光エネルギーで駆動される物質回収・放出カプセルのボトムアップ構築

• Project Area: Bottom-up creation of cell-free molecular systems: surpassing nature
• Project/Area Number: 22H05389
• Research Category: Grant-in-Aid for Transformative Research Areas (A)
• Allocation Type: Single-year Grants
• Review Section: Transformative Research Areas, Section (II)
• Research Institution: Hokkaido University
• Principal Investigator: 菊川 峰志 北海道大学, 先端生命科学研究院, 准教授 (20281842)
• Project Period (FY): 2022-06-16 – 2024-03-31
• Project Status: Completed (Fiscal Year 2023)
• Budget Amount: ¥9,620,000 (Direct Cost: ¥7,400,000、Indirect Cost: ¥2,220,000); Fiscal Year 2023: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2022: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000)
• Keywords: 膜輸送 / イオンポンプ / 微生物ロドプシン / 多剤排出蛋白質

Outline of Research at the Start

リポソームの脂質膜上に「光で駆動されるタンパク質」と「有害な溶質を膜輸送するタンパク質」を、通常の細胞膜中とは逆向きに組み込む。前者には、細胞内からH+やNa+を外向きに輸送する微生物ロドプシンを用いる。後者には、H+やNa+の濃度勾配を消費して、毒物を排出する多剤排出タンパク質を用いる。これらをエネルギー共役させ、太陽光をネルギー源としながら、環境中の毒物をリポソーム内へ効率良く回収する自己完結的な分子システムの構築を目指す。

Outline of Annual Research Achievements

リポソームの脂質膜上に、「光で駆動されるタンパク質」と「有害な溶質を膜輸送するタンパク質」の共役系構築を試みた。前者には、細胞内からH+やNa+を外向きに輸送する微生物ロドプシンを、後者には、H+やNa+の濃度勾配を消費して、有機毒物を排出する多剤排出タンパク質を用いた。これらを細胞膜中とは逆向きにリポソームに組込むことで、太陽光をネルギー源としながら、環境中の有機毒物をリポソーム内へ効率良く回収する自己完結的なシステムの構築を目指した。
本年度は、複数の多剤排出蛋白質の大腸菌での発現系を構築し、大腸菌における有機物質輸送活性と、大腸菌細胞膜からの精製収量を指標に、V. choleraeのNorM_VCを微生物ロドプシンと共役させる蛋白質として決定した。微生物ロドプシンには、H+ポンプ型のバクテリオロドプシン（BR）を採用し、リポソームのプロトタイプを作製した。NorMの基質には、Tetraphenyl phosphonium（TPP+）を用いて、その輸送をTPP+選択膜を用いて検出した。TPP+選択膜は、TPP+を透過させる薄膜である。この膜で二つの溶液を仕切ると、それぞれのTPP+濃度の比に応じたネルンスト電位が溶液間に発生する。一方の溶液を、BRとNorMを同時に再構成したリポソーム懸濁液とし、光照射によってBRを活性化させたところ、TPP+濃度の減少に対応する膜電位の上昇が観測された。BRのみを再構成したリポソームでは膜電位変化が観測されなかった。よって、両者を再構成した場合には、光をエネルギー源として、TPP+のリポソーム内への濃縮を起こせることがわかった。膜電位の変化量から、リポソーム内部には、外部溶液に比べて、TPP+を2.4倍濃縮できることがわかった。

