光照射固体ＮＭＲによる光受容体膜タンパク質の活性中間体の構造機能相関の解明

• Project Area: Integrative understanding of biological processes mediated by transient macromolecular complexes; New technology for visualizing physiologically metastable states.
• Project/Area Number: 24121709
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Complex systems
• Research Institution: Yokohama National University
• Principal Investigator: 内藤 晶 横浜国立大学, 工学研究院, 教授 (80172245)
• Project Period (FY): 2012-04-01 – 2014-03-31
• Project Status: Completed (Fiscal Year 2013)
• Budget Amount: ¥10,140,000 (Direct Cost: ¥7,800,000、Indirect Cost: ¥2,340,000); Fiscal Year 2013: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2012: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000)
• Keywords: 光受容膜タンパク質 / 光走性 / 光中間体 / 高度好塩好アルカリ性菌 / 光照射固体NMR / 光反応サイクル / 信号伝達機能 / トランスデューサー / 走光性 / 固体NMR / 光照射 / 中間体 / 光サイクル

Research Abstract

１）(In-situ光照射固体ＮＭＲシステムの構築) これまでに開発してきた光照射固体ＮＭＲ装置の光照射効率を格段に上げることを試みた。同時にＬＥＤ光源から複数の波長での光照射を可能にした。
２)(ppR-pHtrII複合体における光中間体の捕捉と光反応サイクルの解明)　負の光走性を示すppR-pHtrII複合体は光信号を伝達するM-中間体の寿命が1.7秒と他の中間体に比べて格段に長いことから、in-situ光照射固体ＮＭＲにおいて、520 nmの光を照射することで、標識した[13C-20]retinalのＮＭＲ信号を観測し、M-中間体の捕捉を確認することが出来た。その結果、M-中間体には少なくとも３種類の区別できる中間体が共存していることが明らかになった。次にM-中間体が捕捉された状態で365 nmの青色光を照射した。この結果、３種類の中間体の内１つは紫外線光活性のないことが分かったのでこの中間体がM-中間体ではなくN-中間体であることが判明した。
３）(SRIにおける光中間体の捕捉と光反応サイクルの解明)　SrSRI(Salinibacter ruber sensaryrhodopsin I)は真正細菌由来のロドプシンであり、正と負の両方の光走性機能をもつ光受容膜タンパク質である。光を照射することにより、K,L,M-中間体を経て元に戻る光反応回路に加えて、２光子吸収により、M-からP-中間体を経て元に戻る光反応過程が存在する。SrSRIは大腸菌発現系で発現し、ＰＧ膜に再構成して測定試料とした。光照射固体ＮＭＲの測定はＬＥＤから発生した２波長の光（365 nm, 520 nm）を照射した条件で固体CP-MAS-NMRスペクトルを観測した。この結果、520 nmの光照射でM-中間体が、365 nmの光照射でP-中間体が捕捉され、波長に依存して中間体が入れ替わる現象を観測した

