Updated on 2022/07/02

写真a

 
KOJIMA Keiichi
 
Organization
Faculty of Medicine, Dentistry and Pharmaceutical Sciences Assistant Professor
Position
Assistant Professor
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Degree

  • 博士(理学) ( 2016.3   京都大学 )

Research Interests

  • 光受容タンパク質

  • opsin

  • 光生物

  • 視覚

  • 光操作

  • 生物物理

  • ロドプシン

  • Photobiology

Research Areas

  • Life Science / Animal physiological chemistry, physiology and behavioral biology

  • Life Science / Functional biochemistry

  • Life Science / Biophysics

  • Life Science / Pharmaceutical analytical chemistry and physicochemistry

Education

  • Kyoto University   理学研究科 生物科学専攻 博士後期課程 (理学博士)  

    2013.4 - 2016.3

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    Country: Japan

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  • Kyoto University   理学研究科 生物科学専攻 博士前期課程  

    2011.4 - 2013.3

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  • Kyoto University   理学部  

    2007.4 - 2011.3

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    Country: Japan

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Research History

  • 岡山大学学術研究院医歯薬学域 助教

    2021.4

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  • 岡山大学医歯薬学総合研究科 助教

    2017.12 - 2021.3

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  • 岡山大学医歯薬学総合研究科 助教(特任)

    2017.4 - 2017.11

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Professional Memberships

Committee Memberships

  •   生物物理学会2021年分野別専門委員  

    2021   

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  • 日本生物物理学会 中国四国支部   会計  

    2019.1 - 2020.12   

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    Committee type:Academic society

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  • 日本生物物理学会   平成31年分野別専門委員  

    2019   

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  • 日本生物物理学会   平成30年分野別専門委員  

    2018   

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    Committee type:Academic society

    日本生物物理学会

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Papers

  • Phototriggered apoptotic cell death (PTA) using the light-driven outward proton pump rhodopsin Archaerhodopsin-3 Reviewed

    Shin Nakao, Keiichi Kojima, Yuki Sudo

    Journal of the American Chemical Society   2022.2

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

    DOI: 10.1021/jacs.1c12608

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  • Proton transfer pathway in anion channelrhodopsin-1 Reviewed

    Masaki Tsujimura, Keiichi Kojima, Shiho Kawanishi, Yuki Sudo, Hiroshi Ishikita

    eLife   10   2021.12

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

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

    DOI: 10.7554/elife.72264

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

  • Evolutionary adaptation of visual pigments in geckos for their photic environment Reviewed

    Keiichi Kojima, Yuki Matsutani, Masataka Yanagawa, Yasushi Imamoto, Yumiko Yamano, Akimori Wada, Yoshinori Shichida, Takahiro Yamashita

    Science Advances   7 ( 40 )   2021.10

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    Authorship:Lead author   Publishing type:Research paper (scientific journal)   Publisher:American Association for the Advancement of Science (AAAS)  

    DOI: 10.1126/sciadv.abj1316

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

    Masuzu Kikuchi, Keiichi Kojima, Shin Nakao, Susumu Yoshizawa, Shiho Kawanishi, Atsushi Shibukawa, Takashi Kikukawa, Yuki Sudo

    Scientific Reports   11 ( 1 )   2021.7

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

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

    DOI: 10.1038/s41598-021-94181-w

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

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

    Keiichi Kojima, Susumu Yoshizawa, Masumi Hasegawa, Masaki Nakama, Marie Kurihara, Takashi Kikukawa, Yuki Sudo

    Scientific Reports   10 ( 1 )   20857 - 20857   2020.12

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

    <title>Abstract</title>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-<italic>trans-</italic>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, p<italic>K</italic>a = 12.3) is stabilised by the deprotonated counterion (presumably Asp184, p<italic>K</italic>a = 3.7). The flash-photolysis experiments revealed the presence of two photointermediates, K and M. A proton was released and uptaken from bulk solution upon the formation and decay of the M intermediate. During the M-decay, the Schiff base was reprotonated by the proton from a proton donating residue (presumably Asp172). These properties were compared with other inward (SzRs and xenorhodopsins, XeRs) and outward proton pumps. Notably, LaSzR2 showed acid-induced spectral ‘blue-shift’ due to the protonation of the counterion, whereas outward proton pumps showed opposite shifts (red-shifts). Thus, we can distinguish between inward and outward proton pumps by the direction of the acid-induced spectral shift.

