Visualization of rotation of ATP synthase driven by proton motive force using single-molecule techniques and microdevices

• Project/Area Number: 21770168
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Single-year Grants
• Research Field: Biophysics
• Research Institution: Osaka University
• Principal Investigator: IINO Ryota Osaka University, 産業科学研究所, 助教 (70403003)
• Project Period (FY): 2009 – 2010
• Project Status: Completed (Fiscal Year 2010)
• Budget Amount: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000); Fiscal Year 2010: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2009: ¥2,990,000 (Direct Cost: ¥2,300,000、Indirect Cost: ¥690,000)
• Keywords: 1分子計測・操作 / 回転分子モーター / 分子モーター / 1分子計測 / 1分子イメージング / ATP合成酵素 / 基板支持脂質二重膜 / パッチクランプ / マイクロデバイス / プロトン駆動力 / 膜タンパク質

Research Abstract

We have tried to directly observe the rotation of ATP synthase driven by proton motive force. ATP synthase was reconstituted into the lipid bilayer membrane supported on the solid substrate such as NTA-agarose and NTA-acrylamide. Electric sealing of and voltage allocation to the supported membrane were examined using pipette made by glass or polydimethylsiloxane. Several tens of mega ohm resistance was achieved, and used to seal the membrane containing the rotating ATP synthase driven by ATP hydrolysis. Some of ATP synthase showed change in the rotation speed when voltage was applied. We need elaborate control experiments to confirm whether the change really reflects the behavior of the ATP synthase.

Research Products

• [Journal Article] Activation and stiffness of the inhibited states of F1-ATPase probed by single-molecule manipulation. (2010)
  • Author(s): Saita E, Iino R, Suzuki T, Feniouk BA, Kinosita K.Jr, *^Yoshida M.
  • Journal Title: J Biol Chem. 285 Pages: 11411-11417
  • Peer Reviewed
• [Journal Article] Simple dark-field microscopy with nanometer spatial precision and microsecond temporal resolution. (2010)
  • Author(s): Ueno H, Nishikawa S, Iino R, Tabata KV, Sakakihara S, Yanagida T, *^Noji H.
  • Journal Title: Biophys.J. 98 Pages: 2014-2023
  • Peer Reviewed
• [Journal Article] Fluctuation theorem applied to F1-ATPase. (2010)
  • Author(s): ^*Hayashi K, Ueno H, Iino R, ^*Noji H.
  • Journal Title: Phys.Rev.Lett. 104
  • Peer Reviewed
• [Journal Article] Phosphate-release in F1-ATPase catalytic cycle follows ADP release. (2010)
  • Author(s): Watanabe R, Iino R, *Noji H.
  • Journal Title: Nat.Chem.Biol. 6 Pages: 814-820
  • Peer Reviewed
• [Journal Article] Stiffness of γ subunit of F1-ATPase. (2010)
  • Author(s): Okuno D, Iino R, ^*Noji H.
  • Journal Title: Eur.Biophys.J. 39 Pages: 1589-1596
  • Peer Reviewed
• [Journal Article] A single-molecule enzymatic assay in a directly accessible femtoliter droplet array. (2010)
  • Author(s): Sakakihara S, Araki S, ^*Iino R, ^*Noji H.
  • Journal Title: Lab Chip. 10 Pages: 3355-3362
  • Peer Reviewed
• [Journal Article] Stiffness of γ subunit of F_1-ATPase. (2010)
  • Author(s): Okuno D, Iino R, Noji H.
  • Journal Title: Eur.Biophys.J. Volume: 39 Pages: 1589-1596
  • Peer Reviewed
• [Journal Article] A single-molecule enzymatic assay in a directly accessible femtoliter droplet array (2010)
  • Author(s): Sakakihara S, Araki S, Iino R, Noji H.
