Cooperative Conformational Dynamics of Ring-Shaped ATPase studied by High-Speed AFM/Fluorescence Microscopy

• Project/Area Number: 24241048
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Nanomaterials/Nanobioscience
• Research Institution: Kanazawa University
• Principal Investigator: UCHIHASHI TAKAYUKI 金沢大学, 数物科学系, 教授 (30326300)
• Co-Investigator(Kenkyū-buntansha): IINO Ryota 大学共同利用機関法人自然科学研究機構, 岡崎統合バイオサイエンスセンター, 教授 (70403003) ; WATANABE Yo-hei 甲南大学, 理工学部, 准教授 (40411839)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥46,930,000 (Direct Cost: ¥36,100,000、Indirect Cost: ¥10,830,000); Fiscal Year 2014: ¥14,040,000 (Direct Cost: ¥10,800,000、Indirect Cost: ¥3,240,000); Fiscal Year 2013: ¥13,910,000 (Direct Cost: ¥10,700,000、Indirect Cost: ¥3,210,000); Fiscal Year 2012: ¥18,980,000 (Direct Cost: ¥14,600,000、Indirect Cost: ¥4,380,000)
• Keywords: 一分子イメージング / 走査型プローブ顕微鏡 / 高速原子間力顕微鏡 / タンパク質 / 分子シャペロン / 構造変化 / 一分子計測 / 蛋白質 / ATPase / 走査プローブ顕微鏡 / AAAタンパク質 / シャペロニン / 蛍光顕微鏡

Outline of Final Research Achievements

We observed conformational dynamics of a molecular chaperone ClpB which disaggregates denatured proteins. We found that the hexameric ring of the TClpB dynamically changes between symmetric, asymmetric and broken ring. Also it was indicated that the dynamic fluctuation of the ring structure is essential for the chaperon activity of ClpB.
We also improved combined system high-speed AFM and fluorescent microscopy. The combined system enabled simultaneous single-molecule imaging with high-speed AFM and fluorescence microscopy. We succeeded in observing processive movement of enzymatic chitinase with fluorophores along a chitin fibril and further conformational change of F1-ATPase and binding/dissociation of fluorescent-ATP analogue. We have applied high-speed AFM to investigate cooperative conformational change of V1-ATPase without the shaft (DF subunit). We found that conformational change of the A subunit propagates unidirectionally in the hexameric ring even without the shaft.

Research Products

• [Journal Article] Long-tip high-speed atomic force microscopy for nanometer-scale imaging in live cells (2015)
  • Author(s): M. Shibata, T. Uchihashi, T. Ando and R. Yasuda
  • Journal Title: Scientific Reports Volume: 5 Pages: 8724-8724
  • Peer Reviewed / Open Access
• [Journal Article] Probing structural dynamics of an artificial protein cage using high-speedatomic force microscopy (2015)
  • Author(s): M. Imamura, T. Uchihashi, T. Ando, A. Leifert, U. Simon, A. D. Malay and J. G. Heddle
  • Journal Title: Nano Letters Volume: 15 Issue: 2 Pages: 1331-1335
  • DOI: 10.1021/nl5045617
  • Peer Reviewed
• [Journal Article] Two-way traffic of glycoside hydrolase family 18 processive chitinases on crystalline chitin (2014)
  • Author(s): K. Igarashi, T. Uchihashi, T. Uchiyama, H. Sugimoto, M. Wada, K. Suzuki, S. Sakuda, T. Ando, T. Watanabe, and M. Samejima
  • Journal Title: Nature Communications Volume: 5 Pages: 2975-2975
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] キチナーゼによる結晶性キチンのプロセッシブ（連続的）な分解機構の解明 (2014)
  • Author(s): 杉本華幸, 五十嵐圭日子, 内橋貴之, 鈴木一史, 渡邉剛志
  • Journal Title: 日本応用糖質化学会誌 Volume: 4 Pages: 107-112
• [Journal Article] 高速原子間力顕微鏡による生体試料のダイナミクス観察 (2014)
  • Author(s): 内橋貴之
  • Journal Title: 膜誌 Volume: 39 Pages: 322-328
  • NAID: 130005069445
• [Journal Article] 高速原子間力顕微鏡による生体分子のナノ動体撮影 (2014)
  • Author(s): 古寺哲幸, 内橋貴之, 安藤敏夫
  • Journal Title: 日本物理学会誌 Volume: 69 Pages: 459-464
• [Journal Article] ingle-molecule imaging analysis of elementary reaction steps of Trichoderma Reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose Iα and IIII (2014)
  • Author(s): Y.Shibafuji, A. Nakamura, T. Uchihashi, N. Sugimoto, S. Fukuda, H. Watanabe, M. Samejima, T. Ando, H. Noji, A. Koivula, K. Igarashi, R. Iino
  • Journal Title: Journal of Biological Chemistry Volume: 印刷中
  • Peer Reviewed
• [Journal Article] Trade-off between Processivity and Hydrolytic Velocity of Cellobiohydrolases at the Surface of Crystalline Cellulose (2014)
  • Author(s): Akihiko Nakamura, Watanabe, H., Ishida, T., Uchihashi, T., Wada, M., Ando, T., Igarashi, K. and Samejima, M.
