2021/11/24 更新

写真a

スドウ ユウキ
須藤 雄気
SUDO Yuki
所属
医歯薬学域 教授
職名
教授
外部リンク

学位

  • 博士(薬学) ( 北海道大学 )

研究キーワード

  • Biophysics

  • Photobiology

  • 蛋白質科学

  • 光生物

  • 物理化学

  • 生物物理

  • Protein Science

  • Physical Chemistry

研究分野

  • ライフサイエンス / 薬系分析、物理化学

  • ナノテク・材料 / 基礎物理化学

  • ライフサイエンス / 生物物理学

学歴

  • 北海道大学    

    - 2005年

      詳細を見る

    国名: 日本国

    researchmap

  • 北海道大学   薬学部   総合薬学科

    - 2000年

      詳細を見る

    国名: 日本国

    researchmap

経歴

  • 岡山大学学術研究院医歯薬学域 教授

    2014年 - 現在

      詳細を見る

    国名:日本国

    researchmap

  • 分子科学研究所 客員准教授

    2012年 - 2014年

      詳細を見る

  • 名古屋大学   Associate Professor, Graduate School of Science

    2009年 - 2014年

      詳細を見る

    国名:日本国

    researchmap

  • 科学技術振興機構(JST)・さきがけ 研究員

    2008年 - 2012年

      詳細を見る

  • 名古屋大学   Assistant Professor, Graduate School of Science

    2007年 - 2009年

      詳細を見る

    国名:日本国

    researchmap

  • テキサス大学ヒューストン校   ポスドク   Postdoctoral Fellow

    2005年 - 2007年

      詳細を見る

    国名:アメリカ合衆国

    researchmap

  • 名古屋工業大学大学院工学研究科 研究員   Graduate School of Engineering

    2005年

      詳細を見る

    国名:日本国

    researchmap

▼全件表示

所属学協会

  • 日本薬学会

    2014年 - 現在

      詳細を見る

  • 分子科学会

    2013年 - 現在

      詳細を見る

  • 蛋白質科学会

    2012年 - 現在

      詳細を見る

  • 細胞を創る研究会

    2012年 - 現在

      詳細を見る

  • 生物物理学会

    2000年 - 現在

      詳細を見る

委員歴

  • 日本生物物理学会   副会長  

    2019年 - 2020年   

      詳細を見る

    団体区分:学協会

    researchmap

  • 日本薬学会中国四国支部   支部役員  

    2018年 - 2019年   

      詳細を見る

  • 日本生物物理学会中国四国支部   支部長  

    2018年 - 2019年   

      詳細を見る

  • 日本生物物理学会   理事  

    2017年 - 2018年   

      詳細を見る

  • 生物物理学会   運営委員・理事  

    2010年 - 2014年   

      詳細を見る

    団体区分:学協会

    生物物理学会

    researchmap

 

論文

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

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

    Scientific Reports   11 ( 1 )   2021年12月

     詳細を見る

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

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

    researchmap

    その他リンク: http://www.nature.com/articles/s41598-021-94181-w

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

    Masahiro Hayashi, Keiichi Kojima, Yuki Sudo, Atsuko Yamashita

    Protein Science   2021年7月

     詳細を見る

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

    DOI: 10.1002/pro.4154

    researchmap

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

  • Further thermo-stabilization of thermophilic rhodopsin from Thermus thermophilus JL-18 through engineering in extramembrane regions 査読

    Akiyama T, Kunishima N, Nemoto S, Kazama K, Hirose M, Sudo Y, Matsuura Y, Naitow H, Murata T.

    Proteins   89 ( 3 )   301 - 310   2021年3月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1002/prot.26015

    researchmap

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

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

    Kojima K, Yoshizawa S, Hasegawa M, Nakama M, Kurihara M, Kikukawa T, Sudo Y.

    Sci. Rep.   10 ( 1 )   20857   2020年12月

     詳細を見る

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

    DOI: 10.1038/s41598-020-77936-9

    researchmap

    その他リンク: http://www.nature.com/articles/s41598-020-77936-9

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

    Hasegawa M, Hosaka T, Kojima K, Nishimura Y, Nakajima Y, Kimura-Someya T, Shirouzu M, Sudo Y, Yoshizawa S.

    Sci. Rep.   10 ( 1 )   16752   2020年12月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1038/s41598-020-73606-y

    researchmap

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

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

    Tsujimura M, Noji T, Saito K, Kojima K, Sudo Y, Ishikita H.

    Biochim Biophys Acta (Bioenerg)   148349 - 148349   2020年11月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.bbabio.2020.148349

    researchmap

  • Applicability of Styrene-Maleic Acid Copolymer for Two Microbial Rhodopsins, RxR and HsSRI 査読 国際誌

    Ueta T, Kojima K, Hino T, Shibata M, Nagano S, Sudo Y.

    Biophys. J.   119   1760 - 1770   2020年9月

     詳細を見る

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

    DOI: 10.1016/j.bpj.2020.09.026

    PubMed

    researchmap

  • Interaction of Escherichia coli and its culture supernatant with Vibrio vulnificus during biofilm formation 査読

    Ohn HM, Mizuno T, Sudo Y, Miyoshi SI.

    Microbiol Immunol.   2020年9月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1111/1348-0421.12829

    researchmap

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

    Kojima K, Kurihara R, Sakamoto M, Takanashi T, Kuramochi H, Zhang XM, Bito H, Tahara T, Sudo Y.

    J. Phys. Chem. B   124 ( 34 )   7361 - 7367   2020年8月

     詳細を見る

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

    DOI: 10.1021/acs.jpcb.0c06560

    researchmap

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

    Kojima K, Miyoshi N, Shibukawa A, Chowdhury S, Tsujimura M, Noji T, Ishikita H, Yamanaka A, Sudo Y.

    J. Phys. Chem. Lett.   11 ( 15 )   6214 - 6218   2020年7月

     詳細を見る

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

    DOI: 10.1021/acs.jpclett.0c01406

    researchmap

  • Correction to "How Does a Microbial Rodhopsin RxR Realize Its Exceptionally High Thermostability with the Proton-Pumping Function Being Retained?" 査読 国際誌

    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 ( 14 )   2973 - 2973   2020年4月

     詳細を見る

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

    Yasuda, S., Akiyama, T., Nemoto, S., Hayashi, T., Ueta, T., Kojima, K., Tsukamoto, T., Nagatoishi, S., Tsumoto, K., Sudo, Y., Kinoshita, M., Murata, T.

    Journal of Chemical Information and Modeling   60 ( 3 )   1709 - 1716   2020年3月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1021/acs.jcim.0c00063

    PubMed

    researchmap

  • 神経抑制型光遺伝学ツール開発 長いチャネル開時間を示すステップファンクション型緑色光感受性アニオンチャネルの創成

    小島 慧一, 三好 菜月, 渋川 敦史, チョドリ・スリカンタ, 渡邉 宙志, 石北 央, 山中 章弘, 須藤 雄気

    日本薬学会年会要旨集   140年会   26H - pm07   2020年3月

     詳細を見る

    記述言語:日本語   出版者・発行元:(公社)日本薬学会  

    researchmap

  • Vectorial proton transport mechanism of RxR, a phylogenetically distinct and thermally stable microbial rhodopsin 査読 国際誌

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

    Sci Rep.   10 ( 1 )   282 - 282   2020年1月

     詳細を見る

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

    DOI: 10.1038/s41598-019-57122-2

    PubMed

    researchmap

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

    Nakajima Y, Kojima K, Kashiyama Y, Doi S, Nakai R, Sudo Y, Kogure K, Yoshizawa S.

    Microbes Environ.   35 ( 4 )   n/a - n/a   2020年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1264/jsme2.me20085

    researchmap

  • 微生物ロドプシンの多様性と可能性

    須藤雄気, 小島慧一

    生物物理   60 ( 4 )   209 - 214   2020年

     詳細を見る

    担当区分:筆頭著者, 責任著者  

    DOI: 10.2142/biophys.60.209

    researchmap

  • シアノバクテリアの光合成とは異なる光利用:シアノロドプシンの発見

    長谷川 万純, 保坂 俊彰, 小島 慧一, 西村 陽介, 中島 悠, 染谷 友美, 白水 美香子, 須藤 雄気, 吉澤 晋

    日本地球化学会年会要旨集   67   186 - 186   2020年

     詳細を見る

    記述言語:日本語   出版者・発行元:一般社団法人日本地球化学会  

    <p>微生物による光合成は、地球における一次生産の約5割を担うと試算されており、生態系を支える重要な存在である。一方で、光合成とは全く異なる光受容体であるロドプシン(微生物型ロドプシン)が幅広い微生物分類群に分布することが明らかになり、微生物の光エネルギー利用の常識が大きく揺らいでいる。本研究では大規模な比較ゲノム解析を行い、シアノバクテリアにおけるロドプシンの分布とその系統を明らかにした。さらに、シアノバクテリアが持つロドプシンのみで構成されるグループ(シアノロドプシン)を発見し、その機能・分光学的特性・立体構造を明らかにした。本研究の結果から、シアノロドプシンがクロロフィルを用いる光合成系とともに進化し、シアノバクテリア細胞内の環境に最適化されてきたことが示唆された。</p>

    DOI: 10.14862/geochemproc.67.0_186

    CiNii Article

    researchmap

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

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

    Scientific Reports   9 ( 1 )   2019年3月

     詳細を見る

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

    DOI: 10.1038/s41598-019-44308-x

    researchmap

  • Retinal configuration of ppR intermediates revealed by phot-irradiation solid-state NMR and DFT 査読

    Makino Y, *Kawamura I, Okitsu T, Wada A, Kamo N, Sudo Y, *Ueda K, & *Naito A.

    Biophys. J.   115 ( 1 )   72 - 83   2018年7月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.bpj.2018.05.030

    Scopus

    researchmap

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

    Shionoya T, Mizuno M, Tsukamoto T, Ikeda K, Seki H, Kojima K, Shibata M, Kawamura I, Sudo Y, Mizutani Y

    The journal of physical chemistry. B   122 ( 27 )   6945 - 6953   2018年7月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1021/acs.jpcb.8b04894

    PubMed

    researchmap

  • Production of a light-gated proton channel by replacing the retinal chromophore with its synthetic vinylene derivative

    Takayama R, Kaneko A, Okitsu T, Tsunoda SP, Shimono K, Mizuno M, Kojima K, Tsukamoto T, Kandori H, Mizutani Y, Wada A, & *Sudo Y.

    J. Phys. Chem. Lett.   9 ( 11 )   2857 - 2862   2018年6月

     詳細を見る

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

    DOI: 10.1021/acs.jpclett.8b00879

    Scopus

    researchmap

  • Presence of a haloarchaeal halorhodopsin-like Cl- pump in marine bacteria

    Nakajima Y, Tsukamoto T, Kumagai Y, Ogura Y, Hayashi T, Song J, Kikukawa T, Demura M, Kogure K, Sudo Y, & *Yoshizawa S.

    Microbes Environ.   33 ( 1 )   89 - 97   2018年3月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1264/jsme2.ME17197

    PubMed

    researchmap

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

    Inoue S, Yoshizawa S, Nakajima Y, Kojima K, Tsukamoto T, Kikukawa T, & *Sudo Y.

    Phys. Chem. Chem. Phys.   20 ( 5 )   3172 - 3183   2018年1月

     詳細を見る

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

    DOI: 10.1039/c7cp05033j

    PubMed

    researchmap

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

    Kojima K, Watanabe HC, Doi S, Miyoshi N, Kato M, Ishikita H, Sudo Y

    Biophys. Physicobiol.   15   179 - 188   2018年

     詳細を見る

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

    researchmap

  • Few-cycle pulse generation from noncollinear optical parametric amplifier with static dispersion compensation

    Shunsuke Adachi,Yuya Watanabe,Yuki Sudo*,Toshinori Suzuki

    Chemical Physics Letters   683   7 - 11   2017年9月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.cplett.2017.04.001

    Web of Science

    researchmap

  • Comparative evaluation of the stability of seven-transmembrane microbial rhodopsins to various physicochemical stimuli

    Naoya Honda,Takashi Tsukamoto,Yuki Sudo*

    Chemical Physics Letters   682   6 - 14   2017年8月

     詳細を見る

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

    DOI: 10.1016/j.cplett.2017.05.055

    Web of Science

    researchmap

  • A phylogenetically distinctive and extremely heat stable light-driven proton pump from the eubacterium Rubrobacter xylanophilus DSM 9941T

    Kanehara K, Yoshizawa S, Tsukamoto T, & *Sudo Y.

    Sci. Rep.   7 ( 44427 )   1344 - 1355   2017年3月

     詳細を見る

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

    DOI: 10.1038/srep44427

    Web of Science

    researchmap

  • Demonstration of a light-driven SO42- transporter and its spectroscopic characteristics

    Niho A, Yoshizawa S, Tsukamoto T, Kurihara M, Tahara S, Nakajima Y, Mizuno M, Kuramochi H, Tahara T, Mizutani Y, & *Sudo Y.