  • Journal Title: Lab Chip. Volume: 10 Pages: 3355-3362
  • Peer Reviewed
• [Journal Article] Mechanism of inhibition by C-terminal α-helices of the ε subunit of Escherichia coli FoF1-ATP synthase. (2009)
  • Author(s): ^*Iino R, Hasegawa R, Tabata KV, ^*Noji H.
  • Journal Title: J.Biol.Chem. 284 Pages: 17457-17464
  • Peer Reviewed
• [Journal Article] Single-molecule study on the temperature-sensitive reaction of F1-ATPase with a hybrid F1 carrying a single β(E190D). (2009)
  • Author(s): Enoki S, Watanabe R, Iino R, ^*Noji H.
  • Journal Title: J Biol Chem. 284 Pages: 23169-23176
  • Peer Reviewed
• [Journal Article] Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators. (2009)
  • Author(s): ^*Imamura H, Huynh Nhat KP, Togawa H, Saito K, Iino R, Kato-Yamada Y, Nagai, T, ^*Noji H.
  • Journal Title: Proc.Natl.Acad.Sci.U.S.A. 106 Pages: 15651-15656
  • Peer Reviewed
• [Journal Article] Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators (2009)
  • Author(s): Imamura, H., Huynh Nhat, K.P., Togawa, H., Saito, K., Iino, R., Kato-Yamada, Y., Nagai, T., Noji, H.
  • Journal Title: Proc Natl Acad Sci USA. 106 Pages: 15651-15656
  • Peer Reviewed
• [Journal Article] Single-molecule study on the temperature-sensitive reaction of F_1-ATPase with a hybrid F_1 carrying a single β (E190D) (2009)
  • Author(s): Enoki, S., Watanabe, R., Iino, R., Noji, H.
  • Journal Title: J Biol Chem. 284 Pages: 23169-23176
  • Peer Reviewed
• [Journal Article] Mechanism of inhibition by C-terminal α-helices of the ε subunit of Escherichia coli F_0F_1-ATP synthase (2009)
  • Author(s): Iino, R., Hasegawa, R., Tabata, K.V., Noli, H.
  • Journal Title: J Biol Chem. 284 Pages: 17457-17464
  • Peer Reviewed
• [Journal Article] Preparation and Biological Application of Water-In-Oil Microchamber Array Formed on Hydrophobic/Hydrophilic Patterned Surface. (2009)
  • Author(s): Iino R, Sakakihara S, Noji H
  • Journal Title: Proc μTAS 2 Pages: 1121-1123
  • Peer Reviewed
• [Journal Article] Evaluation of multidrug efflux pump inhibitors by a new method using microfluidic channels.
  • Author(s): ^*Matsumoto Y, Hayama K, Sakakihara S, Nishino K, Noji H, *^Iino R, Yamaguchi A.
  • Journal Title: PLoS ONE 2011 in press
  • Peer Reviewed
• [Presentation] 分子モーターの性質を調べる1分子計測技術 (2011)
  • Author(s): *飯野亮太
  • Organizer: (社)日本電気計測器工業会 関西支部 第29回戦略的基盤技術検討委員会
  • Place of Presentation: 大阪(招待講演)
  • Year and Date: 2011-02-07
• [Presentation] 分子モーターの性質を調べる1分子計測技術 (2011)
  • Author(s): 飯野亮太
  • Organizer: (社)日本電気計測器工業会関西支部第29回戦略的基盤技術検討委員会
  • Place of Presentation: 大阪
  • Year and Date: 2011-02-07
• [Presentation] Subunit rotation in a single FoF1-ATP synthase in a living bacterium monitored by FRET. (2011)
  • Author(s): Seyfert K., Oosaka T., Yaginuma H.,Ernst S., Noji H., *Iino R., *Borsch M.
  • Organizer: SPIE Photonics West 2011.
