic components for RxR and HsBR using our recently developed statistical-mechanical theory. The same type of calculation is independently performed for the portions playing essential roles in the proton-pumping function, helices 3 and 7, and their structural properties are related to the probable roles of water molecules in the proton-transporting mechanism. We succeed in elucidating how RxR realizes its exceptionally high stability with the original function being retained. This study provides an important first step toward the establishment of a method correlating microscopic, geometric characteristics of a protein with its thermodynamic properties and enhancing the thermostability through amino-acid mutations without vitiating the original function.
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http://dx.doi.org/10.1021/acs.jpcb.9b10700DOI Listing
February 2020

Vectorial Proton Transport Mechanism of RxR, a Phylogenetically Distinct and Thermally Stable Microbial Rhodopsin.

Sci Rep 2020 01 14;10(1):282. Epub 2020 Jan 14.

Faculty of Pharmaceutical Sciences, Okayama University, Okayama, 700-8530, Japan.

Rubrobacter xylanophilus rhodopsin (RxR) is a phylogenetically distinct and thermally stable seven-transmembrane protein that functions as a light-driven proton (H) pump with the chromophore retinal. To characterize its vectorial proton transport mechanism, mutational and theoretical investigations were performed for carboxylates in the transmembrane region of RxR and the sequential proton transport steps were revealed as follows: (i) a proton of the retinylidene Schiff base (Lys209) is transferred to the counterion Asp74 upon formation of the blue-shifted M-intermediate in collaboration with Asp205, and simultaneously, a respective proton is released from the proton releasing group (Glu187/Glu197) to the extracellular side, (ii) a proton of Asp85 is transferred to the Schiff base during M-decay, (iii) a proton is taken up from the intracellular side to Asp85 during decay of the red-shifted O-intermediate. This ion transport mechanism of RxR provides valuable information to understand other ion transporters since carboxylates are generally essential for their functions.
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http://dx.doi.org/10.1038/s41598-019-57122-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959264PMC
January 2020

The Unlimited Potential of Microbial Rhodopsins as Optical Tools.

Biochemistry 2020 01 16;59(3):218-229. Epub 2019 Dec 16.

Graduate School of Medicine, Dentistry and Pharmaceutical Sciences , Okayama University , Okayama 700-8530 , Japan.

Microbial rhodopsins, a photoactive membrane protein family, serve as fundamental tools for optogenetics, an innovative technology for controlling biological activities with light. Microbial rhodopsins are widely distributed in nature and have a wide variety of biological functions. Regardless of the many different known types of microbial rhodopsins, only a few of them have been used in optogenetics to control neural activity to understand neural networks. The efforts of our group have been aimed at identifying and characterizing novel rhodopsins from nature and also at engineering novel variant rhodopsins by rational design. On the basis of the molecular and functional characteristics of those novel rhodopsins, we have proposed new rhodopsin-based optogenetics tools to control not only neural activities but also "non-neural" activities. In this Perspective, we introduce the achievements and summarize future challenges in creating optogenetics tools using rhodopsins. The implementation of optogenetics deep inside an brain is the well-known challenge for existing rhodopsins. As a perspective to address this challenge, we introduce innovative optical illumination techniques using wavefront shaping that can reinforce the low light sensitivity of the rhodopsins and realize deep-brain optogenetics. The applications of our optogenetics tools could be extended to manipulate non-neural biological activities such as gene expression, apoptosis, energy production, and muscle contraction. We also discuss the potentially unlimited biotechnological applications of microbial rhodopsins in the future such as in photovoltaic devices and in drug delivery systems. We believe that advances in the field will greatly expand the potential uses of microbial rhodopsins as optical tools.
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http://dx.doi.org/10.1021/acs.biochem.9b00768DOI Listing
January 2020

Conformational Differences among Metarhodopsin I, Metarhodopsin II, and Opsin Probed by Wide-Angle X-ray Scattering.

J Phys Chem B 2019 10 22;123(43):9134-9142. Epub 2019 Oct 22.

Research Organization for Science and Technology , Ritsumeikan University , Kusatsu , Shiga 525-8577 , Japan.