  • Journal Title: Journal of the American Chemical Society Volume: 136 Issue: 12 Pages: 4584-4592
  • DOI: 10.1021/ja4119994
  • Peer Reviewed
• [Journal Article] Filming Biomolecular Processes by High-Speed Atomic Force Microscopy (2014)
  • Author(s): T. Ando, T. Uchihashi, S. Scheuring
  • Journal Title: Chemical Reviews Volume: 114 Issue: 6 Pages: 3120-3188
  • DOI: 10.1021/cr4003837
  • Peer Reviewed / Open Access
• [Journal Article] Real-Time Visualization of Assembling of a Sphingomyelin-Specific Toxin. (2013)
  • Author(s): N. Yilmaz, T. Yamada, P. Greimel, T. Uchihashi, T. Ando, T. Kobayashi
  • Journal Title: Biophys. J. Volume: 105 Issue: 6 Pages: 1397-1405
  • DOI: 10.1016/j.bpj.2013.07.052
  • Peer Reviewed
• [Journal Article] High-speed atomic force microscope combined with single-molecule fluorescence microscope. (2013)
  • Author(s): S. Fukuda, T. Uchihashi, R. Iino, Y. Okazaki, M. Yoshida, K. Igarashi, T. Ando
  • Journal Title: Rev. Sci. Instrum. Volume: 84 Issue: 7 Pages: 73706-73706
  • DOI: 10.1063/1.4813280
  • Peer Reviewed
• [Journal Article] High-speed AFM and applications to biomolecular systems (2013)
  • Author(s): T. Ando, T. Uchihashi, and N. Kodera
  • Journal Title: Annu. Rev. Biophys Volume: 42 Issue: 1 Pages: 393-414
  • DOI: 10.1146/annurev-biophys-083012-130324
  • Peer Reviewed
• [Journal Article] Wide-area scanner for high-speed atomic force microscopy. (2013)
  • Author(s): H. Watanabe, T. Uchihashi, T. Kobashi, M. Shibata, J. Nishiyama, R. Yasuda, T. Ando
  • Journal Title: Rev. Sci. Instrum. Volume: 84 Issue: 5 Pages: 53702-53702
  • DOI: 10.1063/1.4803449
  • NAID: 120005304728
  • Peer Reviewed
• [Journal Article] Role of trimer-trimer interaction of bacteriorhodopsin studied by optical spectroscopy and high-speed atomic force microscopy (2013)
  • Author(s): H. Yamashita, K. Inoue, M. Shibata, T. Uchihashi, J. Sasaki, H. Kandori, and T. Ando
  • Journal Title: J. Struct. Biol. Volume: (accepted) Issue: 1 Pages: 2-11
  • DOI: 10.1016/j.jsb.2013.02.011
  • NAID: 120005253833
  • Peer Reviewed
• [Journal Article] Real Time Single Molecular Imaging of Enzymatic Degradation of Crystalline Cellulose by High-speed Atomic Force Microscopy (2013)
  • Author(s): 五十嵐 圭日子, 内橋 貴之, 鮫島 正浩, 安藤 敏夫
  • Journal Title: Seibutsu Butsuri Volume: 53 Issue: 3 Pages: 140-144
  • DOI: 10.2142/biophys.53.140
  • NAID: 10031159065
  • ISSN: 0582-4052, 1347-4219
  • Peer Reviewed
• [Journal Article] Development of High-speed Atomic Force Microcscopy (2013)
  • Author(s): 内橋貴之, 安藤敏夫
  • Journal Title: Journal of the Japan Society for Precision Engineering Volume: 79 Issue: 3 Pages: 218-222
  • DOI: 10.2493/jjspe.79.218
  • NAID: 130003369799
  • ISSN: 0912-0289, 1882-675X
  • Peer Reviewed
• [Journal Article] リアルタイム原子間力顕微鏡の開発とたんぱく質の機能動態イメ ージング (2013)
  • Author(s): 内橋貴之, 古寺哲幸
  • Journal Title: 光学 Volume: 42 Pages: 89-94
  • NAID: 10031154878
  • Peer Reviewed
• [Journal Article] リアルタイム原子間力顕微鏡の開発とたんぱく質の機能動態イメージング (2013)
  • Author(s): 内橋貴之
  • Journal Title: 光学 Volume: 42 Pages: 89-94
  • NAID: 10031154878
• [Journal Article] Visualization of cellobiohydrolase I from Trichoderma reesei moving on crystalline cellulose using high-speed atomic force microscopy (2012)