  • Place of Presentation: San Francisco, USA
• [Presentation] ATP合成酵素を1分子技術で視る・操る (2010)
  • Author(s): *飯野亮太
  • Organizer: 光塾第2回研究会
  • Place of Presentation: 大阪(招待講演)
  • Year and Date: 2010-12-11
• [Presentation] ATP合成酵素を1分子技術で視る・操る (2010)
  • Author(s): 飯野亮太
  • Organizer: 光塾第2回研究会
  • Place of Presentation: 大阪
  • Year and Date: 2010-12-11
• [Presentation] Single-molecule studies on the fluctuation and function of a rotary motor protein ATP synthase. (2010)
  • Author(s): *Iino R.
  • Organizer: The 4th International Symposium "Molecular Science of Fluctuations toward Biological Functions"
  • Place of Presentation: Shiga. Japan
  • Year and Date: 2010-11-30
• [Presentation] Single-molecule studies on the fluctuation and function of a rotary motor protein ATP synthase (2010)
  • Author(s): Ryota Iino
  • Organizer: The 4th International Symposium "Moleculaz Science of Fluctuations toward Biological Functions"
  • Place of Presentation: 大津
  • Year and Date: 2010-11-30
• [Presentation] 1分子技術で生体回転ナノモーターを視る・操る (2010)
  • Author(s): *飯野亮太
  • Organizer: 特定領域研究「高次系分子科学」若手の会研究会
  • Place of Presentation: 仙台(招待講演)
  • Year and Date: 2010-11-26
• [Presentation] 1分子技術で生体回転ナノモーターを視る・操る (2010)
  • Author(s): 飯野亮太
  • Organizer: 特定領域研究「高次系分子科学」若手の会研究会
  • Place of Presentation: 仙台
  • Year and Date: 2010-11-26
• [Presentation] 中身を出し入れできる細胞サイズのうつわを創る (2010)
  • Author(s): *飯野亮太
  • Organizer: 細胞を創る研究会3.0シンポジウム「器をつくる」
  • Place of Presentation: 東京(招待講演)
  • Year and Date: 2010-11-12
• [Presentation] 中身を出し入れできる細胞サイズのうつわを創る (2010)
  • Author(s): 飯野亮太
  • Organizer: 細胞を創る研究会3.0シンポジウム「器をつくる」
  • Place of Presentation: 東京
  • Year and Date: 2010-11-12
• [Presentation] Fluctuation and function of a rotary motor protein (2010)
  • Author(s): *飯野亮太
  • Organizer: 第48回日本生物物理学会年会シンポジウム「Fluctuation and Function ofBiomolecules」.
  • Place of Presentation: 仙台(招待講演)
  • Year and Date: 2010-09-20
• [Presentation] Fluctuation and function of a rotary motor protein (2010)
  • Author(s): 飯野亮太
  • Organizer: 第48回日本生物物理学会年会シンポジウム「Fluctuation and Function of Biomolecules」
  • Place of Presentation: 仙台
  • Year and Date: 2010-09-20
• [Presentation] Direct observation of steps in c-ring rotation of Escherichia coli FoF1-ATP synthase (Selected for Oral Presentation). (2010)
  • Author(s): Iino R., Khek-Chian Tham, Kazuhito V.Tabata, Hiroshi Ueno, *Hiroyuki Noji.
  • Organizer: 16th European Bioenergetics Conference.
  • Place of Presentation: Warsaw. Poland
  • Year and Date: 2010-07-18
• [Presentation] Direct observation of steps in c-ring rotation of Escherichia coli F_0F_1-ATP synthase (2010)
  • Author(s): Ryota Iino, Khek-Chian Tham, Kazuhito V., Tabata, Hiroshi Ueno, Hiroyuki Noji.