Current Status of Research Progress

Reason

25年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

25年度が最終年度であるため、記入しない。

Research Products

• [Journal Article] Color discriminating retinal configurations of sensory rhodopsin I by photo-irradiation solid state NMR spectroscopy. (2014)
  • Author(s): Yornoda, H., Makino, Y., Tomonaga, Y., Hidaka, T., Kawamura, I., Okitsu, T., Wada, A., Sudo, Y., Naito, A.
  • Journal Title: Angew. Chem. Int. Ed. Volume: (印刷中) Issue: 27 Pages: 7080-7084
  • DOI: 10.1002/ange.201309258
  • Peer Reviewed
• [Journal Article] Grafting of paired 3-aminopropyltrialkoxy silans onto mesoporous silica and adsorptions of isomers of benzenedialdehydes (2013)
  • Author(s): Hideaki Yoshitake, Teruaki Koizumi, Izuru Kawamura, Akira Naito
  • Journal Title: Phys. Chem. Chem. Phys. Volume: 15 Issue: 11 Pages: 3946-3954
  • DOI: 10.1039/c3cp44592e
  • Peer Reviewed
• [Journal Article] Role of aromatic residues in amyloid fibril formation of human calcitonin by solid-state13C NMR and molecular dynamics simulation (2013)
  • Author(s): Hikari Itoh-Watanabe, Miya Kamihira-Ishijima, Namsrai Javkhlantugs, Ryozo Inoue, Yuki Itoh, Hiroshi Endo, Satoru Tuzi, Hazime Saito, Kazuyoshi Ueda, Akira Naito
  • Journal Title: Phys. Chem. Chem. Phys. Volume: 15 Issue: 23 Pages: 8890-8901
  • DOI: 10.1039/c3cp44544e
  • Peer Reviewed
• [Journal Article] Drastic sensitivity enhancement in 29Si MAS NMR of zeolites and mesoporous silica materials by paramagnetic doping of Cu2+ (2013)
  • Author(s): Satoshi Inagaki, Izuru Kawamura, Yukichi Sasaki, Kaname Yoshida, Yoshihiko Kubota, Akira Naito
  • Journal Title: Phys. Chem. Chem. Phys. Volume: 15 Pages: 13523-13531
  • Peer Reviewed
• [Journal Article] Characterization of the spherical intermediates and fibril formation of hCT in HEPES solution using solid-state 13C-NMR and transmission electron microscopy (2013)
  • Author(s): Hikari Itoh-Watanabe, Miya Kamihira-Ishijima, Izuru Kawamura, Masashi Kondoh, Masamichi Nakakoshi, Michio Sato, Akira Naito
  • Journal Title: Phys. Chem. Chem. Phys. Volume: 15 Issue: 39 Pages: 16956-16964
  • DOI: 10.1039/c3cp52810c
  • Peer Reviewed
• [Journal Article] Role of aromatic residues in amyloid bibril formation of human calcitonin by solid state 13C NMR and molecular dynamics simulation. (2013)
  • Author(s): H. Itoh-Watanabe
  • Journal Title: Phys. Chem. Chem. Phys. Volume: -
  • Peer Reviewed
• [Journal Article] NMR characterization of monomer and oligomeric conformations of human calcitonin and its interaction with EGCG. (2012)
  • Author(s): R. Hung
  • Journal Title: J. Mol. Biol. Volume: 416 Issue: 1 Pages: 108-120
  • DOI: 10.1016/j.jmb.2011.12.023
  • Peer Reviewed
• [Journal Article] Solid-state NMR spectroscopy reveals anomer specific transport of galactose in the milk yeast Kluyveromyces lactis (2012)
  • Author(s): Toshio Fukasawa, Akio Abe, Atsusi Nakamura, Miyako Horigome, Akira Naito
  • Journal Title: FEMS Yeast Res Volume: 12 Issue: 4 Pages: 415-422
  • DOI: 10.1111/j.1567-1364.2012.00788.x