Research Progress Status

令和5年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

令和5年度が最終年度であるため、記入しない。

Research Products

• [Journal Article] Configurational Changes of Retinal Schiff Base during Membrane Na⁺ Transport by a Sodium Pumping Rhodopsin (2024)
  • Author(s): Fujisawa Tomotsumi、Kinoue Kouta、Seike Ryouhei、Kikukawa Takashi、Unno Masashi
  • Journal Title: The Journal of Physical Chemistry Letters Volume: 15 Issue: 7 Pages: 1993-1998
  • DOI: 10.1021/acs.jpclett.3c03435
  • Peer Reviewed
• [Journal Article] Multistep conformational changes leading to the gate opening of light-driven sodium pump rhodopsin (2023)
  • Author(s): Sato Yukino、Hashimoto Tsubasa、Kato Koji、Okamura Akiko、Hasegawa Kaito、Shinone Tsukasa、Tanaka Yoshikazu、Tanaka Yoshiki、Tsukazaki Tomoya、Tsukamoto Takashi、Demura Makoto、Yao Min、Kikukawa Takashi
  • Journal Title: Journal of Biological Chemistry Volume: 299 Issue: 12 Pages: 105393-105393
  • DOI: 10.1016/j.jbc.2023.105393
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] The preferential transport of NO3- by full-length Guillardia theta anion channelrhodopsin 1 is enhanced by its extended cytoplasmic domain (2023)
  • Author(s): Ohki Yuya、Shinone Tsukasa、Inoko Sayo、Sudo Miu、Demura Makoto、Kikukawa Takashi、Tsukamoto Takashi
  • Journal Title: Journal of Biological Chemistry Volume: 299 Issue: 11 Pages: 105305-105305
  • DOI: 10.1016/j.jbc.2023.105305
  • Peer Reviewed / Open Access
• [Journal Article] Key determinants for signaling in the sensory rhodopsin <scp>II</scp>/transducer complex are different between <i>Halobacterium salinarum</i> and <i>Natronomonas pharaonis</i> (2023)
  • Author(s): Matsunami‐Nakamura Risa、Tamogami Jun、Takeguchi Miki、Ishikawa Junya、Kikukawa Takashi、Kamo Naoki、Nara Toshifumi
  • Journal Title: FEBS Letters Volume: 597 Issue: 18 Pages: 2334-2344
  • DOI: 10.1002/1873-3468.14711
  • Peer Reviewed
• [Journal Article] Structure and Heterogeneity of Retinal Chromophore in Chloride Pump Rhodopsins Revealed by Raman Optical Activity (2023)
  • Author(s): Ohya Masaiku、Kikukawa Takashi、Matsuo Junpei、Tsukamoto Takashi、Nagaura Ryota、Fujisawa Tomotsumi、Unno Masashi
  • Journal Title: The Journal of Physical Chemistry B Volume: 127 Issue: 21 Pages: 4775-4782
  • DOI: 10.1021/acs.jpcb.3c01801
  • Peer Reviewed
• [Journal Article] Elucidation of a Thermodynamical Feature Attributed to Substrate Binding to the Prokaryotic H+/Oligopeptide Cotransporter YdgR with Calorimetric Analysis: The Substrate Binding Driven by the Change in Entropy Implies the Release of Bound Water Molecules from the Binding Pocket (2023)
  • Author(s): Akiko Omori, Shotaro Sasaki, Takashi Kikukawa, Kazumi Shimono, and Seiji Miyauchi
  • Journal Title: Biochemistry Volume: - Issue: 11 Pages: 1608-1618
  • DOI: 10.1021/acs.biochem.2c00673
  • Peer Reviewed
• [Journal Article] Efficient recombinant production of mouse-derived cryptdin family peptides by a novel facilitation strategy for inclusion body formation (2023)