    J. Am. Chem. Soc.   139 ( 12 )   4376 - 4389   2017年3月

     詳細を見る

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

    DOI: 10.1021/jacs.6b12139

    Web of Science

    researchmap

  • Implications for the light-driven chloride ion transport mechanism of Nonlabens marinus rhodopsin 3 by its photochemical characteristics

    *Tsukamoto T, Yoshizawa S, Kikukawa T, Demura M, & *Sudo Y.

    J. Phys. Chem. B   121 ( 9 )   2027 - 2038   2017年3月

     詳細を見る

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

    DOI: 10.1021/acs.jpcb.6b11101

    Web of Science

    researchmap

  • An inhibitory role of Arg-84 in anion channelrhodopsin-2 expressed in Escherichia coli

    Doi S, Tsukamoto T, Yoshizawa S, & *Sudo Y.

    Sci. Rep.   7 ( 41879 )   621 - 627   2017年2月

     詳細を見る

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

    DOI: 10.1038/srep41879

    Web of Science

    researchmap

  • Hybrid Model Membrane Combining Micropatterned Lipid Bilayer and Hydrophilic Polymer Brush

    Toshiki Nishimura,Fuyuko Tamura,Sawako Kobayashi,Yasushi Tanimoto,Fumio Hayashi,Yuki Sudo*,Yasuhiko Iwasaki,Kenichi Morigaki

    Langmuir   33 ( 23 )   5752 - 5759   2017年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1021/acs.langmuir.7b00463

    Web of Science

    researchmap

  • Live-cell single-molecule imaging of the cytokine receptor MPL for analysis of dynamic dimerization

    *Sakamoto A, Tsukamoto T, Furutani Y, Sudo Y, Shimada K, Tomita A, Kiyoi H, Kato T, & Funatsu T.

    J. Mol. Cell Biol.   8 ( 6 )   553 - 555   2016年12月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1093/jmcb/mjw027

    Web of Science

    researchmap

  • Temperature independence of ultrafast photoisomerization in thermophilic rhodopsin: Assessment versus other microbial proton pumps

    Iyer ES, Misra R, Maity A, Liubashevski O, Sudo Y, Sheves M, & *Ruhman S.

    Journal of the American Chemical Society   138 ( 38 )   12401 - 12407   2016年9月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1021/jacs.6b05002

    Web of Science

    researchmap

  • X-ray crystallographic structure of thermophilic rhodopsin: Implications for high thermal stability and optogenetic function 査読

    Tsukamoto T, Mizutani K, Hasegawa T, Takahashi M, Honda N, Hashimoto N, Shimono K, Yamashita K, Yamamoto M, Miyauchi S, Takagi S, Hayashi S, *Murata T, & *Sudo Y.

    The Journal of BIological Chemistry   291   12223 - 12232   2016年6月

     詳細を見る

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

    researchmap

  • X-ray Crystallographic Structure of Thermophilic Rhodopsin IMPLICATIONS FOR HIGH THERMAL STABILITY AND OPTOGENETIC FUNCTION

    Takashi Tsukamoto, Kenji Mizutani, Taisuke Hasegawa, Megumi Takahashi, Naoya Honda, Naoki Hashimoto, Kazumi Shimono, Keitaro Yamashita, Masaki Yamamoto, Seiji Miyauchi, Shin Takagi, Shigehiko Hayashi, Takeshi Murata, Yuki Sudo

    JOURNAL OF BIOLOGICAL CHEMISTRY   291 ( 23 )   12223 - 12232   2016年6月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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

    DOI: 10.1074/jbc.M116.719815

    Web of Science

    researchmap

  • Functional and photochemical characterization of a light-driven proton pump from the gammaproteobacterium Pantoea vagans 査読

    *Sudo Y, & *Yoshizawa S

    Photochemistry and Photobiology   92   420 - 427   2016年5月

     詳細を見る

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

    researchmap

  • Functional and Photochemical Characterization of a Light-Driven Proton Pump from the Gammaproteobacterium Pantoea vagans

    Yuki Sudo, Susumu Yoshizawa

    Photochemistry and Photobiology   92 ( 3 )   420 - 427   2016年5月

     詳細を見る

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

    DOI: 10.1111/php.12585

    Web of Science

    researchmap

  • Factors affecting the stability and performance of ionic liquid-based planar transient photodetectors

    *Dalgleish S, Reissig L, Hu L, Matsushita MM, Sudo Y, & *Awaga K.

    Langmuir   31 ( 18 )   5235 - 5243   2015年5月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1021/la504972q

    Web of Science

    researchmap

  • Atomistic design of microbial opsin-based blue-shifted optogenetics tools

    Kato HE, Kamiya M, Sugo S, Ito J, Taniguchi R, Orito A, Hirata K, Inutsuka A, Yamanaka A, Maturana AD, Ishitani R, Sudo Y, *Hayashi S, & *Nureki O.

    Nat. Commun.   6 ( 7166 )   2015年5月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1038/ncomms8177

    Web of Science

    researchmap

  • Converting a light-driven proton pump into a light-gated proton channel

    Inoue K, Tsukamoto T, Shimono K, Suzuki Y, Miyauchi S, Hayashi S, Kandori H, & *Sudo Y.

    Journal of the American Chemical Society   137 ( 9 )   3291 - 3299   2015年3月

     詳細を見る

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

    DOI: 10.1021/ja511788f

    Web of Science

    researchmap

  • On-tip photodetection: a simple and universal platform for optoelectronic screening

    *Dalgleish S, *Reissig L, Sudo Y, & *K. Awaga

    Chemical Communications   51 ( 91 )   16401 - 16404   2015年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1039/c5cc06237c

    Web of Science

    researchmap

  • Structural and functional roles of the N- and C-terminal extended modules in channelrhodopsin-1

    Doi S, Mori A, Tsukamoto T, Reissig L, Ihara K, & *Sudo Y.

    Photochemical and Photobiological Sciences   14 ( 9 )   1628 - 1636   2015年

     詳細を見る

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

    DOI: 10.1039/c5pp00213c

    Web of Science

    researchmap

  • Irreversible Trimer to Monomer Transition of Thermophilic Rhodopsin upon Thermal Stimulation

    Tsukamoto T, Demura M, & *Sudo Y.

    The Journal of Physical Chemistry B   118 ( 43 )   12383 - 12394   2014年10月

     詳細を見る

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

    DOI: 10.1021/jp507374q

    Web of Science

    researchmap

  • Color discriminating retinal configurations of sensory rhodopsin I by photo-irradiation solid state NMR spectroscopy

    Yomoda H, Makino Y, Tomonaga Y, Hidaka T, *Kawamura I, Okitsu T, Wada A, *Sudo Y, & *Naito A.

    Angewandte Chemie International Edition   53 ( 27 )   6960 - 6964   2014年7月

     詳細を見る

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

    DOI: 10.1002/anie.201309258

    Web of Science

    researchmap

  • Molecular and evolutionary aspects of microbial sensory rhodopsins

    Keiichi Inoue, Takashi Tsukamoto, Yuki Sudo

    BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS   1837 ( 5 )   562 - 577   2014年5月

     詳細を見る

    記述言語:英語   出版者・発行元:ELSEVIER SCIENCE BV  

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

    DOI: 10.1016/j.bbabio.2013.05.005

    Web of Science

    researchmap

  • The early steps in the photocycle of a photosensor protein sensory rhodopsin I from Salinibacter ruber

    *Sudo Y, Mizuno M, Wei Z, Takeuchi S, *Tahara T, & *Mizutani Y.

    The Journal of Physical Chemistry B   118 ( 6 )   1510 - 1518   2014年2月

     詳細を見る

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

    DOI: 10.1021/jp4112662

    Web of Science

    researchmap

  • 1P235 センサリーロドプシンI-トランスデューサーの一分子FRET観察(18A. 光生物:視覚・光受容,ポスター,第52回日本生物物理学会年会(2014年度))

    Nisimura Ryo, Inoue Keiichi, Yagasaki Jin, Kawamoto Kenichi, Sudo Yuki, Kandori Hideki

    生物物理   54 ( 1 )   S180   2014年

     詳細を見る

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

    DOI: 10.2142/biophys.54.S180_1

    CiNii Article

    researchmap

  • 2P233 高度好熱菌由来サーモフィリックロドプシンの温度依存的な不可逆構造転移(18A. 光生物:視覚・光受容,ポスター,第52回日本生物物理学会年会(2014年度))

    Tsukamoto Takashi, Demura Makoto, Sudo Yuki

    生物物理   54 ( 1 )   S233   2014年

     詳細を見る

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

    DOI: 10.2142/biophys.54.S233_5

    CiNii Article

    researchmap

  • 2P244 固体^<13>C NMRによるファラオニスフォボロドプシンの機能に重要なTyr残基の構造解析(18A. 光生物:視覚・光受容,ポスター,第52回日本生物物理学会年会(2014年度))

    Nishikawa Ryota, Kawamura Izuru, Okitsu Takashi, Wada Akimori, Sudo Yuki, Kamo Naoki, Naito Akira

    生物物理   54 ( 1 )   S235   2014年

     詳細を見る

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

    DOI: 10.2142/biophys.54.S235_4

    CiNii Article

    researchmap

  • 2P240 In situ光照射固体NMRによる光受容膜タンパク質センサリーロドプシンIの光反応過程の解析(18A. 光生物:視覚・光受容,ポスター,第52回日本生物物理学会年会(2014年度))

    Makino Yoshiteru, Yamoda Hiroki, Tomonaga Yuya, Hidaka Tetsurou, Kawamura Izuru, Okitsu Takashi, Wada Akimori, Sudo Yuki, Kamo Naoki, Naito Akira

    生物物理   54 ( 1 )   S234   2014年

     詳細を見る

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

    DOI: 10.2142/biophys.54.S234_6

    CiNii Article

    researchmap

  • Thermal and spectroscopic characterization of a proton pumping rhodopsin from an extreme thermophile 査読

    Takashi Tsukamoto, Keiichi Inoue, Hideki Kandori, Yuki Sudo

    Journal of Biological Chemistry   288 ( 30 )   21581 - 21592   2013年7月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1074/jbc.M113.479394

    Scopus

    PubMed

    researchmap

  • A blue-shifted light-driven proton pump for neural silencing 査読

    Yuki Sudo, Ayako Okazaki, Hikaru Ono, Jin Yagasaki, Seiya Sugo, Motoshi Kamiya, Louisa Reissig, Keiichi Inoue, Kunio Ihara, Hideki Kandori, Shin Takagi, Shigehiko Hayashi

    Journal of Biological Chemistry   288 ( 28 )   20624 - 20632   2013年7月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1074/jbc.M113.475533

    Scopus

    PubMed

    researchmap

  • Large spectral change due to amide modes of a β-sheet upon the formation of an early photointermediate of middle rhodopsin 査読

    Furutani Y, Okitsu T, Reissig L, Mizuno M, Homma M, Wada A, Mizutani Y, Sudo Y

    Journal of Physical Chemistry B   117 ( 13 )   3449 - 3458   2013年4月

     詳細を見る

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

    DOI: 10.1021/jp308765t

    Scopus

    PubMed

    researchmap

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

  • Investigation of the chromophore binding cavity in the 11-cis acceptable microbial rhodopsin MR 査読

    Arisa Mori, Jin Yagasaki, Michio Homma, Louisa Reissig, Yuki Sudo

    Chemical Physics   419   23 - 29   2013年

     詳細を見る

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

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

    DOI: 10.1016/j.chemphys.2012.11.020

    Scopus

    researchmap

  • Expression, purification and biochemical characterization of the cytoplasmic loop of PomA, a stator component of the Na+ driven flagellar motor 査読

    Rei Abe-Yoshizumi, Shiori Kobayashi, Mizuki Gohara, Kokoro Hayashi, Chojiro Kojima, Seiji Kojima, Yuki Sudo, Yasuo Asami, Michio Homma

    Biophysics (Japan)   9   21 - 29   2013年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.2142/biophysics.9.21

    Scopus

    researchmap

  • 1P247 青色光吸収型アーキロドプシン3変異体による内向きプロトン輸送(18A. 光生物:視覚・光受容,ポスター,日本生物物理学会年会第51回(2013年度))

    Inoue Keiichi, Tsukamoto Takashi, Yagasaki Jin, Shimono Kazumi, Miyauchi Seiji, Hayashi Shigehiko, Kandori Hideki, Sudo Yuki

    生物物理   53 ( 1 )   S146   2013年

     詳細を見る

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

    DOI: 10.2142/biophys.53.S146_6

    CiNii Article

    researchmap

  • 2P247 in situ光照射固体NMRによる光受容膜タンパク質ppR/pHtrIIの光励起過程におけるtransducerタンパク質膜貫通領域の構造変化の観測(18A.光生物:視覚・光受容,ポスター,日本生物物理学会年会第51回(2013年度))