Among the photoproducts of vertebrate rhodopsin, only metarhodopsin II (Meta-II) preferentially adopts the active structure in which transmembrane helices are rearranged. Light-induced helical rearrangement of rhodopsin in membrane-embedded form was directly monitored by wide-angle X-ray scattering (WAXS) using nanodiscs. The change in the WAXS curve for the formation of Meta-II was characterized by a peak at 0.2 Å and a valley at 0.6 Å, which were not observed in metarhodopsin I and opsin. However, acid-induced active opsin (Opsin*) showed a 0.2 Å peak, but no 0.6 Å valley. Analyses using the model structures based on the crystal structures of dark state and Meta-II suggest that the outward movement of helix VI occurred in Opsin*. However, the displaced helices III and V in Meta-II resulting from the disruption of cytoplasmic ionic lock were restored in Opsin*, which is likely to destabilize the G-protein-activating structure of opsin.
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http://dx.doi.org/10.1021/acs.jpcb.9b08311DOI Listing
October 2019

A distinct lineage of giant viruses brings a rhodopsin photosystem to unicellular marine predators.

Proc Natl Acad Sci U S A 2019 10 23;116(41):20574-20583. Epub 2019 Sep 23.

Monterey Bay Aquarium Research Institute, Moss Landing, CA 95039;

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 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) 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 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 Our findings indicate that light-dependent energy transfer systems are likely common components of giant viruses of photosynthetic and phagotrophic unicellular marine eukaryotes.
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http://dx.doi.org/10.1073/pnas.1907517116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789865PMC
October 2019

Photochemical Characterization of a New Heliorhodopsin from the Gram-Negative Eubacterium Bellilinea caldifistulae (BcHeR) and Comparison with Heliorhodopsin-48C12.

Biochemistry 2019 07 13;58(26):2934-2943. Epub 2019 Jun 13.

Graduate School of Medicine, Dentistry and Pharmaceutical Sciences , Okayama University , Okayama 700-8530 , Japan.

Many microorganisms express rhodopsins, pigmented membrane proteins capable of absorbing sunlight and harnessing that energy for important biological functions such as ATP synthesis and phototaxis. Microbial rhodopsins that have been discovered to date are categorized as type-1 rhodopsins. Interestingly, researchers have very recently unveiled a new microbial rhodopsin family named the heliorhodopsins, which are phylogenetically distant from type-1 rhodopsins. Among them, only heliorhodopsin-48C12 (HeR-48C12) from a Gram-positive eubacterium has been photochemically characterized [Pushkarev, A., et al. (2018) Nature 558, 595-599]. In this study, we photochemically characterize a purple-colored heliorhodopsin from Gram-negative eubacterium Bellilinea caldifistulae (BcHeR) as a second example and identify which properties are or are not conserved between BcHeR and HeR-48C12. A series of photochemical measurements revealed several conserved properties between them, including a visible absorption spectrum with a maximum at around 550 nm, the lack of ion-transport activity, and the existence of a second-order O-like intermediate during the photocycle that may activate an unidentified biological function. In contrast, as a property that is not conserved, although HeR-48C12 shows the light adaptation state of retinal, BcHeR showed the same retinal configuration under both dark- and light-adapted conditions. These comparisons of photochemical properties between BcHeR and HeR-48C12 are an important first step toward understanding the nature and functional role of heliorhodopsins.
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http://dx.doi.org/10.1021/acs.biochem.9b00257DOI Listing
July 2019

Quantitation of the neural silencing activity of anion channelrhodopsins in Caenorhabditis elegans and their applicability for long-term illumination.

Sci Rep 2019 05 27;9(1):7863. Epub 2019 May 27.

Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, 700-8530, Japan.

Ion pumps and channels are responsible for a wide variety of biological functions. Ion pumps transport only one ion during each stimulus-dependent reaction cycle, whereas ion channels conduct a large number of ions during each cycle. Ion pumping rhodopsins such as archaerhodopsin-3 (Arch) are often utilized as light-dependent neural silencers in animals, but they require a high-density light illumination of around 1 mW/mm. Recently, anion channelrhodopsins -1 and -2 (GtACR1 and GtACR2) were discovered as light-gated anion channels from the cryptophyte algae Guillardia theta. GtACRs are therefore expected to silence neural activity much more efficiently than Arch. In this study, we successfully expressed GtACRs in neurons of the nematode Caenorhabditis elegans (C. elegans) and quantitatively evaluated how potently GtACRs can silence neurons in freely moving C. elegans. The results showed that the light intensity required for GtACRs to cause locomotion paralysis was around 1 µW/mm, which is three orders of magnitude smaller than the light intensity required for Arch. As attractive features, GtACRs are less harmfulness to worms and allow stable neural silencing effects under long-term illumination. Our findings thus demonstrate that GtACRs possess a hypersensitive neural silencing activity in C. elegans and are promising tools for long-term neural silencing.
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http://dx.doi.org/10.1038/s41598-019-44308-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6536681PMC
May 2019

Mutational analysis of the conserved carboxylates of anion channelrhodopsin-2 (ACR2) expressed in and their roles in anion transport.