  • Organizer: 16th European Bioenergetics Conference
  • Place of Presentation: ワルシャワ、ポーランド
  • Year and Date: 2010-07-18
• [Presentation] Single-Molecule Imaging of Ion-Transporting Rotary Motor Protein (2010)
  • Author(s): Ryota Iino, Khek-Chian Tham, Kazuhito V. Tabata, Hiroshi Ueno, Hiroyuki Noji
  • Organizer: International Symposium of Joint Research Network on Advanced Materials and Devices
  • Place of Presentation: Sapporo (Japan)
  • Year and Date: 2010-03-25
• [Presentation] 超微小反応容器を用いた1分子バイオアッセイ (2010)
  • Author(s): 飯野亮太, 野地博行
  • Organizer: 第57同応用物理学関連連合講演会シンポジウム「ナノバイオエンジニアリングの現状と未来像」
  • Place of Presentation: 神奈川県平塚市
  • Year and Date: 2010-03-18
• [Presentation] Fluctuation theorem applied to F_1-ATPase (2010)
  • Author(s): Kumiko Hayashi, Hiroshi Ueno, Ryota Iino, Hiroyuki Noji
  • Organizer: Biophysical Society 54^<th> Annual Meeting
  • Place of Presentation: San Francisco (USA)
  • Year and Date: 2010-02-24
• [Presentation] The role of P_1-release as the main torque generating step of F_1-ATPase (2010)
  • Author(s): Rikiya Watanabe, Hiroshi Ueno, Ryota Iino, Hiroyuki Noji
  • Organizer: Biophysical Society 54^<th> Annual Meeting
  • Place of Presentation: San Francisco (USA)
  • Year and Date: 2010-02-21
• [Presentation] Culture, detection, and recovery of the antibiotic-tolerant persister bacteria in the directly accessible microchamber array. (2010)
  • Author(s): *Iino R., Hayama K., Sakakihara S., Noji H.
  • Organizer: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences (μTAS2010).
  • Place of Presentation: Groningen, Netherlands
• [Presentation] Simple Total Internal Reflection Dirk-field Microscopy with Nanometer spatial precision and Microsecond Temporal Resolution (2009)
  • Author(s): Hiroshi Ueno, So Nishikawa, Ryota Iino, Kazuhito Tabata, Hiroyuki Noji
  • Organizer: The 31^<rd> International Symposium: Molecular Science of Fluctuation toward Biological Functions
  • Place of Presentation: Nagoya (Japan)
  • Year and Date: 2009-12-20
• [Presentation] Direct observation of steps in c-ring rotation of Escherichia coli FoF1-ATP synthase. (2009)
  • Author(s): *Iino R., Khek-Chian Tham, Kazuhito V.Tabata, Hiroshi Ueno, *Hiroyuki Noji.
  • Organizer: International Symposium : Watching Biomolecules in Action (WBMA'09).
  • Place of Presentation: Osaka. Japan
  • Year and Date: 2009-12-15
• [Presentation] Direct observation of steps in c-ring rotation of Escherichia coli F_0F_1-ATP synthase (2009)
  • Author(s): Ryota Iino
  • Organizer: JST/CREST Symposium: WBMA'09 (Watching Biomolecules in Action)
  • Place of Presentation: Osaka (Japan)
  • Year and Date: 2009-12-15
• [Presentation] Simple Total Internal Reflection Dirk-field Microscopy with Nanometer spatial precision and Microsecond Temporal Resolution (2009)
  • Author(s): Hiroshi Ueno, So Nishikawa, Ryota Iino, Kazuhito Tabata, Hiroyuki Noji
  • Organizer: 第47回 (2009年度) 日本生物物理学会年会
  • Place of Presentation: 徳島県
  • Year and Date: 2009-11-01
• [Presentation] Stiffness of Y subunit of F_1-ATPase (2009)
  • Author(s): Daichi Okuno, Ryota Iino, Rie Hasegawa, Hiroyuki Noji
  • Organizer: 第47回 (2009年度) 日本生物物理学会年会
  • Place of Presentation: 徳島県