    DOI: 10.1038/s41598-020-77936-9

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

  • Comparative Studies of the Fluorescence Properties of Microbial Rhodopsins: Spontaneous Emission Versus Photointermediate Fluorescence Reviewed International journal

    Keiichi Kojima, Rika Kurihara, Masayuki Sakamoto, Tsukasa Takanashi, Hikaru Kuramochi, Xiao Min Zhang, Haruhiko Bito, Tahei Tahara, Yuki Sudo

    The Journal of Physical Chemistry B   124 ( 34 )   7361 - 7367   2020.8

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

    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 (R = 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.

    DOI: 10.1021/acs.jpcb.0c06560

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  • Green-Sensitive, Long-Lived, Step-Functional Anion Channelrhodopsin-2 Variant as a High-Potential Neural Silencing Tool Reviewed International journal

    Keiichi Kojima, Natsuki Miyoshi, Atsushi Shibukawa, Srikanta Chowdhury, Masaki Tsujimura, Tomoyasu Noji, Hiroshi Ishikita, Akihiro Yamanaka, Yuki Sudo

    The Journal of Physical Chemistry Letters   11 ( 15 )   6214 - 6218   2020.8

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

    Anion channelrhodopsin-2 (GtACR2) was identified from the alga Guillardia theta 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 Escherichia coli 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 (λmax = 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.

    DOI: 10.1021/acs.jpclett.0c01406

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  • Vectorial proton transport mechanism of RxR, a phylogenetically distinct and thermally stable microbial rhodopsin Reviewed International journal

    Keiichi Kojima, Tetsuya Ueta, Tomoyasu Noji, Keisuke Saito, Kanae Kanehara, Susumu Yoshizawa, Hiroshi Ishikita, Yuki Sudo

    Scientific Reports   10 ( 1 )   282 - 282   2020.1

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

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

    DOI: 10.1038/s41598-019-57122-2

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    Other Link: http://www.nature.com/articles/s41598-019-57122-2

  • Mutational analysis of the conserved carboxylates of anion channelrhodopsin-2 (ACR2) expressed in Escherichia coli and their roles in anion transport Reviewed

    Keiichi Kojima, Hiroshi C. Watanabe, Satoko Doi, Natsuki Miyoshi, Misaki Kato, Hiroshi Ishikita, Yuki Sudo

    Biophysics and Physicobiology   15   179 - 188   2018

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

    DOI: 10.2142/biophysico.15.0_179

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  • Evolutionary steps involving counterion displacement in a tunicate opsin Reviewed

    Keiichi Kojima, Takahiro Yamashita, Yasushi Imamoto, Takehiro G. Kusakabe, Motoyuki Tsuda, Yoshinori Shichida

    Proceedings of the National Academy of Sciences of the United States of America   114 ( 23 )   6028 - 6033   2017.6

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    Authorship:Lead author   Publishing type:Research paper (scientific journal)   Publisher:Proceedings of the National Academy of Sciences  

    Ci-opsin1 is a visible light-sensitive opsin present in the larval ocellus of an ascidian, <italic>Ciona intestinalis</italic>. 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.

    DOI: 10.1073/pnas.1701088114

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  • Adaptation of cone pigments found in green rods for scotopic vision through a single amino acid mutation Reviewed International journal

    Keiichi Kojima, Yuki Matsutani, Takahiro Yamashita, Masataka Yanagawa, Yasushi Imamoto, Yumiko Yamano, Akimori Wada, Osamu Hisatomi, Kanto Nishikawa, Keisuke Sakurai, Yoshinori Shichida

    Proceedings of the National Academy of Sciences of the United States of America   114 ( 21 )   5437 - 5442   2017.5

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Proceedings of the National Academy of Sciences  

    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.