Biophys Physicobiol 2018 7;15:179-188. Epub 2018 Sep 7.

Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan.

Anion channelrhodopsin-2 (ACR2), a light-gated channel recently identified from the cryptophyte alga , exhibits anion channel activity with exclusive selectivity. In addition to its novel function, ACR2 has become a focus of interest as a powerful tool for optogenetics. Here we combined experimental and computational approaches to investigate the roles of conserved carboxylates on the anion transport activity of ACR2 in membrane. First, we replaced six conserved carboxylates with a neutral residue (i.e. E9Q, E56Q, E64Q, E159Q, E219Q and D230N), and measured anion transport activity using expression system. E159Q and D230N exhibited significantly lower anion transport activity compared with wild-type ACR2 (1/12~1/3.4), which suggests that E159 and D230 play important roles in the anion transport. Second, to explain its molecular aspects, we constructed a homology model of ACR2 based on the crystal structure of a cation channelrhodopsin (ChR). The model structure showed a cavity formed by four transmembrane helices (TM1, TM2, TM3 and TM7) similar to ChRs, as a putative anion conducting pathway. Although E159 is not located in the putative pathway, the model structure showed hydrogen bonds between E159 and R129 with a water molecule. D230 is located in the pathway near the protonated Schiff base (PSB) of the chromophore retinal, which suggests that there is an interaction between D230 and the PSB. Thus, we demonstrated the functional importance and the hypothetical roles of two conserved carboxylates, E159 and D230, in the anion transport activity of ACR2 in membrane.
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http://dx.doi.org/10.2142/biophysico.15.0_179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6194965PMC
September 2018

Pinopsin evolved as the ancestral dim-light visual opsin in vertebrates.

Commun Biol 2018 1;1:156. Epub 2018 Oct 1.

Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, 606-8502, Japan.

Pinopsin is the opsin most closely related to vertebrate visual pigments on the phylogenetic tree. This opsin has been discovered among many vertebrates, except mammals and teleosts, and was thought to exclusively function in their brain for extraocular photoreception. Here, we show the possibility that pinopsin also contributes to scotopic vision in some vertebrate species. Pinopsin is distributed in the retina of non-teleost fishes and frogs, especially in their rod photoreceptor cells, in addition to their brain. Moreover, the retinal chromophore of pinopsin exhibits a thermal isomerization rate considerably lower than those of cone visual pigments, but comparable to that of rhodopsin. Therefore, pinopsin can function as a rhodopsin-like visual pigment in the retinas of these lower vertebrates. Since pinopsin diversified before the branching of rhodopsin on the phylogenetic tree, two-step adaptation to scotopic vision would have occurred through the independent acquisition of pinopsin and rhodopsin by the vertebrate lineage.
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http://dx.doi.org/10.1038/s42003-018-0164-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6167363PMC
October 2018

High Thermal Stability of Oligomeric Assemblies of Thermophilic Rhodopsin in a Lipid Environment.

J Phys Chem B 2018 07 27;122(27):6945-6953. Epub 2018 Jun 27.

Department of Chemistry, Graduate School of Science , Osaka University , 1-1 Machikaneyama , Toyonaka , Osaka 560-0043 , Japan.