  • Peer Reviewed
• [Journal Article] Structure and orientation of bovine lactoferrampin in the mimetic bacterial membrane as revealed by solid-state NMR and molecular dynamics simulation (2012)
  • Author(s): A. Tsutsumi
  • Journal Title: Biophys. J. Volume: 103 Pages: 1735-1743
  • Peer Reviewed
• [Journal Article] Change in Local Dynamics of Bacteriorhodopsin with Retinal Isomerization under Pressure as Studied by Fast Magic Angle Spinning NMR (2012)
  • Author(s): I. Kawamura*, S. Yamaguchi, H. Nishikawa, K. Tajima, M. Horigome, S. Tuzi, H. Saito, A. Naito
  • Journal Title: Polymer Journal Volume: 44 Issue: 8 Pages: 863-867
  • DOI: 10.1038/pj.2012.116
  • NAID: 10030931693
  • Peer Reviewed
• [Journal Article] Inhibitory mechanism of pancreatic fibril formation: Formation of the complex between tea catechins and the fragment of residues 22-27. (2012)
  • Author(s): M. Kamihira-Ishijima
  • Journal Title: Biochemistry Volume: 51 Issue: 51 Pages: 10167-10174
  • DOI: 10.1021/bi3012274
  • Peer Reviewed
• [Journal Article] Characterization of Amorphized Zeolite A by Combining High-Energy X-ray Diffraction and High-Resolution Transmission Electron Microscopy (2012)
  • Author(s): 佐藤可久(Kaku Sato)
  • Journal Title: Journal of Physical Chemistry C Volume: 116 Issue: 48 Pages: 25293-25299
  • DOI: 10.1021/jp305359q
  • Peer Reviewed
• [Presentation] マイクロ波加熱分光法―マイクロ波照射下での分子のNMR測定 (2013)
  • Author(s): 内藤 晶
  • Organizer: 日本化学会第９３春季年会
  • Place of Presentation: 立命館大学
  • Year and Date: 2013-03-22
  • Invited
• [Presentation] 光照射固体NMRによる光受容膜タンパク質SrSRIの光反応過程と光中間体の構造解析 (2013)
  • Author(s): 内藤 晶
  • Organizer: 生物物理学会関東支部研究会
  • Place of Presentation: 東京農工大
  • Year and Date: 2013-03-04
• [Presentation] Photo-induced structural changes of photoreceptor membrane proteins as revealed by in-situ photo-irradiated solid-state NMR (2013)
  • Author(s): Akira Naito
  • Organizer: 245th ACS National Meeting
  • Place of Presentation: New Orieans, LA, USA
  • Invited
• [Presentation] Detection of light induced intermediates of photoreceptor membrane proteins by in-situ photo-irradiated solid-state NMR (2013)
  • Author(s): A. Naito, Y. Tomonaga, T. Hidaka, H. Yomoda, T. Makino, I. Kawamura, Y. Sudo, A. wada, T. Okitsu, N. Kamo
  • Organizer: 18th ISMAR Meeting
  • Place of Presentation: Rio De Janeiro, Brazil
• [Presentation] Ｉｎ－ｓｉｔｕ光照射固体ＮＭＲによる光受容膜タンパク質SrSRIの波長依存光中間体形成機構の解析 (2013)
  • Author(s): 内藤 晶、四方田洋紀、槙野義輝、友永雄也、日高徹郎、川村 出、沖津貴志、和田昭盛、須藤雄気
  • Organizer: 第４０回 生体分子科学討論会
  • Place of Presentation: 大阪大学
• [Presentation] 光照射固体ＮＭＲによるバクテリオロドプシンD96N変異体の光中間体の捕捉とタンパク質構造変化の解析 (2013)
  • Author(s): 内藤 晶、宮佐亮太、川村 出、辻 暁
  • Organizer: 第１３回日本蛋白質科学会年会
  • Place of Presentation: とりぎん文化会館、鳥取
• [Presentation] Detection of photoactivated intermediates of photoreceptor membrane proteins by in-situ photo-irradiation solid-state NMR (2013)
  • Author(s): A. Naito, Y. Tomonaga, T. Hidaka, H. Yomoda, T. Makino, I. Kawamura, Y. Sudo, A. Wada, T. Okitsu, N. Kamo, A. Ramamoorthy