    Makino Yoshiteru, Tomonaga Yuya, Shibafuji Yusuke, Hidaka Tetsurou, Kawamura Izuru, Okitsu Takashi, Wada Akimori, Sudo Yuki, Kamo Naoki, Naito Akira

    生物物理   53 ( 1 )   S199   2013年

     詳細を見る

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

    DOI: 10.2142/biophys.53.S199_6

    CiNii Article

    researchmap

  • 1P244 好熱性ロドプシン:高度好熱菌から初めて発見された光駆動イオンポンプ(18A. 光生物:視覚・光受容,ポスター,日本生物物理学会年会第51回(2013年度))

    Tsukamoto Takashi, Sudo Yuki

    生物物理   53 ( 1 )   S146   2013年

     詳細を見る

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

    DOI: 10.2142/biophys.53.S146_3

    CiNii Article

    researchmap

  • 2P108 ナノディスクを用いたセンサリーロドプシンI(03.膜蛋白質,ポスター,日本生物物理学会年会第51回(2013年度))

    Kawamoto Kenichi, Inoue Keiichi, Sasaki Jun, Yagasaki jin, Sudo Yuki, Homma Michio, Kandori Hideki

    生物物理   53 ( 1 )   S176   2013年

     詳細を見る

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

    DOI: 10.2142/biophys.53.S176_6

    CiNii Article

    researchmap

  • 1P243 大腸菌におけるチャネルロドプシン1の発現(18A. 光生物:視覚・光受容,ポスター,日本生物物理学会年会第51回(2013年度))

    Mori Arisa, Tsukamoto Takashi, Yagasaki Zin, Homma Michio, Ihara Kunio, Sudo Yuki

    生物物理   53 ( 1 )   S146   2013年

     詳細を見る

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

    DOI: 10.2142/biophys.53.S146_2

    CiNii Article

    researchmap

  • Influence of halide binding on the hydrogen bonding network in the active site of salinibacter sensory rhodopsin i 査読

    Louisa Reissig, Tatsuya Iwata, Takashi Kikukawa, Makoto Demura, Naoki Kamo, Hideki Kandori, Yuki Sudo

    Biochemistry   51 ( 44 )   8802 - 8813   2012年11月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:44  

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

    DOI: 10.1021/bi3009592

    Web of Science

    Scopus

    PubMed

    J-GLOBAL

    researchmap

  • Photo-induced regulation of the chromatic adaptive gene expression by Anabaena sensory rhodopsin 査読

    Hiroki Irieda, Teppei Morita, Kimika Maki, Michio Homma, Hiroji Aiba, Yuki Sudo

    Journal of Biological Chemistry   287 ( 39 )   32485 - 32493   2012年9月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1074/jbc.M112.390864

    Scopus

    PubMed

    researchmap

  • Optical silencing of c. elegans cells with arch proton pump 査読

    Ayako Okazaki, Yuki Sudo, Shin Takagi

    PLoS ONE   7 ( 5 )   2012年5月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1371/journal.pone.0035370

    Scopus

    PubMed

    researchmap

  • Absorption spectra and photochemical reactions in a unique photoactive protein, middle rhodopsin MR 査読

    Keiichi Inoue, Louisa Reissig, Makoto Sakai, Shiori Kobayashi, Michio Homma, Masaaki Fujii, Hideki Kandori, Yuki Sudo

    Journal of Physical Chemistry B   116 ( 20 )   5888 - 5899   2012年3月

     詳細を見る

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

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

    DOI: 10.1021/jp302357m

    Scopus

    researchmap

  • 1F1558 In-situ光照射固体NMRによる光受容膜蛋白質の光活性状態の解明(光生物-視覚,光受容I,口頭発表,日本生物物理学会第50回年会(2012年度))

    Naito Akira, Tomonaga Yuya, Hidaka Tetsurou, Shibafuji Yusuke, Makino Yoshiteru, Kawamura Izuru, Sudo Yuki, Wada Akimori, Okitsu Takashi, Kamo Naoki

    生物物理   52   S30   2012年

     詳細を見る

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

    DOI: 10.2142/biophys.52.S30_5

    CiNii Article

    researchmap

  • 光受容タンパク質による微生物の光センシングの理解とその利用

    須藤 雄気, 本間 道夫

    YAKUGAKU ZASSHI   132 ( 4 )   407 - 416   2012年

     詳細を見る

    記述言語:日本語   出版者・発行元:公益社団法人 日本薬学会  

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

    DOI: 10.1248/yakushi.132.407

    CiNii Article

    CiNii Books

    J-GLOBAL

    researchmap

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

  • Structural characteristics around the β-ionone ring of the retinal chromophore in Salinibacter sensory rhodopsin i 査読

    Hiroki Irieda, Louisa Reissig, Akira Kawanabe, Michio Homma, Hideki Kandori, Yuki Sudo

    Biochemistry   50 ( 22 )   4912 - 4922   2011年6月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1021/bi200284s

    Scopus

    PubMed

    researchmap

  • Direct observation of the structural change of Tyr174 in the primary reaction of sensory Rhodopsin II 査読

    Misao Mizuno, Yuki Sudo, Michio Homma, Yasuhisa Mizutani

    Biochemistry   50 ( 15 )   3170 - 3180   2011年4月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1021/bi101817y

    Scopus

    PubMed

    researchmap

  • Spectral tuning in sensory rhodopsin I from Salinibacter ruber 査読

    Yuki Sudo, Yasufumi Yuasa, Jun Shibata, Daisuke Suzuki, Michio Homma

    Journal of Biological Chemistry   286 ( 13 )   11328 - 11336   2011年4月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Organisms utilize light as energy sources and as signals. Rhodopsins, which have seven transmembrane α-helices with retinal covalently linked to a conserved Lys residue, are found in various organisms as distant in evolution as bacteria, archaea, and eukarya. One of the most notable properties of rhodopsin molecules is the large variation in their absorption spectrum. Sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII) function as photosensors and have similar properties (retinal composition, photocycle, structure, and function) except for their λmax (SRI, ∼560 nm
    SRII, ∼500 nm). An expression system utilizing Escherichia coli and the high protein stability of a newly found SRI-like protein, SrSRI, enables studies of mutant proteins. To determine the residue contributing to the spectral shift from SRI to SRII, we constructed various SRI mutants, in which individual residues were substituted with the corresponding residues of SRII. Three such mutants of SrSRI showed a large spectral blue-shift (&gt
    14 nm) without a large alteration of their retinal composition. Two of them, A136Y and A200T, are newly discovered color tuning residues. In the triple mutant, the λmax was 525 nm. The inverse mutation of SRII (F134H/Y139A/T204A) generated a spectral-shifted SRII toward longer wavelengths, although the effect is smaller than in the case of SRI, which is probably due to the lack of anion binding in the SRII mutant. Thus, half of the spectral shift from SRI to SRII could be explained by only those three residues taking into account the effect of Cl- binding. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

    DOI: 10.1074/jbc.M110.187948

    Scopus

    PubMed

    researchmap

  • Spectrally Silent Intermediates during the Photochemical Reactions of Salinibacter Sensory Rhodopsin I 査読

    Keiichi Inoue, Yuki Sudo, Michio Homma, Hideki Kandori

    JOURNAL OF PHYSICAL CHEMISTRY B   115 ( 15 )   4500 - 4508   2011年4月

     詳細を見る

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

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

    DOI: 10.1021/jp2000706

    Web of Science

    researchmap

  • A Microbial Rhodopsin with a Unique Retinal Composition Shows Both Sensory Rhodopsin II and Bacteriorhodopsin-like Properties 査読

    Yuki Sudo, Kunio Ihara, Shiori Kobayashi, Daisuke Suzuki, Hiroki Irieda, Takashi Kikukawa, Hideki Kandori, Michio Homma

    JOURNAL OF BIOLOGICAL CHEMISTRY   286 ( 8 )   5967 - 5976   2011年2月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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

    DOI: 10.1074/jbc.M110.190058

    Web of Science

    PubMed

    researchmap

  • Spectroscopic studies of a sensory rhodopsin I homologue from the archaeon Haloarcula vallismortis 査読

    Jin Yagasaki, Daisuke Suzuki, Kunio Ihara, Keiichi Inoue, Takashi Kikukawa, Makoto Sakai, Masaaki Fujii, Michio Homma, Hideki Kandori, Yuki Sudo

    Biochemistry   49 ( 6 )   1183 - 1190   2010年2月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1021/bi901824a

    Scopus

    PubMed

    researchmap

  • Corrigendum to “Effects of Chloride Ion Binding on the Photochemical Properties of Salinibacter Sensory Rhodopsin I” [J. Mol. Biol. (2009) 392, 48-61] 査読

    Daisuke Suzuki, Yuji Furutani, Keiichi Inoue, Takashi Kikukawa, Makoto Sakai, Masaaki Fujii, Hideki Kandori, Michio Homma, Yuki Sudo

    Journal of Molecular Biology   395 ( 1 )   220 - 221   2010年1月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.jmb.2009.10.046

    Scopus

    researchmap

  • Protein-protein interaction changes in an archaeal light-signal transduction 査読

    Hideki Kandori, Yuki Sudo, Yuji Furutani

    Journal of Biomedicine and Biotechnology   2010   Article ID 424760   2010年

     詳細を見る

    記述言語:英語   出版者・発行元:Hindawi Publishing Corporation  

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

    DOI: 10.1155/2010/424760

    Scopus

    PubMed

    researchmap

  • Interaction between Na+ Ion and Carboxylates of the PomA-PomB Stator Unit Studied by ATR-FTIR Spectroscopy 査読

    Yuki Sudo, Yuya Kitade, Yuji Furutani, Masaru Kojima, Seiji Kojima, Michio Homma, Hideki Kandori

    BIOCHEMISTRY   48 ( 49 )   11699 - 11705   2009年12月

     詳細を見る

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

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

    DOI: 10.1021/bi901517n

    Web of Science

    researchmap

  • Characterization of a signaling complex composed of sensory rhodopsin I and its cognate transducer protein from the eubacterium Salinibacter ruber 査読

    Yuki Sudo, Akiko Okada, Daisuke Suzuki, Keiichi Inoue, Hiroki Irieda, Makoto Sakai, Masaaki Fujii, Yuji Furutani, Hideki Kandori, Michio Homma

    Biochemistry   48 ( 42 )   10136 - 10145   2009年10月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1021/bi901338d

    Scopus

    PubMed

    researchmap

  • Effects of Chloride Ion Binding on the Photochemical Properties of Salinibacter Sensory Rhodopsin I 査読

    Daisuke Suzuki, Yuji Furutani, Keiichi Inoue, Takashi Kikukawa, Makoto Sakai, Masaaki Fujii, Hideki Kandori, Michio Homma, Yuki Sudo

    Journal of Molecular Biology   392 ( 1 )   48 - 62   2009年9月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1016/j.jmb.2009.06.050

    Scopus

    PubMed

    researchmap

  • Stator assembly and activation mechanism of the flagellar motor by the periplasms region of MotB 査読

    Seiji Kojima, Katsumi Imada, Mayuko Sakuma, Yuki Sudo, Chojiro Kojima, Tohru Minamino, Michio Homma, Keiichi Namba

    Molecular Microbiology   73 ( 4 )   710 - 718   2009年8月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1111/j.1365-2958.2009.06802.x

    Scopus

    PubMed

    researchmap

  • Comparative study of the ion flux pathway in stator units of proton- and sodium-driven flagellar motors 査読

    Yuki Sudo, Hiroyuki Terashima, Rei Abe-Yoshizumi, Seiji Kojima, Michio Homma

    Biophysics   5   45 - 52   2009年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.2142/biophysics.5.45

    Scopus

    researchmap

  • Structural changes of Salinibacter sensory rhodopsin I upon formation of the K and M photointermediates 査読

    Daisuke Suzuki, Yuki Sudo, Yuji Furutani, Hazuki Takahashi, Michio Homma, Hideki Kandori

    Biochemistry   47 ( 48 )   12750 - 12759   2008年12月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1021/bi801358b

    Scopus

    PubMed

    researchmap

  • Salinibacter sensory rhodopsin: Sensory rhodopsin I-like protein from a eubacterium 査読

    Tomomi Kitajima-Ihara, Yuji Furutani, Daisuke Suzuki, Kunio Ihara, Hideki Kandori, Michio Homma, Yuki Sudo