Thermophilic rhodopsin (TR) is a light-driven proton pump from the extreme thermophile Thermus thermophilus JL-18. Previous studies on TR solubilized with detergent showed that the protein exhibits high thermal stability and forms a trimer at room temperature but irreversibly dissociates into monomers when incubated at physiological temperature (75 °C). In the present study, we used resonance Raman (RR) spectroscopy, solid-state NMR spectroscopy, and high-speed atomic force microscopy to analyze the oligomeric structure of TR in a lipid environment. The obtained spectra and microscopic images demonstrate that TR adopts a pentameric form in a lipid environment and that this assembly is stable at the physiological temperature, in contrast to the behavior of the protein in the solubilized state. These results indicate that the thermal stability of the oligomeric assembly of TR is higher in a lipid environment than in detergent micelles. The observed RR spectra also showed that the retinal chromophore is strongly hydrogen bonded to an internal water molecule via a protonated Schiff base, which is characteristic of proton-pumping rhodopsins. The obtained data strongly suggest that TR functions in the pentameric form at physiological temperature in the extreme thermophile T. thermophilus JL-18. We utilized the high thermal stability of the monomeric form of solubilized TR and here report the first RR spectra of the monomeric form of a microbial rhodopsin. The observed RR spectra revealed that the monomerization of TR alters the chromophore structure: there are changes in the bond alternation of the polyene chain and in the hydrogen-bond strength of the protonated Schiff base. The present study revealed the high thermal stability of oligomeric assemblies of TR in the lipid environment and suggested the importance of using TR embedded in lipid membrane for elucidation of its functional mechanism.
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http://dx.doi.org/10.1021/acs.jpcb.8b04894DOI Listing
July 2018

Production of a Light-Gated Proton Channel by Replacing the Retinal Chromophore with Its Synthetic Vinylene Derivative.

J Phys Chem Lett 2018 Jun 16;9(11):2857-2862. Epub 2018 May 16.

Faculty of Pharmaceutical Sciences , Okayama University , Okayama 700-8530 , Japan.

Rhodopsin is widely distributed in organisms as a membrane-embedded photoreceptor protein, consisting of the apoprotein opsin and vitamin-A aldehyde retinal, A1-retinal and A2-retinal being the natural chromophores. Modifications of opsin (e.g., by mutations) have provided insight into the molecular mechanism of the light-induced functions of rhodopsins as well as providing tools in chemical biology to control cellular activity by light. Instead of the apoprotein opsin, in this study, we focused on the retinal chromophore and synthesized three vinylene derivatives of A2-retinal. One of them, C(14)-vinylene A2-retinal (14V-A2), was successfully incorporated into the opsin of a light-driven proton pump archaerhodopsin-3 (AR3). Electrophysiological experiments revealed that the opsin of AR3 (archaeopsin3, AO3) with 14V-A2 functions as a light-gated proton channel. The engineered proton channel showed characteristic photochemical properties, which are significantly different from those of AR3. Thus, we successfully produced a proton channel by replacing the chromophore of AR3.
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http://dx.doi.org/10.1021/acs.jpclett.8b00879DOI Listing
June 2018

Conversion of microbial rhodopsins: insights into functionally essential elements and rational protein engineering.

Biophys Rev 2017 Dec 25;9(6):861-876. Epub 2017 Nov 25.

Faculty of Pharmaceutical Sciences, Okayama University, Okayama, 700-8530, Japan.

Technological progress has enabled the successful application of functional conversion to a variety of biological molecules, such as nucleotides and proteins. Such studies have revealed the functionally essential elements of these engineered molecules, which are difficult to characterize at the level of an individual molecule. The functional conversion of biological molecules has also provided a strategy for their rational and atomistic design. The engineered molecules can be used in studies to improve our understanding of their biological functions and to develop protein-based tools. In this review, we introduce the functional conversion of membrane-embedded photoreceptive retinylidene proteins (also called rhodopsins) and discuss these proteins mainly on the basis of results obtained from our own studies. This information provides insights into the molecular mechanism of light-induced protein functions and their use in optogenetics, a technology which involves the use of light to control biological activities.
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http://dx.doi.org/10.1007/s12551-017-0335-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5711702PMC
December 2017

Spectroscopic characteristics of Rubricoccus marinus xenorhodopsin (RmXeR) and a putative model for its inward H transport mechanism.

Phys Chem Chem Phys 2018 Jan;20(5):3172-3183

Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan.