  • Author(s): Song Yuchi、Wang Yi、Yan Shaonan、Nakamura Kiminori、Kikukawa Takashi、Ayabe Tokiyoshi、Aizawa Tomoyasu
  • Journal Title: Microbial Cell Factories Volume: 22 Issue: 1 Pages: 9-9
  • DOI: 10.1186/s12934-023-02016-2
  • Peer Reviewed / Open Access
• [Journal Article] Phenylazothiazoles as Visible-Light Photoswitches (2023)
  • Author(s): Runze Lin, P. K. Hashim, Saugata Sahu, Ammathnadu S. Amrutha, Nusaiba Madappuram Cheruthu, Shakkeeb Thazhathethil, Kiyonori Takahashi, Takayoshi Nakamura, Takashi Kikukawa, Nobuyuki Tamaoki
  • Journal Title: Journal of the American Chemical Society Volume: － Issue: 16 Pages: 9072-9080
  • DOI: 10.1021/jacs.3c00609
  • Peer Reviewed
• [Journal Article] Mutations conferring SO42- pumping ability on the cyanobacterial anion pump rhodopsin and the resultant unique features of the mutant (2022)
  • Author(s): Yuhei Doi, Jo Watanabe, Rytota Nii, Takashi Tsukamoto, Makoto Demura, Yuki Sudo, Takashi Kikukawa
  • Journal Title: Scientific Reports Volume: 12 Issue: 1 Pages: 16422-16422
  • DOI: 10.1038/s41598-022-20784-6
  • Peer Reviewed / Open Access
• [Journal Article] Reisomerization of retinal represents a molecular switch mediating Na+ uptake and release by a bacterial sodium-pumping rhodopsin (2022)
  • Author(s): Fujisawa, T., Kinoue, K. Seike, R., Kikukawa, T., Unno, M.
  • Journal Title: Journal of biological chemistry Volume: 298 Issue: 9 Pages: 102366-102366
  • DOI: 10.1016/j.jbc.2022.102366
  • Peer Reviewed / Open Access
• [Journal Article] Three-Dimensional Structure of the Antimicrobial Peptide Cecropin P1 in Dodecylphosphocholine Micelles and the Role of the C-Terminal Residues (2022)
  • Author(s): Hao Gu, Takasumi Kato, Hiroyuki Kumeta, Yasuhiro Kumaki, Takashi Tsukamoto, Takashi Kikukawa, Makoto Demura, Hiroaki Ishida, Hans J. Vogel, Tomoyasu Aizawa
  • Journal Title: ACS Omega Volume: 7 Issue: 36 Pages: 31924-31934
  • DOI: 10.1021/acsomega.2c02778
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Unknown role of the extended cytoplasmic domain of anion channelrhodopsin (2023)
  • Author(s): Yuya Ohki, Tsukasa Shinone, Sayo Inoko, Miu Sudo, Makoto Demura, Takashi Kikukawa, Takashi Tsukamoto
  • Organizer: 第61回 日本生物物理学会年会
  • Invited
• [Presentation] Structural and functional properties of bacteriorhodopsin reconstituted in highly fluorinated dipalmitoylphosphatidylcholine membranes (2023)
  • Author(s): Daiki Kojima, Ai Nakagawara, Takafumi Shimoaka, Takashi Kikukawa, Toshiyuki Takagi, Hiroshi Takahashi, Hideki Amii, Masashi Sonoyama
  • Organizer: 第61回 日本生物物理学会年会
• [Presentation] Synchronicity of gate closing and transient proton release from Asp234 in Guillardia theta anion channelrhodopsin 1 (2023)
  • Author(s): Miu Sudo, Sayo Inoko, Takuma Watanabe, Makoto Demura, Takashi Kikukawa, Takashi Tsukamoto
  • Organizer: 第61回 日本生物物理学会年会