  • Year and Date: 2009-11-01
• [Presentation] Mechanism of inhibition by C-terminal a-helices of the ε subunit of Escherichia coli F_0F_1-ATP synthase (2009)
  • Author(s): Ryota Iino, Rie Hasegawa, Kazuhito Tabata, Hiroyuki Noji
  • Organizer: 第47回 (2009年度) 日本生物物理学会年会
  • Place of Presentation: 徳島県
  • Year and Date: 2009-11-01
• [Presentation] The completion of chemo-mechanical coupling scheme of F_1-ATPase ; The determination of the timing of P_1-release (2009)
  • Author(s): Rikiya Watanabe, Hiroshi Ueno, Rvota Iino. Hirovuki Noji
  • Organizer: 第47回 (2009年度) 日本生物物理学会年会
  • Place of Presentation: 徳島県
  • Year and Date: 2009-11-01
• [Presentation] Torque measurements of F_1-ATPase by using the fluctuation theorem (2009)
  • Author(s): Kumiko Hayashi, Hiroshi Ueno, Ryota Iino, Hiroyuki Noji
  • Organizer: 第47回 (2009年度) 日本生物物理学会年会
  • Place of Presentation: 徳島県
  • Year and Date: 2009-10-30
• [Presentation] 36-degree stepping rotation of FoF1-ATP synthase (2009)
  • Author(s): *Hiroyuki Noji, *Iino R.
  • Organizer: International Symposium : Innovative Nanoscience of Supermolecular Motor Proteins Working in Biomembrane.
  • Place of Presentation: Kyoto. Japan(招待講演)
  • Year and Date: 2009-09-09
• [Presentation] 36-degree stepping rotation of F_0F_1-ATP synthase (2009)
  • Author(s): Hiroyuki Noji, Ryota Iino
  • Organizer: International Symposium "Innovative Nanoscience of Supermolecular Motor Proteins Working in Biomembranes"
  • Place of Presentation: Kyoto (Japan)
  • Year and Date: 2009-09-09
• [Presentation] Preparation and biological application of water-in-oil microchamber array formed on hydrophobic/hydrophilic patterned surface. (2009)
  • Author(s): *Iino R., Sakakihara S., Noji H.
  • Organizer: 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences (μTAS2009).
  • Place of Presentation: Jeju, Korea
• [Book] Fundamental Properties and Structure of F_1-ATPase. In "Encyclopedia of Biophysics" edited by Roberts G.C. (2011)
  • Author(s): Okuno D, Iino R, Noji H.
  • Publisher: Springer (India)(In press)
• [Book] ATP synthase-Structure and dynamics of the smallest rotary motor proteins. In "Molecular Biology of Assemblies and Machines" edited by Baumeister W, Johnson L, Perham R, and Steven A. (2011)
  • Author(s): Noji H, Iino R.
  • Publisher: Garland(England)(In press)
• [Book] In "Encyclopedia of Biophysics" (edited by Roberts G.C.)(Fundamental Properties and Structure of F1-ATPase.)(In press) (2010)
  • Author(s): Okuno D, Iino R, ^*Noji H.
  • Publisher: Springer (India).
• [Book] In "Molecular Biology of Assemblies and Machines"(ATP synthase-Structure and dynamics of the smallest rotary motor proteins.)(edited by Baumeister W, Johnson L,Perham R, Steven A.)(In press) (2010)
  • Author(s): ^*Noji H, Iino R.
  • Publisher: Garland (England).
• [Book] 「第4章第1節第1項生体膜超分子モーター-細胞の同転エネルギー変換装置:ATP合成酵素」超分子サイエンス&テクノロジー (2009)
  • Author(s): 飯野亮太、田端和仁、野地博行
  • Publisher: NTS
• [Patent(Industrial Property Rights)] ビーズ封入方法、ターゲット分子を検出する方法、アレイ、キット及びターゲット分子検出装置 (2011)
  • Inventor(s): *^野地博行, 新木卓, 飯野亮太
  • Industrial Property Rights Holder: 科学技術振興機構
  • Industrial Property Number: 2011-050629
  • Filing Date: 2011-03-08