A new group of microbial rhodopsins named xenorhodopsins (XeR), which are closely related to the cyanobacterial Anabaena sensory rhodopsin, show a light-driven "inward" proton transport activity, as reported for one representative of this group from Parvularcula oceani (PoXeR). In this study, we functionally and spectroscopically characterized a new member of the XeR clade from a marine bacterium Rubricoccus marinus SG-29 (RmXeR). Escherichia coli cells expressing recombinant RmXeR showed a light-induced alkalization of the cell suspension, which was strongly impaired by a protonophore, suggesting that RmXeR is a light-driven "inward" proton pump as is PoXeR. The spectroscopic properties of purified RmXeR were investigated and compared with those of PoXeR and a light-driven "outward" proton pump, bacteriorhodopsin (BR) from the archaeon Halobacterium salinarum. Action spectroscopy revealed that RmXeR with all-trans retinal is responsible for the light-driven inward proton transport activity, but not with 13-cis retinal. From pH titration experiments and mutational analysis, we estimated the pK values for the protonated Schiff base of the retinal chromophore and its counterion as 11.1 ± 0.07 and 2.1 ± 0.07, respectively. Of note, the direction of both the retinal composition change upon light-dark adaptation and the acid-induced spectral shift was opposite that of BR, which is presumably related to the opposite directions of ion transport (from outside to inside for RmXeR and from inside to outside for BR). Flash photolysis experiments revealed the appearances of three intermediates (L, M and O) during the photocycle. The proton uptake and release were coincident with the formation and decay of the M intermediate, respectively. Together with associated findings from other microbial rhodopsins, we propose a putative model for the inward proton transport mechanism of RmXeR.
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http://dx.doi.org/10.1039/c7cp05033jDOI Listing
January 2018

Evolutionary steps involving counterion displacement in a tunicate opsin.

Proc Natl Acad Sci U S A 2017 06 22;114(23):6028-6033. Epub 2017 May 22.

Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan;

Ci-opsin1 is a visible light-sensitive opsin present in the larval ocellus of an ascidian, This invertebrate opsin belongs to the vertebrate visual and nonvisual opsin groups in the opsin phylogenetic tree. Ci-opsin1 contains candidate counterions (glutamic acid residues) at positions 113 and 181; the former is a newly acquired position in the vertebrate visual opsin lineage, whereas the latter is an ancestral position widely conserved among invertebrate opsins. Here, we show that Glu113 and Glu181 in Ci-opsin1 act synergistically as counterions, which imparts molecular properties to Ci-opsin1 intermediate between those of vertebrate- and invertebrate-type opsins. Synergy between the counterions in Ci-opsin1 was demonstrated by E113Q and E181Q mutants that exhibit a pH-dependent spectral shift, whereas only the E113Q mutation in vertebrate rhodopsin yields this spectral shift. On absorbing light, Ci-opsin1 forms an equilibrium between two intermediates with protonated and deprotonated Schiff bases, namely the MI-like and MII-like intermediates, respectively. Adding G protein caused the equilibrium to shift toward the MI-like intermediate, indicating that Ci-opsin1 has a protonated Schiff base in its active state, like invertebrate-type opsins. Ci-opsin1's G protein activation efficiency is between the efficiencies of vertebrate- and invertebrate-type opsins. Interestingly, the E113Y and E181S mutations change the molecular properties of Ci-opsin1 into those resembling invertebrate-type or bistable opsins and vertebrate ancient/vertebrate ancient-long or monostable opsins, respectively. These results strongly suggest that acquisition of counterion Glu113 changed the molecular properties of visual opsin in a vertebrate/tunicate common ancestor as a crucial step in the evolution of vertebrate visual opsins.
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http://dx.doi.org/10.1073/pnas.1701088114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5468630PMC
June 2017

Adaptation of cone pigments found in green rods for scotopic vision through a single amino acid mutation.

Proc Natl Acad Sci U S A 2017 05 8;114(21):5437-5442. Epub 2017 May 8.

Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan;

Most vertebrate retinas contain a single type of rod for scotopic vision and multiple types of cones for photopic and color vision. The retinas of certain amphibian species uniquely contain two types of rods: red rods, which express rhodopsin, and green rods, which express a blue-sensitive cone pigment (M1/SWS2 group). Spontaneous activation of rhodopsin induced by thermal isomerization of the retinal chromophore has been suggested to contribute to the rod's background noise, which limits the visual threshold for scotopic vision. Therefore, rhodopsin must exhibit low thermal isomerization rate compared with cone visual pigments to adapt to scotopic condition. In this study, we determined whether amphibian blue-sensitive cone pigments in green rods exhibit low thermal isomerization rates to act as rhodopsin-like pigments for scotopic vision. Anura blue-sensitive cone pigments exhibit low thermal isomerization rates similar to rhodopsin, whereas Urodela pigments exhibit high rates like other vertebrate cone pigments present in cones. Furthermore, by mutational analysis, we identified a key amino acid residue, Thr47, that is responsible for the low thermal isomerization rates of Anura blue-sensitive cone pigments. These results strongly suggest that, through this mutation, anurans acquired special blue-sensitive cone pigments in their green rods, which could form the molecular basis for scotopic color vision with normal red rods containing green-sensitive rhodopsin.
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http://dx.doi.org/10.1073/pnas.1620010114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5448186PMC
May 2017