  • Author(s): Kiyohiko Igarashi
  • Journal Title: Methods in Enzymology Volume: 510 Pages: 169-182
  • DOI: 10.1016/b978-0-12-415931-0.00009-4
  • Peer Reviewed
• [Journal Article] Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy (2012)
  • Author(s): Takayuki Uchihashi
  • Journal Title: Nature Protocols Volume: 7 Pages: 1193-1206
  • Peer Reviewed
• [Journal Article] Single molecule imaging on living bacterial cell surface by high-speed AFM (2012)
  • Author(s): Hayato Yamashita
  • Journal Title: Journal of Molecular Biology Volume: 422 Issue: 2 Pages: 300-309
  • DOI: 10.1016/j.jmb.2012.05.018
  • Peer Reviewed
• [Journal Article] High-speed atomic force microscopy (2012)
  • Author(s): T. Ando, T. Uchihashi, and N. Kodera
  • Journal Title: Jpn. J. Appl. Phys Volume: 51 Issue: 8S3 Pages: 08KA02-08KA02
  • DOI: 10.1143/jjap.51.08ka02
  • NAID: 210000141129
  • Peer Reviewed
• [Presentation] High-speed atomic force microscope for imaging of biomolecular dynamics at solid surface (2015)
  • Author(s): Takayuki Uchihashi
  • Organizer: 10th Annual International Electromaterials Science Symposium
  • Place of Presentation: Wollongong, Australia
  • Year and Date: 2015-02-11
  • Invited
• [Presentation] Visualization of single molecule dynamics at work with high-speed atomic force microscopy (2015)
  • Author(s): Takayuki Uchihashi
  • Organizer: The 7th Biennial Australian Colloid & Interface Symposium
  • Place of Presentation: Hobart, Tasmania, Australia
  • Year and Date: 2015-02-03
  • Invited
• [Presentation] 高速原子間力顕微鏡によるタンパク質のダイナミクスと物性計測 (2015)
  • Author(s): 内橋貴之
  • Organizer: NIBBバイオイメージングフォーラム「物理特性のイメージング」
  • Place of Presentation: 基礎生物学研究所, 愛知県
  • Year and Date: 2015-01-27
  • Invited
• [Presentation] 高速原子間力顕微鏡の開発とバイオ応用 (2015)
  • Author(s): 内橋貴之
  • Organizer: 新世代研究所水和ナノ構造・界面ナノ科学合同研究会「固液界面の水和ナノ構造と生体高分子ダイナミクス」
  • Place of Presentation: 伊豆長岡温泉 天坊, 静岡県
  • Year and Date: 2015-01-25
  • Invited
• [Presentation] High-speed atomic force microscope for studying dynamic interactions in biomolecular system (2015)
  • Author(s): Takayuki Uchihashi
  • Organizer: The 3rd International Symposium on Dynamical Ordering of Biomolecular Systems for Creation of Integrated Functions
  • Place of Presentation: 合歓の郷,三重県
  • Year and Date: 2015-01-10
  • Invited
• [Presentation] Single-molecule imaging of proteins at work with high-speed atomic force microscopy (2014)
  • Author(s): Takayuki Uchihashi
  • Organizer: 16th International Conference on Retinal Proteins
  • Place of Presentation: 長浜ロイヤルホテル, 滋賀県
  • Year and Date: 2014-10-05
  • Invited
• [Presentation] Cooperative Conformational Change in Ring-Shaped ATPase Observed by High-Speed AFM (2014)
  • Author(s): 内橋貴之
  • Organizer: 第52回日本生物物理学会年会 Japan-China-Taiwan Joint Symposium on Cooperativity in Supramolecular Machine
  • Place of Presentation: 札幌国際会議場, 北海道
  • Year and Date: 2014-09-26
  • Invited
• [Presentation] High-speed atomic force microscopy for imaging of protein dynamics (2014)
  • Author(s): Takayuki Uchihashi
  • Organizer: Agilent Nanomeasure 2014
  • Place of Presentation: Beijing, China