    Journal of Biological Chemistry   283 ( 35 )   23533 - 23541   2008年8月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Halobacterium salinarum sensory rhodopsin I (HsSRI), a dual receptor regulating both negative and positive phototaxis in haloarchaea, transmits light signals through changes in protein-protein interactions with its transducer, halobacterial transducer protein I (HtrI). Haloarchaea also have another sensor pigment, sensory rhodopsin II (SRII), which functions as a receptor regulating negative phototaxis. Compared with HsSRI, the signal relay mechanism of SRII is well characterized because SRII from Natronomonus pharaonis (NpSRII) is much more stable than HsSRI and HsSRII, especially in dilute salt solutions and is much more resistant to detergents. Two genes encoding SRI homologs were identified from the genome sequence of the eubacterium Salinibacter ruber. Those sequences are distantly related to HsSRI (∼40% identity) and contain most of the amino acid residues identified as necessary for its function. To determine whether those genes encode functional protein(s), we cloned and expressed them in Escherichia coli. One of them (SrSRI) was expressed well as a recombinant protein having all-trans retinal as a chromophore. UV-Vis, low-temperature UVVis, pH-titration, and flash photolysis experiments revealed that the photochemical properties of SrSRI are similar to those of HsSRI. In addition to the expression system, the high stability of SrSRI makes it possible to prepare large amounts of protein and enables studies of mutant proteins that will allow new approaches to investigate the photosignaling process of SRI-HtrI. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

    DOI: 10.1074/jbc.M802990200

    Scopus

    PubMed

    researchmap

  • A long-lived M-like state of phoborhodopsin that mimics the active state 査読

    Yuki Sudo, Tatsuya Nishihori, Masayuki Iwamoto, Kazumi Shimono, Chojiro Kojima, Naoki Kamo

    Biophysical Journal   95 ( 2 )   753 - 760   2008年7月

     詳細を見る

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

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

    DOI: 10.1529/biophysj.107.125294

    Scopus

    PubMed

    researchmap

  • Protein-protein interaction of a pharaonis halorhodopsin mutant forming a complex with pharaonis halobacterial transducer protein ii detected by fourier-transform infrared spectroscopy 査読

    Yuji Furutani, Motohiro Ito, Yuki Sudo, Naoki Kamo, Hideki Kandori

    Photochemistry and Photobiology   84 ( 4 )   874 - 879   2008年7月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(国際会議プロシーディングス)   出版者・発行元:WILEY-VCH  

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

    DOI: 10.1111/j.1751-1097.2008.00317.x

    Scopus

    PubMed

    researchmap

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

  • Steric constraint in the primary photoproduct of sensory rhodopsin II is a prerequisite for light-signal transfer to HtrII 査読

    Motohiro Ito, Yuki Sudo, Yuji Furutani, Takashi Okitsu, Akimori Wada, Michio Homma, John L. Spudich, Hideki Kandori

    Biochemistry   47 ( 23 )   6208 - 6215   2008年6月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1021/bi8003507

    Scopus

    PubMed

    researchmap

  • Structural changes of sensory rhodopsin I and its transducer protein are dependent on the protonated state of Asp76 査読

    Yuji Furutani, Hazuki Takahashi, Jun Sasaki, Yuki Sudo, John L. Spudich, Hideki Kandori

    Biochemistry   47 ( 9 )   2875 - 2883   2008年3月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1021/bi702050c

    Scopus

    PubMed

    researchmap

  • Structural changes in the O-decay accelerated mutants of pharaonis phoborhodopsin 査読

    Yuki Sudo, Yuji Furutani, Masayuki Iwamoto, Naoki Kamo, Hideki Kandori

    Biochemistry   47 ( 9 )   2866 - 2874   2008年3月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1021/bi701885k

    Scopus

    PubMed

    researchmap

  • FTIR Studies of Protein-Protein Interaction Changes between pharaonis Phoborhodopsin and its Cognate Transducer Protein 査読

    Yuji Furutani, Yuki Sudo, Hideki Kandori

    Current topics in biochemical research   10 ( 2 )   63 - 77   2008年

     詳細を見る

    記述言語:英語   出版者・発行元:Trivandrum : Research Trends  

    researchmap

  • Structural analysis of the phototactic transducer protein HtrII linker region from Natronomonas pharaonis 査読

    Kokoro Hayashi, Yuki Sudo, JunGoo Jee, Masaki Mishima, Hideyuki Hara, Naoki Kamo, Chojiro Kojima

    Biochemistry   46 ( 50 )   14380 - 14390   2007年12月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1021/bi701837n

    Scopus

    PubMed

    researchmap

  • Early photocycle structural changes in a bacteriorhodopsin mutant engineered to transmit photosensory signals 査読

    Yuki Sudo, Yuji Furutani, John L. Spudich, Hideki Kandori

    Journal of Biological Chemistry   282 ( 21 )   15550 - 15558   2007年5月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:American Society for Biochemistry and Molecular Biology Inc. (ASBMB)  

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

    DOI: 10.1074/jbc.M701271200

    Scopus

    PubMed

    researchmap

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

  • Interaction of the halobacterial transducer to a halorhodopsin mutant engineered so as to bind the transducer: Cl- circulation within the extracellular channel 査読

    Chisa Hasegawa, Takashi Kikukawa, Seiji Miyauchi, Akiteru Seki, Yuki Sudo, Megumi Kubo, Makoto Demura, Naoki Kamo

    Photochemistry and Photobiology   83 ( 2 )   293 - 302   2007年3月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(国際会議プロシーディングス)   出版者・発行元:2  

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

    DOI: 10.1562/2006-06-09-RA-916

    Scopus

    PubMed

    researchmap

  • Participation of the surface structure of Pharaonis phoborhodopsin, ppR and its A149S and A149V mutants, consisting of the C-terminal α-helix and E-F loop, in the complex-formation with the cognate transducer pHtrII, as revealed by site-directed 13C solid-state NMR 査読

    Izuru Kawamura, Yoichi Ikeda, Yuki Sudo, Masayuki Iwamoto, Kazumi Shimono, Satoru Yamaguchi, Satoru Tuzi, Hazime Saitô, Naoki Kamo, Akira Naito

    Photochemistry and Photobiology   83 ( 2 )   339 - 345   2007年3月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(国際会議プロシーディングス)  

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

    DOI: 10.1562/2006-06-20-RA-940

    Scopus

    PubMed

    researchmap

  • Functional importance of the interhelical hydrogen bond between Thr 204 and Tyr174 of sensory rhodopsin II and its alteration during the signaling process 査読

    Yuki Sudo, Yuji Furutani, Hideki Kandori, John L. Spudich

    Journal of Biological Chemistry   281 ( 45 )   34239 - 34245   2006年11月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:American Society for Biochemistry and Molecular Biology Inc. (ASBMB)  

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

    DOI: 10.1074/jbc.M605907200

    Scopus

    PubMed

    researchmap

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

  • Assignment of the hydrogen-out-of-plane and -in-plane vibrations of the retinal chromophore in the K intermediate of pharaonis phoborhodopsin 査読

    Yuji Furutani, Yuki Sudo, Akimori Wada, Masayoshi Ito, Kazumi Shimono, Naoki Kamo, Hideki Kandori

    Biochemistry   45 ( 39 )   11836 - 11843   2006年10月

     詳細を見る

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

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

    DOI: 10.1021/bi0610597

    Scopus

    PubMed

    researchmap

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

  • Temperature-dependent interactions between photoactivated Pharaonis phoborhodopsin and its transducer 査読

    Kentaro Kamada, Yuji Furutani, Yuki Sudo, Naoki Kamo, Hideki Kandori

    Biochemistry   45 ( 15 )   4859 - 4866   2006年4月

     詳細を見る

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

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

    DOI: 10.1021/bi060047i

    Scopus

    PubMed

    researchmap

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

  • Importance of specific hydrogen bonds of archaeal rhodopsins for the binding to the transducer protein 査読

    Yuki Sudo, Masaki Yamabi, Shinnosuke Kato, Chisa Hasegawa, Masayuki Iwamoto, Kazumi Shimono, Naoki Kamo

    Journal of Molecular Biology   357 ( 4 )   1274 - 1282   2006年4月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1016/j.jmb.2006.01.061

    Scopus

    PubMed

    researchmap

  • Steric constraint in the primary photoproduct of an archaeal rhodopsin from regiospecific perturbation of C-D stretching vibration of the retinyl chromophore 査読

    Yuki Sudo, Yuji Furutani, Akimori Wada, Masayoshi Ito, Naoki Kamo, Hideki Kandori

    Journal of the American Chemical Society   127 ( 46 )   16036 - 16037   2005年11月

     詳細を見る

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

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

    DOI: 10.1021/ja056203a

    Scopus

    PubMed

    researchmap

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

  • 1P270 トランスジューサタンパクがハロロドプシンの光化学反応に及ぼす影響(光生物 A) 視覚・光受容))

    長谷川 千紗, 須藤 雄気, 下野 和実, 宮内 正二, 出村 誠, 加茂 直樹

    生物物理   45 ( 1 )   2005年10月

     詳細を見る

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

    CiNii Article

    researchmap

  • Linker region of a halobacterial transducer protein interacts directly with its sensor retinal protein 査読

    Yuki Sudo, Hideyasu Okuda, Masaki Yamabi, Yuta Fukuzaki, Masaki Mishima, Naoki Kamo, Chojiro Kojima

    Biochemistry   44 ( 16 )   6144 - 6152   2005年4月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1021/bi047573z

    Scopus

    PubMed

    researchmap

  • Structural changes of the complex between pharaonis phoborhodopsin and its cognate transducer upon formation of the M photointermediate 査読

    Yuji Furutani, Kentaro Kamada, Yuki Sudo, Kazumi Shimono, Naoki Kamo, Hideki Kandori

    Biochemistry   44 ( 8 )   2909 - 2915   2005年3月

     詳細を見る

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

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

    DOI: 10.1021/bi047893i

    Scopus

    PubMed

    researchmap

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

  • Correlation of the O-intermediate rate with the pKa of Asp-75 in the dark, the counterion of the Schiff base of pharaonis phoborhodopsin (sensory rhodopsin II) 査読

    Masayuki Iwamoto, Yuki Sudo, Kazumi Shimono, Tsunehisa Araiso, Naoki Kamo

    Biophysical Journal   88 ( 2 )   1215 - 1223   2005年

     詳細を見る

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

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

    DOI: 10.1529/biophysj.104.045583

    Scopus

    PubMed

    researchmap

  • Role of charged residues of pharaonis phoborhodopsin (sensory rhodopsin II) in its interaction with the transducer protein 査読

    Yuki Sudo, Masayuki Iwamoto, Kazumi Shimono, Naoki Kamo

    Biochemistry   43 ( 43 )   13748 - 13754   2004年11月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1021/bi048803c

    Scopus

    PubMed

    researchmap

  • Transient movement of helix F revealed by photo-induced inactivation by reaction of a bulky SH-reagent to cysteine-introduced pharaonis phoborhodopsin (sensory rhodopsin II) 査読

    Hideaki Yoshida, Yuki Sudo, Kazumi Shimono, Masayuki Iwamoto, Naoki Kamo

    Photochemical and Photobiological Sciences   3 ( 6 )   537 - 542   2004年6月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1039/b315454h

    Scopus

    PubMed

    researchmap

  • Proton Release and Uptake of pharaonis Phoborhodopsin (Sensory Rhodopsin II) Reconstituted into Phospholipids 査読

    Masayuki Iwamoto, Chisa Hasegawa, Yuki Sudo, Kazumi Shimono, Tsunehisa Araiso, Naoki Kamo

    Biochemistry   43 ( 11 )   3195 - 3203   2004年3月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1021/bi035960n

    Scopus

    PubMed

    researchmap

  • Role of Arg-72 of pharaonis Phoborhodopsin (Sensory Rhodopsin II) on its Photochemistry 査読

    Yukako Ikeura, Kazumi Shimono, Masayuki Iwamoto, Yuki Sudo, Naoki Kamo

    Biophysical Journal   86 ( 5 )   3112 - 3120   2004年

     詳細を見る

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

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

    DOI: 10.1016/S0006-3495(04)74359-3

    Scopus

    PubMed

    researchmap

  • Conformation and Dynamics of the [3-13C]Ala,[1- 13C]Val-Labeled Truncated pharaonis Transducer, pHtrll(1-159), as Revealed by Site-Directed 13C Solid-State NMR: Changes Due to Association with Phoborhodopsin (Sensory Rhodopsin II) 査読

    Satoru Yamaguchi, Kazumi Shimono, Yuki Sudo, Satoru Tuzi, Akira Naito, Naoki Kamo, Hazime Saitô

    Biophysical Journal   86 ( 5 )   3131 - 3140   2004年

     詳細を見る

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

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

    DOI: 10.1016/S0006-3495(04)74361-1

    Scopus

    PubMed

    researchmap

  • Hydrogen Bonding Alteration of Thr-204 in the Complex between pharaonis Phoborhodopsin and Its Transducer Protein 査読

    Yuki Sudo, Yuji Furutani, Kazumi Shimono, Naoki Kamo, Hideki Kandori

    Biochemistry   42 ( 48 )   14166 - 14172   2003年12月

     詳細を見る

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

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

    DOI: 10.1021/bi035678g

    Scopus

    PubMed

    researchmap

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

  • Interaction of Natronobacterium pharaonis Phoborhodopsin (Sensory Rhodopsin II) with its Cognate Transducer Probed by Increase in the Thermal Stability