Helical rearrangement of photoactivated rhodopsin in monomeric and dimeric forms probed by high-angle X-ray scattering.

Photochem Photobiol Sci 2015 Nov;14(11):1965-73

Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.

Light-induced helical rearrangement of vertebrate visual rhodopsin was directly monitored by high-angle X-ray scattering (HAXS), ranging from Q (= 4π sin θ/λ) = 0.03 Å(-1) to Q = 1.5 Å(-1). HAXS of nanodiscs containing a single rhodopsin molecule was performed before and after photoactivation of rhodopsin. The intensity difference curve obtained by HAXS agreed with that calculated from the crystal structure of dark state rhodopsin and metarhodopsin II, indicating that the conformational change of monomeric rhodopsin in the membrane is consistent with that occurring in the crystal. On the other hand, the HAXS intensity difference curve of nanodiscs containing two rhodopsin molecules was significantly reduced, similar to that calculated from the crystal structure of the deprotonated intermediate, without a large conformational change. These results suggest that rhodopsin is dimerized in the membrane and that the interaction between rhodopsin molecules modulates structural changes.
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http://dx.doi.org/10.1039/c5pp00175gDOI Listing
November 2015

Origin of the low thermal isomerization rate of rhodopsin chromophore.

Sci Rep 2015 Jun 10;5:11081. Epub 2015 Jun 10.

Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.

Low dark noise is a prerequisite for rod cells, which mediate our dim-light vision. The low dark noise is achieved by the extremely stable character of the rod visual pigment, rhodopsin, which evolved from less stable cone visual pigments. We have developed a biochemical method to quickly evaluate the thermal activation rate of visual pigments. Using an isomerization locked chromophore, we confirmed that thermal isomerization of the chromophore is the sole cause of thermal activation. Interestingly, we revealed an unexpected correlation between the thermal stability of the dark state and that of the active intermediate MetaII. Furthermore, we assessed key residues in rhodopsin and cone visual pigments by mutation analysis and identified two critical residues (E122 and I189) in the retinal binding pocket which account for the extremely low thermal activation rate of rhodopsin.
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http://dx.doi.org/10.1038/srep11081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4462023PMC
June 2015

Rod visual pigment optimizes active state to achieve efficient G protein activation as compared with cone visual pigments.

J Biol Chem 2014 Feb 27;289(8):5061-73. Epub 2013 Dec 27.

From the Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.

Most vertebrate retinas contain two types of photoreceptor cells, rods and cones, which show different photoresponses to mediate scotopic and photopic vision, respectively. These cells contain different types of visual pigments, rhodopsin and cone visual pigments, respectively, but little is known about the molecular properties of cone visual pigments under physiological conditions, making it difficult to link the molecular properties of rhodopsin and cone visual pigments with the differences in photoresponse between rods and cones. Here we prepared bovine and mouse rhodopsin (bvRh and mRh) and chicken and mouse green-sensitive cone visual pigments (cG and mG) embedded in nanodiscs and applied time-resolved fluorescence spectroscopy to compare their Gt activation efficiencies. Rhodopsin exhibited greater Gt activation efficiencies than cone visual pigments. Especially, the Gt activation efficiency of mRh was about 2.5-fold greater than that of mG at 37 °C, which is consistent with our previous electrophysiological data of knock-in mice. Although the active state (Meta-II) was in equilibrium with inactive states (Meta-I and Meta-III), quantitative determination of Meta-II in the equilibrium showed that the Gt activation efficiency per Meta-II of bvRh was also greater than those of cG and mG. These results indicated that efficient Gt activation by rhodopsin, resulting from an optimized active state of rhodopsin, is one of the causes of the high amplification efficiency of rods.
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http://dx.doi.org/10.1074/jbc.M113.508507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3931065PMC
February 2014
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