    DOI: 10.1073/pnas.1620010114

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  • Origin of the low thermal isomerization rate of rhodopsin chromophore Reviewed

    Masataka Yanagawa, Keiichi Kojima, Takahiro Yamashita, Yasushi Imamoto, Take Matsuyama, Koji Nakanishi, Yumiko Yamano, Akimori Wada, Yasushi Sako, Yoshinori Shichida

    Scientific Reports   5 ( 1 )   2015.9

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

    DOI: 10.1038/srep11081

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    Other Link: http://www.nature.com/articles/srep11081.pdf

  • Rod Visual Pigment Optimizes Active State to Achieve Efficient G Protein Activation as Compared with Cone Visual Pigments Reviewed

    Keiichi Kojima, Yasushi Imamoto, Ryo Maeda, Takahiro Yamashita, Yoshinori Shichida

    Journal of Biological Chemistry   289 ( 8 )   5061 - 5073   2014.2

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    Authorship:Lead author   Publishing type:Research paper (scientific journal)   Publisher:Elsevier BV  

    DOI: 10.1074/jbc.m113.508507

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  • Proton-pumping rhodopsins in marine diatoms

    Susumu Yoshizawa, Tomonori Azuma, Keiichi Kojima, Keisuke Inomura, Masumi Hasegawa, Yosuke Nishimura, Masuzu Kikuchi, Gabrielle Armin, Hideaki Miyashita, Kentaro Ifuku, Takashi Yamano, Adrian Marchetti, Hideya Fukuzawa, Yuki Sudo, Ryoma Kamikawa

    2022.1

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    Publisher:Cold Spring Harbor Laboratory  

    Abstract

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

    DOI: 10.1101/2022.01.18.476826

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  • Exploring the Retinal Binding Cavity of Archaerhodopsin-3 by Replacing the Retinal Chromophore With a Dimethyl Phenylated Derivative

    Taichi Tsuneishi, Masataka Takahashi, Masaki Tsujimura, Keiichi Kojima, Hiroshi Ishikita, Yasuo Takeuchi, Yuki Sudo

    Frontiers in Molecular Biosciences   8   2021.12

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    Publishing type:Research paper (scientific journal)   Publisher:Frontiers Media SA  

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

    DOI: 10.3389/fmolb.2021.794948

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

    Masahiro Hayashi, Keiichi Kojima, Yuki Sudo, Atsuko Yamashita

    Protein Science   2021.7

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

    DOI: 10.1002/pro.4154

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

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

    Masaki Tsujimura, Tomoyasu Noji, Keisuke Saito, Keiichi Kojima, Yuki Sudo, Hiroshi Ishikita

    Biochimica et Biophysica Acta (BBA) - Bioenergetics   148349 - 148349   2020.11

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

    DOI: 10.1016/j.bbabio.2020.148349

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

    Masumi Hasegawa, Toshiaki Hosaka, Keiichi Kojima, Yosuke Nishimura, Yu Nakajima, Tomomi Kimura-Someya, Mikako Shirouzu, Yuki Sudo, Susumu Yoshizawa

    Scientific reports   10 ( 1 )   16752 - 16752   2020.10

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

    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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  • Applicability of Styrene-Maleic Acid Copolymer for Two Microbial Rhodopsins, RxR and HsSRI Reviewed International journal

    Tetsuya Ueta, Keiichi Kojima, Tomoya Hino, Mikihiro Shibata, Shingo Nagano, Yuki Sudo

    Biophysical Journal   2020.9

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

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

    DOI: 10.1016/j.bpj.2020.09.026

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

    Satoshi Yasuda, Tomoki Akiyama, Sayaka Nemoto, Tomohiko Hayashi, Tetsuya Ueta, Keiichi Kojima, Takashi Tsukamoto, Satoru Nagatoishi, Kouhei Tsumoto, Yuki Sudo, Masahiro Kinoshita, Takeshi Murata

    Journal of chemical information and modeling   60 ( 3 )   1709 - 1716   2020.3

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

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

    DOI: 10.1021/acs.jcim.0c00063

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

    Tomohiko Hayashi, Satoshi Yasuda, Kano Suzuki, Tomoki Akiyama, Kanae Kanehara, Keiichi Kojima, Mikio Tanabe, Ryuichi Kato, Toshiya Senda, Yuki Sudo, Takeshi Murata, Masahiro Kinoshita

    The journal of physical chemistry. B   124 ( 6 )   990 - 1000   2020.2

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    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 Rubrobacter xylanophilus rhodopsin (RxR) and long-known bacteriorhodopsin from Halobacterium salinarum (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 Cα 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.