  • Organizer: Atomic View of Biomolecular Function
  • Place of Presentation: U of Michigan, MI, USA
  • Invited
• [Presentation] Ｉｎ－ｓｉｔｕ光照射固体ＮＭＲによるバクテリオロドプシンD96N変異体の光中間体の捕捉とタンパク質構造変化の解析 (2013)
  • Author(s): 内藤 晶、宮佐亮太、重田安里寿、川村 出、辻 暁
  • Organizer: 第８６回 日本生化学会大会
  • Place of Presentation: パシフィコ横浜
• [Presentation] 固体ＮＭＲとＴＥＭによるヒトカルシトニンとグルカゴンにおけるアミロイド線維形成と阻害機構の解明 (2013)
  • Author(s): 内藤 晶
  • Organizer: 日本生物物理学会第５１回年会
  • Place of Presentation: 国立京都国際会館、京都
  • Invited
• [Presentation] Microwave Heating NMR Spectroscopy. Application to Liquid Crystalline Systems (2013)
  • Author(s): A. Naito, Y. Tasei, T. Fujito, I. Kawamura
  • Organizer: 第５２回NMR討論会
  • Place of Presentation: 石川県立音楽堂、金沢
• [Presentation] 光照射固体ＮＭＲによる光受容膜タンパク質の活性中間体の捕捉と構造変化の解明 (2013)
  • Author(s): 内藤 晶
  • Organizer: 分子研研究会「ロドプシン研究の故きを温ねて新しきを知る」
  • Place of Presentation: 分子研、岡崎
  • Invited
• [Presentation] Amyloid Fibril Formation and Inhibition Mechanisms of Human Calcitonin as Revealed by 13C Solid State NMR and Transmission Electron Microscopy (2013)
  • Author(s): Akira Naito
  • Organizer: Indo-Japan Bilateral Symposium on Nanotechnology based innovation for Environmental, Energy & Biomedical Application
  • Place of Presentation: Bangalor, India
  • Invited
• [Presentation] In-situ光照射固体NMRによる光受容膜タンパク質の光反応サイクルと光励起構造変化の解析 (2012)
  • Author(s): 内藤 晶
  • Organizer: 第51回NMR討論会
  • Place of Presentation: ウインクあいち
  • Year and Date: 2012-11-08
• [Presentation] Pressure and heat induced structural change of bacteriorhodopsin as studied by fast magic angle spinning soli state NMR (2012)
  • Author(s): A. Naito
  • Organizer: 7th IPBB
  • Place of Presentation: Otsu, Japan
  • Year and Date: 2012-10-29
  • Invited
• [Presentation] Light induced structural changes of photoreceptor membrane ptroteins as revealed by in-situ photo-irradiated solid-state NMR (2012)
  • Author(s): A. Naito
  • Organizer: 15th ICRP
  • Place of Presentation: AScona, Switzerland
  • Year and Date: 2012-09-30
  • Invited
• [Presentation] In-situ 光照射固体NMRによる光受容膜タンパク質の光活性状態の解明 (2012)
  • Author(s): A. Naito
  • Organizer: 日本生物物理学会第49回年会
  • Place of Presentation: 名古屋大学
  • Year and Date: 2012-09-22
• [Presentation] Light induced intermediate of photoreceptor membrane proteins as revealed by in situ photo-irradiated solid-state NMR spectroscopy (2012)
  • Author(s): A. Naito
  • Organizer: 25th ICMRBS
  • Place of Presentation: Lyon, France
  • Year and Date: 2012-08-19
• [Presentation] 熱および圧力に応答するバクテリオロドプシンの構造変化の解析 (2012)
  • Author(s): 内藤 晶
  • Organizer: 第１２回蛋白質科学会年会
  • Place of Presentation: 名古屋国際会議場
  • Year and Date: 2012-06-20
• [Presentation] 光照射固体NMRによる光受容膜タンパク質の光中間体の捕捉と光励起構造変化の解析 (2012)
  • Author(s): 内藤 晶
  • Organizer: 第３９回生体分子科学討論会
  • Place of Presentation: 東北大学
  • Year and Date: 2012-06-08
• [Book] Advances in Biological Solid-State NMR. Proteins and Membrane-Active Peptides. (2014)
  • Author(s): A. Naito, I. Kawamura
  • Total Pages: 608
  • Publisher: Royal Society of Chemistry
• [Remarks] 内藤研究室紹介
  • URL: http://www.chem.ynu.ac.jp/lab/naitolab/