    Yuki Sudo, Masaki Yamabi, Masayuki Iwamoto, Kazumi Shimono, Naoki Kamo

    Photochemistry and Photobiology   78 ( 5 )   511 - 516   2003年11月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

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

    Scopus

    PubMed

    researchmap

  • 7回膜貫通型膜蛋白質phR及びppRの溶液NMR

    奥田 秀泰, 須藤 雄気, 三島 正規, 佐藤 麻希, 出村 誠, 新田 勝利, 加茂 直樹, 児嶋 長次郎

    生物物理   43 ( 1 )   2003年8月

     詳細を見る

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

    CiNii Article

    researchmap

  • Importance of the broad regional interaction for spectral Tuning in natronobacterium pharaonis phoborhodopsin (sensory rhodopsin II) 査読

    Kazumi Shimono, Takanori Hayashi, Yukako Ikeura, Yuki Sudo, Masayuki Iwamoto, Naoki Kamo

    Journal of Biological Chemistry   278 ( 26 )   23882 - 23889   2003年7月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1074/jbc.M301200200

    Scopus

    PubMed

    researchmap

  • FTIR spectroscopy of the complex between pharaonis phoborhodopsin and its transducer protein 査読

    Yuji Furutani, Yuki Sudo, Naoki Kamo, Hideki Kandori

    Biochemistry   42 ( 17 )   4837 - 4842   2003年5月

     詳細を見る

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

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

    DOI: 10.1021/bi034317y

    Scopus

    PubMed

    researchmap

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

  • Dynamic structure of pharaonis phoborhodopsin (sensory rhodopsin II) and complex with a cognate truncated transducer as revealed by site-directed 13C solid-state NMR 査読

    Tadashi Arakawa, Kazumi Shimono, Satoru Yamaguchi, Satoru Tuzi, Yuki Sudo, Naoki Kamo, Hazime Saitô

    FEBS Letters   536 ( 1-3 )   237 - 240   2003年2月

     詳細を見る

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

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

    DOI: 10.1016/S0014-5793(03)00065-6

    Scopus

    PubMed

    researchmap

  • Arg-72 of pharaonis Phoborhodopsin (Sensory Rhodopsin II) is Important for the Maintenance of the Protein Structure in the Solubilized State 査読

    Yukako Ikeura, Kazumi Shimono, Masayuki Iwamoto, Yuki Sudo, Naoki Kamo

    Photochemistry and Photobiology   77 ( 1 )   96 - 100   2003年1月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

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

    Scopus

    PubMed

    researchmap

  • Illumination accelerates the decay of the O-intermediate of pharaonis phoborhodopsin (sensory rhodopsin II) 査読

    Masayuki Iwamoto, Yuki Sudo, Kazumi Shimono, Naoki Kamo

    Photochemistry and Photobiology   76 ( 4 )   462 - 466   2002年10月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

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

    Scopus

    PubMed

    researchmap

  • Association between a photo-intermediate of a M-lacking mutant D75N of pharaonis phoborhodopsin and its cognate transducer 査読

    Yuki Sudo, Masayuki Iwamoto, Kazumi Shimono, Naoki Kamo

    Journal of Photochemistry and Photobiology B: Biology   67 ( 3 )   171 - 176   2002年7月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1016/S1011-1344(02)00322-6

    Scopus

    PubMed

    researchmap

  • Association of pharaonis phoborhodopsin with its cognate transducer decreases the photo-dependent reactivity by water-soluble reagents of azide and hydroxylamine 査読

    Yuki Sudo, Masayuki Iwamoto, Kazumi Shimono, Naoki Kamo

    Biochimica et Biophysica Acta - Biomembranes   1558 ( 1 )   63 - 69   2002年1月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1016/S0005-2736(01)00423-0

    Scopus

    PubMed

    researchmap

  • Tyr-199 and charged residues of pharaonis phoborhodopsin are important for the interaction with its transducer 査読

    Yuki Sudo, Masayuki Iwamoto, Kazumi Shimono, Naoki Kamo

    Biophysical Journal   83 ( 1 )   427 - 432   2002年

     詳細を見る

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

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

    DOI: 10.1016/S0006-3495(02)75180-1

    Scopus

    PubMed

    researchmap

  • Role of Asp193 in chromophore-protein interaction of pharaonis phoborhodopsin (sensory rhodopsin II) 査読

    Masayuki Iwamoto, Yuji Furutani, Yuki Sudo, Kazumi Shimono, Hideki Kandori, Naoki Kamo

    Biophysical Journal   83 ( 2 )   1130 - 1135   2002年

     詳細を見る

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

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

    DOI: 10.1016/S0006-3495(02)75236-3

    Scopus

    PubMed

    researchmap

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

  • Environment around the chromophore in pharaonis phoborhodopsin: Mutation analysis of the retinal binding site 査読

    Kazumi Shimono, Yukako Ikeura, Yuki Sudo, Masayuki Iwamoto, Naoki Kamo

    Biochimica et Biophysica Acta - Biomembranes   1515 ( 2 )   92 - 100   2001年12月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1016/S0005-2736(01)00394-7

    Scopus

    PubMed

    researchmap

  • Selective reaction of hydroxylamine with chromophore during the photocycle of pharaonis phoborhodopsin 査読

    Masayuki Iwamoto, Yuki Sudo, Kazumi Shimono, Naoki Kamo

    Biochimica et Biophysica Acta - Biomembranes   1514 ( 1 )   152 - 158   2001年9月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1016/S0005-2736(01)00380-7

    Scopus

    PubMed

    researchmap

  • Pharaonis phoborhodopsin binds to its cognate truncated transducer even in the presence of a detergent with a 1:1 stoichiometry 査読

    Yuki Sudo, Masayuki Iwamoto, Kazumi Shimono, Naoki Kamo

    Photochemistry and Photobiology   74 ( 3 )   489 - 494   2001年9月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

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

    Scopus

    PubMed

    researchmap

  • Photo-induced proton transport of pharaonis phoborhodopsin (sensory rhodopsin II) is ceased by association with the transducer 査読

    Yuki Sudo, Masayuki Iwamoto, Kazumi Shimono, Masato Sumi, Naoki Kamo

    Biophysical Journal   80 ( 2 )   916 - 922   2001年

     詳細を見る

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

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

    DOI: 10.1016/S0006-3495(01)76070-5

    Scopus

    PubMed

    researchmap

▼全件表示

MISC

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

    Shin Nakao, Keiichi Kojima, Yuki Sudo

    Biological and Pharmaceutical Bulletin   44 ( 10 )   1357 - 1363   2021年10月

     詳細を見る

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

    DOI: 10.1248/bpb.b21-00544

    researchmap

  • マルチタレント光受容タンパク質「ロドプシン 招待

    須藤雄気, 小島慧一

    現代化学   5 ( 602 )   50 - 53   2021年5月

     詳細を見る

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

    J-GLOBAL

    researchmap

  • 微生物ロドプシンの多様性と可能性

    須藤雄気, 小島慧一

    生物物理(Web)   60 ( 4 )   2020年

     詳細を見る

  • 微生物型ロドプシンTRのX線結晶構造解析

    水谷健二, 橋本直記, 塚本卓, 須藤雄気, 村田武士

    KEK Progress Report (Web)   ( 2016-8 )   ROMBUNNO.242 (WEB ONLY)   2017年1月

     詳細を見る

    記述言語:日本語  

    J-GLOBAL

    researchmap

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

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

    Biophys. Rev.   9 ( 6 )   861 - 876   2017年

     詳細を見る

  • 光エネルギー変換の新常識! – 光駆動2価多原子アニオン輸送体の発見とその分光特性

    仁保亜希子, 須藤雄気

    academist Journal   2017年

     詳細を見る

    担当区分:最終著者, 責任著者  

    researchmap

  • Structural and functional studies on photoactive retinal proteins

    Yuki Sudo*

    Yakugaku Zasshi, Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan   136 ( 2 )   185 - 189   2016年2月

     詳細を見る

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

    DOI: 10.1248/yakushi.15-00229-3

    Web of Science

    J-GLOBAL

    researchmap

  • オプトジェネティクス

    須藤雄気

    光と生命の事典 日本光生物学協会「光と生命の事典」編集委員会 編 [朝倉書店]   372 - 373   2016年

     詳細を見る

  • 光受容レチナールタンパク質の構造・機能解析:光+タンパク質=薬!?

    須藤雄気

    薬学雑誌   136   185 - 189   2016年

  • Color tuning in retinylidene proteins

    Kota Katayama, Sivakumar Sekharan, Yuki Sudo

    Optogenetics: Light-Sensing Proteins and their Applications   89 - 109   2015年1月

     詳細を見る

    記述言語:英語   出版者・発行元:Springer Japan  

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

    DOI: 10.1007/978-4-431-55516-2_7

    Scopus

    researchmap

  • Microbial rhodopsins: wide distribution, rich diversity and great potential

    Kurihara M, Sudo Y

    BIophysics and Physicobiology   12   121 - 129   2015年

  • ビタミンAアルデヒドを発色団とするレチナールタンパク質の多様性と可能性

    土井聡子, 須藤雄気

    ビタミン学会誌   89 ( 2 )   83 - 86   2015年

  • 好熱性細菌のレチナールタンパク質

    塚本卓, 須藤雄気

    生物物理   55 ( 2 )   092-094 (J-STAGE) - 94   2015年

     詳細を見る

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

    DOI: 10.2142/biophys.55.092

    CiNii Article

    J-GLOBAL

    researchmap

  • Sensory rhodopsins

    Tsukamoto, T, Sudo, Y

    eLS (Encyclopedia of Life Sciences)   2014年

  • 膜タンパク質の可溶化(抽出)(2)

    須藤雄気、塚本卓

    蛋白質科学会・アーカイブ   7   e079   2014年

     詳細を見る

    担当区分:筆頭著者, 責任著者  

    researchmap

  • オプトジェネティクス(光遺伝学)の原理と基礎

    須藤雄気, 神取秀樹

    ファルマシア   50 ( 10 )   958 - 962   2014年

     詳細を見る

    担当区分:筆頭著者  

    J-GLOBAL

    researchmap

  • 正方形の古細菌が持つ光受容タンパク質の特徴的な構造変化

    須藤雄気

    分子研レターズ   68   2013年

     詳細を見る

  • 高度好塩性微生物の“目”:センサリーロドプシンへのCl<sup>-</sup>イオン結合の役割

    須藤雄気, 須藤雄気, 井原邦夫, 本間道夫, 加茂直樹

    極限環境生物学会誌   10 ( 1 )   2011年

     詳細を見る

  • Phototactic and chemotactic signal transduction by transmembrane receptors and transducers in microorganisms

    Daisuke Suzuki, Hiroki Irieda, Michio Homma, Ikuro Kawagishi, Yuki Sudo

    Sensors   10 ( 4 )   4010 - 4039   2010年4月

     詳細を見る

    記述言語:英語   掲載種別:書評論文,書評,文献紹介等  

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

    DOI: 10.3390/s100404010

    Scopus

    PubMed

    researchmap

  • 光で/を動かす技術 光で動きを変える微生物-ロドプシン分子による光受容と情報伝達機構-

    割石学, 本間道夫, 須藤雄気

    O plus E   ( 366 )   2010年

     詳細を見る

  • 真正細菌から得られた2つの機能をもつフォトクロミック光受容体ホモログ

    須藤雄気

    生物物理   49 ( 1 )   2009年

     詳細を見る

  • 光駆動イオンポンプから光情報伝達への機能変換の試み

    須藤雄気

    生物物理   46 ( 6 )   2006年

     詳細を見る

  • Close Up実験法 Series144 無細胞タンパク質合成系を用いた膜タンパク質発現の新規手法

    須藤雄気, 須藤雄気, 河野俊之, 児嶋長次郎

    実験医学   23 ( 12 )   2005年

     詳細を見る

▼全件表示

講演・口頭発表等

  • Molecular-based rational design and engineering of microbial retinal proteins for optogenetics

    The 16th International Conference on Retinal Proteins  2014年 

     詳細を見る

  • レチナールタンパク質による光合成モドキが 世界を救う!?