    DOI: 10.1021/acs.jpcb.9b10700

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  • Bacterium Lacking a Known Gene for Retinal Biosynthesis Constructs Functional Rhodopsins Reviewed

    Yu Nakajima, Keiichi Kojima, Yuichiro Kashiyama, Satoko Doi, Ryosuke Nakai, Yuki Sudo, Kazuhiro Kogure, Susumu Yoshizawa

    Microbes and Environments   35 ( 4 )   n/a - n/a   2020

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

    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 KNCT, 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 KNCT 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.

    DOI: 10.1264/jsme2.me20085

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

    Taro Yamanashi, Misayo Maki, Keiichi Kojima, Atsushi Shibukawa, Takashi Tsukamoto, Srikanta Chowdhury, Akihiro Yamanaka, Shin Takagi, Yuki Sudo

    Scientific Reports   9 ( 1 )   2019.12

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    DOI: 10.1038/s41598-019-44308-x

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    Other Link: http://www.nature.com/articles/s41598-019-44308-x

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

    Yasushi Imamoto, Keiichi Kojima, Toshihiko Oka, Ryo Maeda, Yoshinori Shichida

    The Journal of Physical Chemistry B   123 ( 43 )   9134 - 9142   2019.10

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    DOI: 10.1021/acs.jpcb.9b08311

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  • A distinct lineage of giant viruses brings a rhodopsin photosystem to unicellular marine predators. Reviewed International journal

    David M Needham, Susumu Yoshizawa, Toshiaki Hosaka, Camille Poirier, Chang Jae Choi, Elisabeth Hehenberger, Nicholas A T Irwin, Susanne Wilken, Cheuk-Man Yung, Charles Bachy, Rika Kurihara, Yu Nakajima, Keiichi Kojima, Tomomi Kimura-Someya, Guy Leonard, Rex R Malmstrom, Daniel R Mende, Daniel K Olson, Yuki Sudo, Sebastian Sudek, Thomas A Richards, Edward F DeLong, Patrick J Keeling, Alyson E Santoro, Mikako Shirouzu, Wataru Iwasaki, Alexandra Z Worden

    Proceedings of the National Academy of Sciences of the United States of America   116 ( 41 )   20574 - 20583   2019.10

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    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 (VirRDTS) 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 VirRDTS 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

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

    Shibukawa A, Kojima K, Nakajima Y, Nishimura Y, Yoshizawa S, Sudo Y

    Biochemistry   58 ( 26 )   2934 - 2943   2019.7

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

    Tomomi Shionoya, Misao Mizuno, Takashi Tsukamoto, Kento Ikeda, Hayato Seki, Keiichi Kojima, Mikihiro Shibata, Izuru Kawamura, Yuki Sudo, Yasuhisa Mizutani

    The Journal of Physical Chemistry B   122 ( 27 )   6945 - 6953   2018.7

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    DOI: 10.1021/acs.jpcb.8b04894

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

    Riho Takayama, Akimasa Kaneko, Takashi Okitsu, Satoshi P. Tsunoda, Kazumi Shimono, Misao Mizuno, Keiichi Kojima, Takashi Tsukamoto, Hideki Kandori, Yasuhisa Mizutani, Akimori Wada, Yuki Sudo

    Journal of Physical Chemistry Letters   9 ( 11 )   2857 - 2862   2018.6

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

    DOI: 10.1021/acs.jpclett.8b00879

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  • Pinopsin evolved as the ancestral dim-light visual opsin in vertebrates. Reviewed International journal

    Keita Sato, Takahiro Yamashita, Keiichi Kojima, Kazumi Sakai, Yuki Matsutani, Masataka Yanagawa, Yumiko Yamano, Akimori Wada, Naoyuki Iwabe, Hideyo Ohuchi, Yoshinori Shichida

    Communications biology   1   156 - 156   2018

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    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.