• [Presentation] Structure and Heterogeneity of Retinal Chromophore in Chloride Pump Rhodopsins Revealed by Raman Optical Activity (2023)
  • Author(s): Masashi Unno, Masaiku Ohya, Takashi Kikukawa, Junpei Matsuo, Takashi Tsukamoto, Ryota Nagaura, Tomotsumi Fujisawa
  • Organizer: 第61回 日本生物物理学会年会
• [Presentation] Configurational change of retinal Schiff base chromophore in a sodium pump rhodopsin (2023)
  • Author(s): Tomotsumi Fujisawa, Kouta Kinoue, Ryouhei Seike, Takashi Kikukawa, Masashi Unno
  • Organizer: 第61回 日本生物物理学会年会
• [Presentation] Attempts to strategic functional modifications of ion pump rhodopsins (2023)
  • Author(s): Kaito Hasegawa, Tomoya Maeda, Satoru Fukiya, Takashi Tsukamoto, Makoto Demura, Takashi Kikukawa
  • Organizer: 第61回 日本生物物理学会年会
• [Presentation] Effect of the phytanyl groups and membrane-spanning cross-linkage on structure and photocycle of bR in ether-linked phospholipid membrane (2023)
  • Author(s): Ami Harasawa, Ai Nakagawara, Takafumi Shimoaka, Toshiyuki Takagi, Takashi Kikukawa, Hiroshi Takahashi, Masashi Sonoyama
  • Organizer: 第61回 日本生物物理学会年会
• [Presentation] Actinobacteria-derived novel outward proton-pumping rhodopsins having long photocycle (2023)
  • Author(s): Shota Takahashi, Mako Ueno, Fumio Hayashi, Takashi Kikukawa, Ichiro Kasano, Yusuke Inoue, Masashi Sonoyama
  • Organizer: 第61回 日本生物物理学会年会
• [Presentation] The cytoplasmic domain regulates the photocurrent lifetime of anion channelrhodopsin GtACR1 (2023)
  • Author(s): Hana Maruyama, Shoko Hososhima, Satoshi Tsunoda, Yuya Ohki, Takashi Kikukawa, Takashi Tsukamoto, Hideki Kandori
  • Organizer: 第61回 日本生物物理学会年会
• [Presentation] 光駆動Cl-ポンプ蛋白質が起こす過渡的な基質放出・取込み過程の直接観測 (2023)
  • Author(s): 濱田 知快, 村部 圭祐, 塚本 卓, 出村 誠, 菊川 峰志
  • Organizer: 第23回日本蛋白質科学会年会
• [Presentation] 光駆動型Cl-ポンプ蛋白質の疎水性チャネルにおける高速Cl-輸送メカニズム (2023)
  • Author(s): ザイ 郁博, 下坂 杏奈, 塚本 卓, 出村誠, 菊川峰志
  • Organizer: 第23回日本蛋白質科学会年会
• [Presentation] Structure and Heterogeneity of Retinal Chromophore in Chloride Pump Rhodopsins Revealed by Raman Optical Activity (2023)
  • Author(s): Masaiku Ohya, Junpei Matsuo, Takashi Kikukawa, Takashi Tsukamoto, Tomotsumi Fujisawa, Masashi Unno
  • Organizer: 19th International Conference on Chiroptical Spectroscopy
  • Int'l Joint Research
• [Presentation] クライオ時間分解ラマン分光法によるナトリウムポンプロドプシンの研究：O中間体における発色団構造変化 (2023)
  • Author(s): 藤澤 知績, 木ノ上 晃汰, 清家 陵平, 菊川 峰志, 海野 雅司
  • Organizer: 第17回分子科学討論会
• [Presentation] 好冷性細菌由来の新規微生物ロドプシンにおける特徴的な光物性 (2023)
  • Author(s): 高橋 渉太, 上野 真琴, 林 史夫, 菊川 峰志, 園山 正史
  • Organizer: 第23回日本蛋白質科学会年会
• [Presentation] 一方の疎水鎖末端をフッ素化した新規リン脂質F4-Hybridに再構成した膜タンパク質バクテロリオロドプシンの特徴 (2023)
  • Author(s): 中川原 亜依, 高木 俊之, 菊川 峰志, 高橋 浩, 網井 秀樹, 下赤卓史, 園山 正史
  • Organizer: 第49回生体分子科学討論会
• [Presentation] A possible gate-opening mechanism of Indibacter alkaliphilus sodium pump rhodopsin (2022)
  • Author(s): Takashi Kikukawa
  • Organizer: 19th International Conference on Retinal Proteins
  • Int'l Joint Research / Invited