    自然科学研究機構 分間連携ワークショップ  2014年 

     詳細を見る

  • Rational Design and Engineering of Photoactive Retinal Proteins

    The 2nd Awaji International Workshop on “Electron Spin Science & Technology: Biological and Materials Science Oriented Applications (2nd AWEST 2014)  2014年 

     詳細を見る

  • Converting a Light-driven Proton Pump into a Light-gated Ion Channel

    The 52th Annual Meeting of the Biophysical Society of Japa  2014年 

     詳細を見る

産業財産権

  • 膜電位センサー

    坂本 雅行, チョウ シャオミン, 尾藤 晴彦, 須藤 雄気, 小島 慧一

     詳細を見る

    出願人:国立大学法人 東京大学

    出願番号:特願2020-070136  出願日:2020年4月9日

    公開番号:特開2021-167731  公開日:2021年10月21日

    J-GLOBAL

    researchmap

受賞

  • 文部科学大臣表彰 若手科学者賞

    2016年4月  

     詳細を見る

    受賞国:日本国

    researchmap

  • 研究者表彰

    2010年3月   財団法人光科学技術研究振興財団  

     詳細を見る

共同研究・競争的資金等の研究

  • ロドプシン基底関数の理解と利用

    研究課題/領域番号:21H02446  2021年04月 - 2024年03月

    日本学術振興会  科学研究費助成事業 基盤研究(B)  基盤研究(B)

    須藤 雄気

      詳細を見る

    配分額:17420000円 ( 直接経費:13400000円 、 間接経費:4020000円 )

    researchmap

  • ロドプシンを起動分子とした「化学・力学・光」エネルギー発動機構の理解と利用

    研究課題/領域番号:21H00404  2021年04月 - 2023年03月

    日本学術振興会  科学研究費助成事業 新学術領域研究(研究領域提案型)  新学術領域研究(研究領域提案型)

    須藤 雄気

      詳細を見る

    配分額:5980000円 ( 直接経費:4600000円 、 間接経費:1380000円 )

    researchmap

  • 光誘起崩壊リポソーム(LiDL)の開発による新奇薬物送達手法の確立

    研究課題/領域番号:20K21482  2020年07月 - 2023年03月

    日本学術振興会  科学研究費助成事業 挑戦的研究(萌芽)  挑戦的研究(萌芽)

    須藤 雄気, 山田 勇磨

      詳細を見る

    配分額:6500000円 ( 直接経費:5000000円 、 間接経費:1500000円 )

    researchmap

  • ロドプシンによる葉緑体プロトン勾配制御システムの確立と植物応答解析への展開

    研究課題/領域番号:19H04727  2019年04月 - 2021年03月

    日本学術振興会  科学研究費助成事業 新学術領域研究(研究領域提案型)  新学術領域研究(研究領域提案型)

    須藤 雄気

      詳細を見る

    配分額:7540000円 ( 直接経費:5800000円 、 間接経費:1740000円 )

    植物による光合成は、水と二酸化炭素から炭素固定・酸素発生・ATP産生を行う反応であある。ここで、生物のエネルギー通貨とも呼ばれるATPは、光合成タンパク質における細胞内から細胞外にプロトン(H+)の輸送により実現している。また、植物には光強度にあわせて余剰なエネルギーを熱として放出する機構(Non Photochemical Quenching:NPQ)が備わっており、効率的な光合成を実現している。これらは光合成に伴う葉緑体ルーメン側の酸性化(プロトン濃度上昇)が引き金になることがわかっているが、その制御機構の詳細は不明である。
    本研究では、光合成色素クロロフィルがほとんど吸収しない緑色光で働くロドプシンを緑藻(クラミドモナス)および陸上植物(シロイヌナズナ)の葉緑体に異種発現させる組み換え体を創出する。次に、ロドプシンを光により励起し、人為的に膜を介したプロトン移動を誘起する。これにより、擬似的に強・弱光条件を作り出し、その際に起こる植物応答(ATP合成、NPQ制御、成長、形態、その他)を光で制御し、それらのメカニズムの解明を目指す。
    <BR>
    本年度は、以下の生化学的・細胞生物学的解析を行った。
    ①生化学的解析:クラミドモナスおよびシロイヌナズナにおけるロドプシンの発現を検討する。加えて、葉緑体への局在を中心に確認する。具体的には、確認用の抗体の検討および細かな実験条件の設定を行った。クラミドモナスおよびシロイヌナズナともに、ロドプシンの葉緑体への局在を示唆する結果が得られ、計画は順調に進んでいる。
    ②細胞生物学的解析:NPQをはじめとした応答解析を進めた。(1)クラミドモナスについては、NPQ誘導の確認に加え、細胞形態や生育などへのロドプシンおよび光の影響を検討した。(2)シロイヌナズナについては、上記に加えレチナールの添加法の検討と、レチナールが及ぼす細胞毒性について検討した。

    researchmap

  • ロドプシンを起動分子とした「化学・力学・光」エネルギー発動機構の理解と利用

    研究課題/領域番号:19H05396  2019年04月 - 2021年03月

    日本学術振興会  科学研究費助成事業 新学術領域研究(研究領域提案型)  新学術領域研究(研究領域提案型)

    須藤 雄気

      詳細を見る

    配分額:5850000円 ( 直接経費:4500000円 、 間接経費:1350000円 )

    ロドプシンは光エネルギーを吸収し、レチナールの異性化を介して化学エネルギーへと変換する。化学エネルギーは、タンパク質の構造変化として力学エネルギーに変換され、分子機能が発現する。また、ロドプシンは、蛍光を発する特性を有し、光エネルギーにも変換可能である。このように、本領域における『発動分子』の定義(外部エネルギーを別エネルギーに変えるもの)から、ロドプシンはまさに“発動分子”そのものと言える。このような背景のもと、本研究では、ロドプシンによる『光-->化学・力学・光』エネルギーへの変換機構の理解と光遺伝学的利用を行うことで、ロドプシン型『発動分子』の基礎学理構築を行うことを目的とした。
    <BR>
    本年度は、以下に示す多様なロドプシンのマルチ『光-->化学・力学・光』発動機構(エネルギー変換)の理解を進めた。 さらに、これらを基盤に、分子機能(速度,収率,構造変化,生理応答,発光)の合理的改変の試みと光遺伝学への展開を行った。
    1)『光-->化学』:多様なロドプシンに対し、“時間”分解測定(過渡吸収,ラマン・赤外)及び“空間”分解測定(ラマン・赤外,X線結晶構造,NMR)を行い異性化速度,量子収率,異性化に伴うレチナール及びタンパク質の構造変化を調べた。得られた定量的な数値とその比較から、『光-->化学』エネルギー変換の分子機構の理解を進めた。
    (2)『光-->(化学)-->力学』:レチナールの異性化を引き金するロドプシンの構造変化,他分子との相互作用とその変化を、“時間”分解及び“空間”分解測定により明らかにした。加えて、生化学的・細胞生物学的解析を行い、構造と機能に関わる『化学-->力学』エネルギー変換機構を明らかにした。
    (3)『光-->光』:定常蛍光分光法と各種時間分解分光法を組み合わせ、多様なロドプシンの蛍光特性の定量的比較解析と発光機構の解明を行った。

    researchmap

  • ロドプシンの多様性の探求と可能性の追求

    研究課題/領域番号:18H02411  2018年04月 - 2021年03月

    日本学術振興会  科学研究費助成事業 基盤研究(B)  基盤研究(B)

    須藤 雄気

      詳細を見る

    配分額:17420000円 ( 直接経費:13400000円 、 間接経費:4020000円 )

    ロドプシンは、生物の三大ドメイン(真核生物・真正細菌・古細菌)に分布する光受容膜タンパク質の総称で、光エネルギー・情報変換を介し生命機能の根幹を司る:基礎的重要性。また、光遺伝学:オプトジェネティクスを生み出した分子として知られ、脳神経科学に利用されている:応用的重要性。このような重要性にもかかわらず、研究が行われているロドプシンはわずかであり、大部分は手つかずのまま残されている。そこで本研究は「ロドプシンの多様性の探究と可能性の追求」を目的とした。すなわち未解析ロドプシンの発現・精製(1. 探索)と精密解析(2. 解析)で多様性を探求し、基礎を強化する。さらに、それらの機能・特性を利用した新奇オプトジェネティクス(3. 操作)を実現し応用の可能性を追求することを目的とした。
    <BR>
    本年度は、昨年度に引き続き以下の3項目に取り組んだ。
    1.探索:(1)公開および未公開遺伝子情報から、様々な推定新規ロドプシン遺伝子を絞り込んだ。(2)これらの情報から分子系統樹を作成し、グループに分類した。(3)各グループ中央に位置する数種類と末端に位置する数種類を選抜した。(4)絞り込んだ遺伝子について、組み換え生物(大腸菌,古細菌,酵母,動物細胞)のコドンに最適化した遺伝子を合成し、発現プラスミドを作成した。タンパク質発現は宿主細胞の色により確認し、新奇ロドプシンの発現・精製系を構築した。
    2.解析:探索により発現・精製系を構築した新規ロドプシンの分子機能を様々な時空間領域「フェムト秒-ペタ秒・Å-ミリメートル」で、解析した。
    3.操作:解析が終了したロドプシンについて、その新奇機能や特性を生かした新しい光操作を実現した。特に4つのロドプシン(RmXeR,ACR2,SyHR, AR3)について、その特性を生かした新奇オプトジェネティクスを実現した。

    researchmap

  • ロドプシンによる葉緑体プロトン勾配制御システムの確立と植物応答解析への展開

    研究課題/領域番号:17H05726  2017年04月 - 2019年03月

    日本学術振興会  科学研究費助成事業 新学術領域研究(研究領域提案型)  新学術領域研究(研究領域提案型)

    須藤 雄気

      詳細を見る

    配分額:6500000円 ( 直接経費:5000000円 、 間接経費:1500000円 )

    植物による光合成は、水と二酸化炭素から、炭素固定・酸素発生・ATP産生を行う反応で、生命活動の源でもある。植物には、光強度にあわせて余剰なエネルギーを熱として放出する機構(Non photochemical quenching:NPQ)が備わっており、効率的な光合成を実現している。これらは、光合成に伴うルーメン側の酸性化(プロトン濃度上昇)が引き金になることがわかっているが、その制御機構はわかっていない。本研究では、光合成色素クロロフィルがほとんど吸収しない緑色光で働くロドプシンを植物の葉緑体に異種発現させ、人為的に膜を介したプロトン移動を誘起する。これにより、擬似的に強・弱光条件を再現し、その際に起こる植物応答を解析することで、NPQ制御メカニズムを解明することを目的としている。
    本年(H30年度)は、(1)内向きプロトンポンプロドプシンと外向きプロトンポンプロドプシンを植物モデルとしてのクラミドモナスおよびシロイヌナズナへの遺伝子導入を行い、その組み換え体においてそれぞれのロドプシン発現を確認した。植物細胞における異種ロドプシンの発現は世界初となる成果である。(2)クラミドモナスについては、NPQ測定を行い内向きプロトンポンプを発現した際に狙い通り光照射によりNPQが有意に増大することを確認した。外向きプロトンポンプでは若干の低下がみられた。このようにロドプシンを用いてNPQを制御することに世界で初めて成功した。シロイヌナズナについては、内向きプロトンポンプの発現は確認できたが、外向きプロトンポンプの発現は確認できなかった。
    以上のように、植物にロドプシンを発現させその生理応答(NPQ)を調節するという当初の目的は達成された。

    researchmap

  • カロテノイドを光捕集系とするレチナールタンパク質の創出と展開

    研究課題/領域番号:15H00878  2015年06月 - 2017年03月

    日本学術振興会  科学研究費助成事業 新学術領域研究(研究領域提案型)  新学術領域研究(研究領域提案型)

    須藤 雄気

      詳細を見る

    配分額:3900000円 ( 直接経費:3000000円 、 間接経費:900000円 )

    ロドプシン(レチナールタンパク質)は、動物から微生物まで幅広い生物が共通にもっている光受容体タンパク質であり、アポタンパク質を構成する7本の膜貫通αヘリックスの中央部に、アルデヒド型ビタミンAである発色団レチナールが結合した構造を持つ。ロドプシンは、クロロフィルの吸収がほとんどない領域の光(450-600 nm)を吸収することで機能する。具体的には、光吸収によりレチナールの異性化が起こり、続いて様々な特徴を持った光中間体(活性型)となることで、多彩な機能(視覚、ATP合成、膜電位の調節など)が発現する。本研究では、ロドプシンの反応を生物の光応答のモデルと捉え、新規分子の探索・解析とその高機能化を実現することで、人工光合成の可能性を拡げることを目的とした研究を行い、以下の3つの成果をあげた。
    <BR>
    [1] ロドプシンは一般に熱に不安定で、その不安定性は高機能化を行う上で支障になる。そこで、安定な分子の探索と解析を行い、安定化分子の取得に成功した。またそれらの高分解能構造を明らかにし、安定化機構の解明に成功した。
    [2] 新しい機能を持ったロドプシンの発見や創成は、新しい光生物応答制御を可能とする。そこで、新規ロドプシンの単離・取得・創成を行った。その結果、二価アニオン・SO42-を輸送する新規分子の発見・解析と輸送方向・基質の変換に成功した。
    [3] ロドプシンの光反応素子および光遺伝学ツールとしての有用性を高めるため、その高機能化を試みた。アミノ酸変異によるイオン輸送力の10倍の向上、カロテノイドアンテナ結合型分子の創成に成功した。
    これらの成果は、JACS誌2報、Sci. Rep.誌2報を含む原著論文として報告するなど、H27-28年の公募期間2年間の間に、当初想定した予想を超える成果をあげることが出来たと自負している。

    researchmap

  • 固体NMRによる光受容蛋白質ロドプシンのレチナール結合部位の精密構造解析法の開発

    研究課題/領域番号:15H04336  2015年04月 - 2018年03月

    日本学術振興会  科学研究費助成事業 基盤研究(B)  基盤研究(B)