    DOI: 10.1038/s42003-018-0164-x

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

    Saki Inoue, Susumu Yoshizawa, Yu Nakajima, Keiichi Kojima, Takashi Tsukamoto, Takashi Kikukawa, Yuki Sudo

    Physical Chemistry Chemical Physics   20 ( 5 )   3172 - 3183   2018

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    Publishing type:Research paper (scientific journal)   Publisher:Royal Society of Chemistry (RSC)  

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

    DOI: 10.1039/c7cp05033j

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

    Yasushi Imamoto, Keiichi Kojima, Toshihiko Oka, Ryo Maeda, Yoshinori Shichida

    Photochemical & Photobiological Sciences   14 ( 11 )   1965 - 1973   2015

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    <p>The light-induced conformational change of monomeric and dimeric rhodopsin in the nanodisc membrane was directly monitored by high-angle solution X-ray scattering.</p>

    DOI: 10.1039/c5pp00175g

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MISC

  • Expression of microbial rhodopsins in Escherichia coli and their extraction and purification using styrene-maleic acid copolymers

    Keiichi Kojima, Yuki Sudo

    STAR Protocols   3 ( 1 )   101046 - 101046   2022.3

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    Authorship:Lead author   Publishing type:Article, review, commentary, editorial, etc. (scientific journal)   Publisher:Elsevier BV  

    DOI: 10.1016/j.xpro.2021.101046

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  • Microbial Rhodopsins as Multi-functional Photoreactive Membrane Proteins for Optogenetics Reviewed

    Shin Nakao, Keiichi Kojima, Yuki Sudo

    Biological and Pharmaceutical Bulletin   44 ( 10 )   1357 - 1363   2021.10

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

    DOI: 10.1248/bpb.b21-00544

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  • Biophysical and Biochemical Research of Animal Rhodopsins

    Keiichi Kojima

    YAKUGAKU ZASSHI   141 ( 10 )   1155 - 1160   2021.10

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

    DOI: 10.1248/yakushi.21-00144

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  • The Unlimited Potential of Microbial Rhodopsins as Optical Tools

    Keiichi Kojima, Atsushi Shibukawa, Yuki Sudo

    Biochemistry   59 ( 3 )   218 - 229   2020.1

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    Authorship:Lead author   Publishing type:Article, review, commentary, editorial, etc. (scientific journal)   Publisher:American Chemical Society (ACS)  

    DOI: 10.1021/acs.biochem.9b00768

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  • Diversity and Potential of Microbial Rhodopsins

    Yuki SUDO, Keiichi KOJIMA

    Seibutsu Butsuri   60 ( 4 )   209 - 214   2020

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

    DOI: 10.2142/biophys.60.209

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  • RESEARCH:光受容タンパク質から眼の進化を追う

    小島 慧一

    季刊「生命誌」   ( 97号 )   2018

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  • “カエルは暗がりで色を見分けられる? ~暗所視を司る錐体視物質の低い熱雑音の 進化的獲得~”

    小島 慧一

    生物物理   58   209 - 210   2018

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

    Kaneko A, Inoue K, Kojima K, Kandori H, Sudo Y

    Biophysical Reviews   9 ( 6 )   861 - 876   2017

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  • マルチタレント光受容タンパク質「ロドプシン」

    須藤雄気, 小島慧一

    現代化学   50 - 53   2021.4

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  • Diversity and Potential of Microbial Rhodopsins

    須藤雄気, 小島慧一

    生物物理(Web)   60 ( 4 )   2020

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Presentations

  • Phosphate ion binding modulates photochemical properties of a light-driven SO42- transporter, SyHR

    第56回日本生物物理学会  2018 

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  • Impact and mechanism of phosphate binding to a light-driven anion transporter SyHR

    18th International Conference on Retinal Proteins  2018 

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  • Bottom-up approach for microbial rhodopsin-based optogenetic tools

    第56回日本生物物理学会  2018 

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  • Optimization mechanism of vertebrate visual pigments for scotopic vision

    International Symposium on Biophysics of Rhodopsins  2017 

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  • Evolutionary acquisition of low thermal noise of cone pigments for scotopic vision

    第55回日本生物物理学会  2017 

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  • Comparative analysis of thermal activation rate in vertebrate cone visual pigments

    第87回 日本動物学会  2016 

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  • Thermal activation rates of visual pigments expressed in rods

    第54回日本生物物理学会  2016 

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  • Thermal isomerization rate of retinal chromophore in blue-sensitive cone pigments expressed in amphibian green rods

    17th International Conference on Retinal Proteins  2016 

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Awards

  • 日本薬学会 中国四国支部 奨励賞

    2020  

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  • 日本生物物理学会若手奨励賞

    2017  

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    Country:Japan

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  • Basic Physics (2020academic year) 1st semester  - 火4,火5,火6

  • Senses for Organisms (2020academic year) Fourth semester  - 金3,金4

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