• [Presentation] Retinal conformations in microbial rhodopsins probed by Raman optical activity (2022)
  • Author(s): Masashi Unno, Tomotsumi Fujisawa, Takashi Kikukawa
  • Organizer: 19th International Conference on Retinal Proteins
  • Int'l Joint Research / Invited
• [Presentation] Essential residues for fast chloride ion transport of Natronomonas pharaonis halorhodopsin (2022)
  • Author(s): Zhai Yubo, Anna Shimosaka, Takashi Tsukamoto, Makoto Demura, Takashi Kikukawa
  • Organizer: 19th International Conference on Retinal Proteins
  • Int'l Joint Research
• [Presentation] Biological role and molecular mechanism of preferential NO3- transport by full-length Guillardia theta anion channelrhodopsin 1 (2022)
  • Author(s): Yuya Ohki, Ryouhei Ohtake, Yukino Sato, Takuma Watanabe, Jo Watanabe, Makoto Demura, Takashi Kikukawa, Takashi Tsukamoto
  • Organizer: 19th International Conference on Retinal Proteins
  • Int'l Joint Research
• [Presentation] Comparative study in thermodynamics of microbial proton-pumping rhodopsins identified in various temperature environments (2022)
  • Author(s): Ryouhei Ohtake, Kaori Kondo, Makoto Demura, Takashi Kikukawa, Takashi Tsukamoto
  • Organizer: 19th International Conference on Retinal Proteins
  • Int'l Joint Research
• [Presentation] Gate closing mechanism of anion channelrhodopsin synchronized with transient proton release (2022)
  • Author(s): Sayo Inoko, Takuma Watanabe, Tsukasa Shinone, Makoto Demura, Takashi Kikukawa, Takashi Tsukamoto
  • Organizer: 19th International Conference on Retinal Proteins
  • Int'l Joint Research
• [Presentation] 多様な温度環境に分布するプロトンポンプ型ロドプシンの熱力学的性質の網羅的解析 (2022)
  • Author(s): Ryouhei Ohtake, Kaori Kondo, Makoto Demura, Takashi Kikukawa, Takashi Tsukamoto
  • Organizer: 第60回 日本物物理学会年会
• [Presentation] Time-resolved cryo-Raman study of Na+ uptake and release by a sodium pumping rhodopsin from Indibacter alkaliphilus (2022)
  • Author(s): Tomotsumi Fujisawa, Kouta Kinoue, Ryouhei Seike, Takashi Kikukawa, Masashi Unno
  • Organizer: 第60回 日本物物理学会年会
• [Presentation] 多孔質ハイドロゲル固体試料中における紫膜積層に適した孔サイズ分布 (2022)
  • Author(s): Yasunori Yokoyama, Morise Karasawa, Kingo Takiguchi, Hiroshi Takahashi, Takashi Kikukawa, Masashi Sonoyama, Koshi Takenaka
  • Organizer: 第60回 日本物物理学会年会
• [Presentation] 光駆動型Cl－ポンプ・ハロロドプシンの細胞質型ハーフチャンネルにおける高速Cl－輸送のメカニズム解明 (2022)
  • Author(s): Zhai Yubo, Anna Shimosaka, Takashi Tsukamoto, Makoto Demura, Takashi Kikukawa
  • Organizer: 第60回 日本物物理学会年会
• [Presentation] 光駆動Cl－ポンプhalorhodopsin の基質放出・取込み中間体の同定 (2022)
  • Author(s): Chihaya Hamada, Keisuke Murabe, Takashi Tukamoto, Makoto Demura, Takashi Kikukawa
  • Organizer: 第60回 日本物物理学会年会
• [Presentation] 低温ラマン分光法による光駆動Cl-ポンプNMR3 の発色団構造変化の研究 (2022)
  • Author(s): Natsuki Ejima, Tomotsumi Fujisawa, Takashi Kikukawa, Masashi Unno
  • Organizer: 第60回 日本物物理学会年会
• [Presentation] 陸生アクチノバクテリア由来の新規微生物ロドプシン群 (2022)
  • Author(s): Mako Ueno, Fumio Hayashi, Takashi Kikukawa, Masashi Sonoyama
  • Organizer: 第60回 日本物物理学会年会