    川村 出, 五東 弘昭, 須藤 雄気, 内藤 晶, 重田 安里寿, 槇野 義輝

      詳細を見る

    配分額:16380000円 ( 直接経費:12600000円 、 間接経費:3780000円 )

    本研究では、微生物型タンパク質であるロドプシンの発色団レチナールの結合部位の精密構造解析のために、レチナールオキシム体からアルデヒドへの変換反応の探索と高度好塩菌由来のバクテリオロドプシンから13C標識レチナールの抽出および固体NMRを用いたレチナール結合部位の構造解析を行った。その結果、オキシム体からレチナールへの効率的な変換反応の構築を達成し、13Cセグメント標識レチナールの生成に成功した。また、固体NMRを用いてセンサリーロドプシンIIやクロキノバクターロドプシン2などのレチナール結合部位の構造を明らかにした。さらに、光照射固体NMRによるセンサリーロドプシンIIの光中間体を観測した。

    researchmap

  • レチナールタンパク質を「知る・変える・役立てる」

    研究課題/領域番号:15H04363  2015年04月 - 2018年03月

    日本学術振興会  科学研究費助成事業 基盤研究(B)  基盤研究(B)

    須藤 雄気, 塚本 卓

      詳細を見る

    配分額:16380000円 ( 直接経費:12600000円 、 間接経費:3780000円 )

    ビタミンAのアルデヒド型であるレチナールを発色団とするタンパク質は、総称してレチナールタンパク質と呼ばれる。生物の三大ドメイン(動物・細菌・古細菌)に万を越える分子が広く分布し、様々な光依存性機能を担っている。このような生物学的興味に加え、近年レチナールタンパク質を利用し、細胞や個体の機能を光で操作する技術(光遺伝学)が確立してきた。本研究では、これまでの研究を礎に、レチナールタンパク質を様々な手法により根源的に理解し(知る)、その知見に基づいた分子機能の改変・創成を行い(変える)、さらには様々な生命科学研究に利用できる光操作ツールを開発する(役立てる)ことを目的とした研究を行い成果を挙げた。

    researchmap

  • 光照射固体NMRによる細菌型センサリーロドプシンの光活性構造変化の解明

    研究課題/領域番号:15K06963  2015年04月 - 2018年03月

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    内藤 晶, 川村 出, 須藤 雄気, 加茂 直樹, 和田 昭盛

      詳細を見る

    配分額:5200000円 ( 直接経費:4000000円 、 間接経費:1200000円 )

    細菌型光受容膜タンパク質は物質輸送や信号伝達に重要な役割を果たしている。発色団レチナールが光サイクルを回る間に光受容体としての機能が発現する。本研究では光サイクル中に生成する光中間体を固体NMRによって観測するため、光照射固体NMR装置の開発を行った。この装置を用いて、プロトン輸送活性をもつバクテリオロドプシンの変異体Y185F-bRを用いて、光サイクル中に生成するO-中間体やCS*-中間体のNMR信号の観測に成功した。次に負の光走性を示すppR/pHtrII複合体について光照射を行った結果、M-中間体からO-中間体が生成し、N’-中間体がO-中間体から平衡反応で生成することが判明した。

    researchmap

  • 先端的な超高速分光と非線形分光による多自由度複雑分子系の研究

    研究課題/領域番号:25104005  2013年06月 - 2018年03月

    日本学術振興会  科学研究費助成事業 新学術領域研究(研究領域提案型)  新学術領域研究(研究領域提案型)

    田原 太平, 竹内 佐年, 石井 邦彦, 山口 祥一, 須藤 雄気, 二本柳 聡史, 倉持 光

      詳細を見る

    配分額:141050000円 ( 直接経費:108500000円 、 間接経費:32550000円 )

    最高の分光計測を開発・駆使して多自由度複雑分子系の研究を行った。領域の研究者との共同研究を強力に進めながら多くの研究成果をあげた。特に(1)超高速分光では独自の時間分解インパルシブ誘導ラマン分光法の極限化を推し進めるとともに、種々の生体分子、超分子、機能性分子の超高速過程を解明した。(2)界面選択的非線形分光では、我々が開発したヘテロダイン検出振動和周波発生分光法を駆使して界面水構造を解明し、また二次元分光法や紫外励起時間分解測定に発展させて界面のダイナミクス研究を実現した。(3)単分子分光では、独自の二次元蛍光寿命相関分光法を開発してタンパク質の折り畳み過程を研究し、新しい重要な知見を得た。

    researchmap

  • 光応答性タンパク質の機能転換が明らかにする柔らかな構造機能相関

    研究課題/領域番号:25104009  2013年06月 - 2018年03月

    日本学術振興会  科学研究費助成事業 新学術領域研究(研究領域提案型)  新学術領域研究(研究領域提案型)

    神取 秀樹, 須藤 雄気, 井上 圭一, 岩田 達也, 片山 耕大, 山田 大智

      詳細を見る

    配分額:131300000円 ( 直接経費:101000000円 、 間接経費:30300000円 )

    多くの生体分子は共通の構造をもとに多彩な機能を演出している。本課題で我々は、ロドプシンやフラビンタンパク質などを対象として機能の発見・転換・創成をテーマに柔らかさと機能との関わりを研究した。その結果、内向きプロトンポンプや新規チャネルロドプシン、環状ヌクレオチドを光で分解する酵素ロドプシンなどの発見を報告した。一方、機能転換については、ロドプシンやDNA光回復酵素に対して限られた変異導入により機能転換に成功したが逆方向は成功せず、非対称な機能転換が明らかになった。機能の創成に関しては、光駆動ナトリウムポンプの構造基盤に基づき、カリウムやセシウムをポンプするタンパク質を創成することができた。

    researchmap

  • レチナールタンパク質の生物物理化学的解析

    研究課題/領域番号:13F03076  2013年04月 - 2015年03月

    日本学術振興会  科学研究費助成事業 特別研究員奨励費  特別研究員奨励費

    阿波賀 邦夫, 須藤 雄気, REISSIG Louisa, REISISIG Louisa

      詳細を見る

    配分額:2300000円 ( 直接経費:2300000円 )

    これまでの研究により超好熱菌から新しいレチナールタンパク質を発見し、サーモフィリックロドプシン・TRと命名している。この分子は、これまで見つかっているレチナールタンパク質の中で最も熱に対して安定な分子であった。この分子について、構造および構造変化を過渡吸収スペクトル(可視、赤外、ラマン、蛍光)によって追跡し、さらにNMR分光法を用いた発色団構造の決定に成功した。さらに今年度は、過渡光電流の発生が期待される [電極1(M)|電荷分離層(S)|絶縁分極層(I)|電極2(M’)] なる構造をもつ光学セルにおいて、その安定作動の最適化を行った後に、電荷分離層を光活性生体物質とすることによってより、環境応答型の光応答を求めた。
    [金属(M)|電荷分離層(S)|絶縁分極層(I)|金属(M’)]光電セルにおいて、絶縁分極層としてイオン液体(IL)を用いた系において、実用化に向けた検討を行った。この光電セルでは、界面電気二重層の形成による巨大電場によって電荷分離が促進されることが期待されている。近赤外外部に吸収をもつVOナフタロシアニンとC60の固溶体膜を電荷分離層とし、二つの電極を平行に同一基板上に配置したIL-MSIM光電セルの特性を調べところ、電気二重層の生成が電極間距離に依存しないことを利用し、電極間距離を7 mmに広げても過渡光電流を取り出せることが分かった。このように、透明電極を必要とせず、また電極の位置を厭わない柔軟性は、IL-MISM光電セル光検出器としての実用性を保証する。さらに、[M|S|I|M’]光電セルのM’電極を光ファイバーのジャケットとすることによって、On-tip型の光センサー構造を実現し、S層に生体物質を用い過渡光電流の検出に成功した。

    researchmap

  • 振動分光法による過渡的膜タンパク質複合体の解析

    研究課題/領域番号:24121712  2012年04月 - 2014年03月

    日本学術振興会  科学研究費助成事業 新学術領域研究(研究領域提案型)  新学術領域研究(研究領域提案型)

    須藤 雄気

      詳細を見る

    配分額:11310000円 ( 直接経費:8700000円 、 間接経費:2610000円 )

    細胞内外の情報や物質のやり取りを行う”膜タンパク質”は、全タンパク質分子の約20%を占め、生命活動に必須である。一方で、細胞膜中で機能するため取り扱いが難しい、発現量が少ないなどの理由からそのメカニズムの理解は遅れている。我々は、光受容体・ロドプシンと伝達膜タンパク質の“膜分子複合体の過渡的変化”を様々な手法により解析している。このうち、赤外分光(FTIR) 法やラマン分光法に代表される振動分光法は、分子振動を鋭敏に捉える手法で、側鎖, 主鎖, 低分子(イオンや水など)の微細構造変化を捉えることができ、X線結晶構造解析やNMR解析では得ることが難しい情報を取得することができる。本研究では以下の4点について研究を行い、成果を得た。本研究を通じて、膜蛋白質解析のボトルネックである、試料調製法や解析手法を確立できたと考えている。
    (1) 試料調製法の確立 [Sudo et al. 2013, J. Biol. Chem., Tsukamoto et al., 2013, J. Biol. Chem.], (2) 時間分解FTIR法による分子構造変化 [Furutani et al. 2013, J. Phys. Chem. B], (3) 時間分解ラマン分光法による分子構造変化 [Sudo et al. 2014, J. Phys. Chem. B], (4) 光照射固体NMR分光法による発色団構造変化 [Yomoda et al., 2014, Angew. Chem. Int. Ed.], (5) 全反射型FTIR法による2次構造測定 [東京大学船津研究室との共同研究]。

    researchmap

  • 光情報伝達のサブÅ・ピコ秒分解能での全経路詳細解析

    研究課題/領域番号:23687019  2011年04月 - 2015年03月

    日本学術振興会  科学研究費助成事業 若手研究(A)  若手研究(A)

    須藤 雄気, 塚本 卓

      詳細を見る

    配分額:27300000円 ( 直接経費:21000000円 、 間接経費:6300000円 )

    生体中に存在するタンパク質分子は、時々刻々とその形を変化させることで、様々な生理機能を担っている。そのため、タンパク質分子の本質的な理解には、「時々刻々=時間」・「形=空間」の2つの側面からの理解が重要となる。本研究では、光受容体の1種であるレチナールタンパク質による光情報伝達を、様々な「時間」および「空間」分解能で理解することを目的とした。

    researchmap

  • 色を知り、色を作る:ロドプシンタンパク質群の挑戦

    研究課題/領域番号:23657100  2011年 - 2013年

    日本学術振興会  科学研究費助成事業 挑戦的萌芽研究  挑戦的萌芽研究

    須藤 雄気, 林 重彦

      詳細を見る

    配分額:3900000円 ( 直接経費:3000000円 、 間接経費:900000円 )

    本研究は、実験及び理論の両面から、光受容レチナールタンパク質の発色機構を探り、得られた知見を基盤に、様々な色を呈する分子を創成することを目的とした。主な成果は以下の通りである。(1) 発色機構の実験的解明(4報)、(2) 発色機構の理論的解明(2報)、(3)様々な色を呈する分子の創成(2報)。このように実験と理論の融合により、色素タンパク質の新しい発色機構の解明とそれをもとにした分子創成は、様々な分野の研究者に取って有用な情報を提供するだろう。

    researchmap

  • センサー型ロドプシンの分子科学:機能と構造変化の連関性

    研究課題/領域番号:22018010  2010年 - 2011年

    日本学術振興会  科学研究費助成事業 特定領域研究  特定領域研究

    須藤 雄気

      詳細を見る

    配分額:4200000円 ( 直接経費:4200000円 )

    ロドプシン類は、光で反応のオン・オフを容易に調節できる点から、現在の生命科学研究の主題である構造-機能相関を分子論的に理解できる有用なツールである。本研究では微生物の光センサー型ロドプシンに着目し、機能と構造変化の連関性を明らかにすることを目的とした。当該年度の主な研究成果は以下の通りである。
    [1]新しいロドプシン類の単離・同定・発現・精製
    不安定で解析が困難だった誘因光受容体・センサリーロドプシンI(SRI)について、安定な分子を見いだした。また、新規ロドプシン分子を見いだしミドルロドプシン(MR)と名付けた。さらに、これら光センサータンパク質の下流分子(情報伝達に関わる分子群)についても単離・同定に成功し、発現・精製系を構築した。これにより、これまで困難であった測定が可能となった。
    [2]構造・構造変化・機能解析
    SRIや忌避光受容体・センサリーロドプシンII(SRH)を中心に、種々の分光法や生化学的・生物物理学的・分子生理学的手法を駆使して、タンパク質分子の形(構造)、やその変化(構造変化)を明らかにし、機能との連関性をアミノ酸レベルで明らかにした。SRHのTyr174の構造変化、SRIの体積変化など、物理化学的性質から、細菌運動解析など細胞生物学的解析までをうまく融合することができた。MRについては、光反応が極めて特徴的であることを見いだし、さらに光反応中における構造変化を時間分解分光法により明らかにした。

    researchmap

  • 全反射型赤外分光法による過渡的複合体の解析

    研究課題/領域番号:22121508  2010年 - 2011年

    日本学術振興会  科学研究費助成事業 新学術領域研究(研究領域提案型)  新学術領域研究(研究領域提案型)

    須藤 雄気

      詳細を見る

    配分額:11440000円 ( 直接経費:8800000円 、 間接経費:2640000円 )

    細胞内外の情報や物質のやり取りを行う"膜タンパク質"は、生命活動に重要である。我々は、光受容体・ロドプシンと伝達膜タンパク質の"膜分子複合体の過渡的変化"を様々な手法により解析している。このうち、赤外分光(FTIR)法やラマン分光法に代表される振動分光法は、分子振動を鋭敏に捉える手法で、側鎖,主鎖,低分子(イオンや水など)の微細構造変化を捉えることができ、X線結晶構造解析やNMR解析では得ることが難しい情報を取得することができる。本研究では以下の4点について研究を行い、成果を得た。本研究を通じて、膜蛋白質解析のボトルネックである、試料調製法や解析手法を確立できたと考えている。
    1)FTIR解析に適した試料調製[Sudo et al.2011a,J.Biol.Chem.,Sudo et al.2011,Biophysics]
    2)低温/時間分解赤外分光法によるロドプシンタンパク質の構造変化解析[Sudo et al.2011b,J.Biol.Chem.,Irieda et al.2011,Biochemistry]
    3)ラマン分光法など他の分光測定[Mizuno et al.,2011,Biochemistry,Inoue et al.,2011,J.Phys.Chem.B]
    4)全反射型FTIR(ATR-FTIR)におけるイオンと膜タンパク質の相互作用解析を行い、解析の難しい膜タンパク質の微細構造変化を明らかにした[投稿準備中]。

    researchmap

  • 2段階励起過渡回折格子法の開発と情報伝達タンパク質の光反応機構の解明と制御

    研究課題/領域番号:21770165  2009年 - 2010年

    日本学術振興会  科学研究費助成事業 若手研究(B)  若手研究(B)

    井上 圭一, 須藤 雄気

      詳細を見る

    配分額:4420000円 ( 直接経費:3400000円 、 間接経費:1020000円 )

    過渡回折格子法を用い、真正細菌Salinibacter ruber由来の細胞の走光性を引き起こすための光受容タンパク質、Sensory Rhodopsin Iの光反応ダイナミクスを調べた。その結果これまで知られていなかった中間体の存在を明らかにした。そしてさらにそれぞれの中間体のエンタルピーを決定することにも成功し、それらが塩化物イオンの結合によって大きく影響を受けることを突き止めた。

    researchmap

  • べん毛モーター固定子複合体の相互作用・イオン透過・構造変化の解析

    研究課題/領域番号:21770166  2009年 - 2010年

    日本学術振興会  科学研究費助成事業 若手研究(B)  若手研究(B)

    須藤 雄気

      詳細を見る

    配分額:4550000円 ( 直接経費:3500000円 、 間接経費:1050000円 )

    細菌は環境を認識するレセプターと運動器官(べん毛モーター)により過酷な環境を生き抜く。本研究では、全反射型赤外分光法により、これまで謎とされてきた固定子複合体(PomAB)とNa^+の結合にAsp24が直接関与すること(Sudo et al.2009b,Biochemistry)、その親和性と他に2つのカルボン酸が結合に関与することを明らかにした。変異体解析からCys31-PomBがイオン透過を制御すること(Sudo et al.2009,Biophysics)、サルモネラ菌の固定子・MotBのC末端の構造を明らかにした(Kojima et al.2009,Mol.Microbiol.)。さらに本研究の一部として、新規受容体を単離し物理化学的性質を明らかにした(Suzuki et al.2009,J.Mol.Biol.,Sudo et al.2009a,Biochemistry,Yagasaki et al.2010,Biochemistry,Sudo et al.2011,J.Biol.Chem.)。

    researchmap

  • 膜蛋白質の機能変換から観る機能-構造変化の連関性と分子論的理解

    研究課題/領域番号:20050012  2008年 - 2010年

    日本学術振興会  科学研究費助成事業 特定領域研究  特定領域研究

    須藤 雄気

      詳細を見る

    配分額:3400000円 ( 直接経費:3400000円 )

    2-1. 忌避応答光センサー蛋白質、SRIIの機能発現分子メカニズム
    現在阪大/水谷グループと共同で、機能・構造変化に必須のアミノ酸残基(Tyr174)の変化を検出するため、時間分解紫外共鳴ラマン測定を行っている。
    2-2. デュアル光センサー蛋白質、SRIの機能発現メカニズム
    (1)新しいSRI分子、SrSRIのCl^-依存的光反応変化を見出した[J.Mol.Biol. 2009](A01:藤井グループとの共同研究)。(2)新しいSRI分子、HvSRIの発現・精製と分光解析[Biochemistry 2010](A01:藤井グループとの共同研究)。(3)SrSRIと伝達タンパク質、SrHtrIの複合体の機能発現系構築に成功し、その分光学的解析を行った[Biochemistry 2009a](A01:藤井グループとの共同研究)。
    2-3. SRI-HtrIが制御するべん毛蛋白質の解析
    SRI-HtrIが受けた光情報は、最終的にべん毛モーターの回転方向の制御として出力される。ここでは、べん毛固定子タンパク質MotBの部分結晶構造と[Mol.Microbiol. 2009]、赤外分光法によるNa^+透過経路の検討を行った[Biochemistry 2009b]
    このように光センサータンパク質の機能発現機構について、分子科学的に迫ることが出来た。

    researchmap

  • 分子生理学的解析から探る膜蛋白質複合体の機能発現機構

    研究課題/領域番号:19870010  2007年 - 2008年

    日本学術振興会  科学研究費助成事業 若手研究(スタートアップ)  若手研究(スタートアップ)

    須藤 雄気

      詳細を見る

    配分額:3125000円 ( 直接経費:2720000円 、 間接経費:405000円 )

    1)SRII-HtrII膜蛋白質複合体を介した光情報伝達(忌避応答)
    代表者らは、これまでSRII-HtrIIを介した細菌の光忌避行動について、生化学的・生理学的検討を行ってきた。今年度は、固体NMRを用いたSRII-HtrIIの相互作用解析(雑誌論文7)、溶液NMRを用いたHtrIIの部分構造の決定(雑誌論文6)、安定な活性型中間体の発見(雑誌論文2)、赤外分光法(FTIR)を用いた構造変化の解析(雑誌論文5)を行った。さらに得られた結果を基に、様々な変異体での比較解析から構造変化と忌避応答性に相関があることを見出した(雑誌論文1)。この結果は、Biochemistry誌の注目論文(Hot Article)として掲載される予定である。
    2)SRI-HtrI膜蛋白質複合体を介した光情報伝達(誘因応答)
    上記SRII-HtrIIを介した忌避応答機構に比べ、SRI-HtrIを介した光誘因応答に関する解析は著しく遅れている。今年度は、構造変化解析に優れたFTIR分光法を用いて、SRI-HtrI複合体の構造変化解析を行った(雑誌論文4)。また、これまで用いてきたSRIが極めて不安定であることから、古細菌、真正細菌で新たなSRI遺伝子を探索し、発現を試みたところ、真正細菌であるSalinibacter ruberから極めて安定なSRIを単離することに成功した(論文投稿中)。このように、誘因応答メカニズムについてもその分子機構を理解できる目処がたってきた。
    3その他光受容体解析
    イオンポンプであるバクテリオロドプシン(BR)をSRII型へ機能転換した論文(2006年Sudo and Spudich, PNAS)が注目され、機能変換を通じた機能「創出」から蛋白質由来の人工光素子構築を目指している。今年度は、クロライドポンプであるハロロドプシン(HR)からSRIIへの機能変換を試みた(雑誌論文3)。上述の通り誘因レセプターも研究対象と出来る目処がつき、これまで知られているレセプター間での機能改変を行っている。

    researchmap

▼全件表示

 

担当授業科目

  • 先端薬学研究 (2021年度) 夏季集中  - その他

  • 分析科学・物理化学 (2021年度) 前期  - その他

  • 基礎物理学 (2021年度) 第1学期  - 火5,火6,金3,金4

  • 基礎物理学 (2021年度) 第1学期  - 火5,火6,金3,金4

  • 基礎物理学 (2021年度) 第1学期  - 火5,火6,金3,金4

  • 基礎物理学 (2021年度) 第1学期  - 火5,火6,金3,金4

  • 感じる科学 (2021年度) 第3学期  - 月3~4

  • 感じる科学 (2021年度) 第4学期  - 金3~4

  • 数理・データサイエンスの基礎 (2021年度) 第3学期  - 月5~6

  • 物理化学1 (2021年度) 第2学期  - 金5,金6

  • 物理化学1 (2021年度) 第2学期  - 金5,金6

  • 物理化学3 (2021年度) 第3学期  - 火7,火8

  • 物理化学3 (2021年度) 第3学期  - 火7~8

  • 物理化学4 (2021年度) 第1学期  - 木3,木4

  • 物理化学4 (2021年度) 第1学期  - 木3,木4

  • 物理化学5 (2021年度) 第2学期  - 木3,木4

  • 物理化学5 (2021年度) 第2学期  - 木3,木4

  • 物理化学A (2021年度) 2・3学期  - [第2学期]金5,金6, [第3学期]火1,火2

  • 物理化学A (2021年度) 2・3学期  - [第2学期]金5,金6, [第3学期]火1,火2

  • 物理化学B (2021年度) 3・4学期  - [第3学期]火7,火8, [第4学期]火1,火2

  • 物理化学B (2021年度) 3・4学期  - [第3学期]火7,火8, [第4学期]火1,火2

  • 生体分子解析学A (2021年度) 特別  - その他

  • 生体分子解析学A演習 (2021年度) 特別  - その他

  • 生体分子解析学I (2021年度) 特別  - その他

  • 生体分子解析学II (2021年度) 特別  - その他

  • 薬学ガイダンス (2021年度) 1・2学期  - [第1学期]水4, [第2学期]月1

  • 薬学ガイダンス (2021年度) 1・2学期  - [第1学期]水4, [第2学期]月1

  • 薬学ガイダンス (2021年度) 1・2学期  - [第1学期]水4, [第2学期]月1

  • 薬学ガイダンス (2021年度) 1・2学期  - [第1学期]水4, [第2学期]月1

  • 薬学基礎実習Ⅰ (2021年度) 第1学期  - その他6~9

  • 薬学基礎実習Ⅰ (2021年度) 第1学期  - その他6~9

  • 薬学基礎実習I (2021年度) 第1学期  - その他6~9

  • 薬学基礎実習I (2021年度) 第1学期  - その他6~9

  • 先端薬学特論 (2020年度) 特別  - その他

  • 先端薬学研究 (2020年度) 夏季集中  - その他

  • 分析科学・物理化学 (2020年度) 前期  - その他

  • 基礎物理学 (2020年度) 第1学期  - その他

  • 基礎物理学 (2020年度) 第1学期  - 火4,火5,火6

  • 基礎物理学 (2020年度) 第1学期  - 火4,火5,火6

  • 感じる科学 (2020年度) 第3学期  - 月3,月4

  • 感じる科学 (2020年度) 第4学期  - 金3,金4

  • 数理・データサイエンスの基礎 (2020年度) 第3学期  - 月5,月6

  • 物理化学Ⅲ (2020年度) 第2学期  - 木3,木4

  • 物理化学1 (2020年度) 第2学期  - 火5,火6

  • 物理化学1 (2020年度) 第2学期  - 火5,火6

  • 物理化学4 (2020年度) 第1学期  - 木3,木4

  • 物理化学4 (2020年度) 第1学期  - 木3,木4

  • 物理化学5 (2020年度) 第2学期  - 木3,木4

  • 物理化学5 (2020年度) 第2学期  - 木3,木4

  • 生体分子解析学A (2020年度) 特別  - その他

  • 生体分子解析学A演習 (2020年度) 特別  - その他

  • 生体分子解析学I (2020年度) 特別  - その他

  • 生体分子解析学II (2020年度) 特別  - その他

  • 薬学ガイダンス (2020年度) 1・2学期  - [第1学期]水4, [第2学期]月1

  • 薬学ガイダンス (2020年度) 1・2学期  - 月1,水4

  • 薬学ガイダンス (2020年度) 1・2学期  - 月1,水4

  • 薬学ガイダンス (2020年度) 1・2学期  - [第1学期]水4, [第2学期]月1

  • 薬学基礎実習I (2020年度) 特別  - その他

  • 薬学基礎実習I (2020年度) 特別  - その他

▼全件表示