  • Year and Date: 2014-09-16
  • Invited
• [Presentation] 高速AFMによる生体試料のダイナミクス観察 (2014)
  • Author(s): 内橋貴之
  • Organizer: 日本膜学会第36回年会境界領域シンポジウム
  • Place of Presentation: 早稲田大学, 東京都
  • Year and Date: 2014-05-13
  • Invited
• [Presentation] Visualization of single molecule dynamics at work with high-speed atomic force microscopy (2014)
  • Author(s): Takayuki Uchihashi
  • Organizer: Single Protein Dynamics in Cellulo 2014: Spatio-Temporal, Structural and Quantitative Analyses
  • Place of Presentation: 沖縄科学技術大学院大学、沖縄県
  • Year and Date: 2014-04-23
  • Invited
• [Presentation] High-speed AFM observation of the FliI/ FliJ complex (2013)
  • Author(s): D. Carriou1, T. Uchihashi, Y. Uchida, H. Yanagawa, T. Minamino, K. Imada
  • 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
• [Presentation] Nanoscale investigation on bacterial cell surface using high-speed AFM (2013)
  • Author(s): H. Watanabe, C. David, T. Uchihashi, T. Ando
  • 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
• [Presentation] Combined system of high-speed-AFM and optical microscopy (2013)
  • Author(s): S. Fukuda1, T. Uchihashi, R. Iino, T. Ando
  • 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
• [Presentation] Conformations and dynamics of ClpB hexameric ring observed by high-speed AFM (2013)
  • Author(s): T. Uchihashi, Y. Watanabe, R. Iino, H. Watanabe, T. Yamasaki, T. Ando
  • 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
• [Presentation] 高速原子間力顕微鏡によるタンパク質の一分子ダイナミクス計測 (2013)
  • Author(s): 内橋貴之
  • Organizer: 第９０回日本生理学会大会 日本生物物理学会連携シンポジウ ”新しい計測で生命科学を切り拓く”
  • Place of Presentation: 船堀
  • Invited
• [Presentation] HIGH-SPEED ATOMIC FORCE MICROSCOPY FOR OBSERVING DYNAMIC BIOMOLECULAR PROCESSES (2012)
  • Author(s): Takayuki Uchihashi
  • Organizer: XXI International Materials Research Congress
  • Place of Presentation: Cancun, Mexico
  • Invited
• [Presentation] 高速原子間力顕微鏡で可視化するタンパク質の機能動態 (2012)
  • Author(s): 内橋貴之
  • Organizer: 第52回生命科学夏の学校
  • Place of Presentation: 蒲郡
  • Invited
• [Presentation] High-speed atomic force microscopy: A tool for nanobionnics (2012)
  • Author(s): Takayuki Uchihashi
  • Organizer: 3rd Asia-Pacific Symposium on Nanobionics
  • Place of Presentation: Wollongong, Australia
  • Invited
• [Presentation] Image analysis of HS-AFM movies for dynamic events on biological molecules: F1-ATPase and cellulase (2012)
  • Author(s): Takayuki Uchihashi
  • Organizer: 3rd Kanazawa Bio-AFM Workshop
  • Place of Presentation: Kanazawa
  • Invited
• [Presentation] 高速原子間力顕微鏡でイメージ ングするタンパク質の一分子ダイナミクス (2012)
  • Author(s): 内橋貴之
  • Organizer: 第１２回名古屋大学遺伝子実験施設公開セミナー
  • Place of Presentation: 名古屋
  • Invited
• [Presentation] 高速AFMによるバイオ分子の液中動的観察 (2012)
  • Author(s): 内橋貴之
  • Organizer: 生体分子機能解析のための走査型プローブ顕微鏡手法研究部会
  • Place of Presentation: 越後湯沢
  • Invited
• [Presentation] 高速AFMを用いた1分子ダイナミクス (2012)
  • Author(s): 内橋貴之
  • Organizer: 第８５回日本生化学会大会, シンポジウム”少数性・生化学の新たな視点”
  • Place of Presentation: 福岡
  • Invited
• [Presentation] Combined High-Speed AFM and Optical Microscopy (2012)
  • Author(s): Takayuki Uchihashi
  • Organizer: 20th International Colloquium on Scanning Probe Microscopy (ICSPM20)
  • Place of Presentation: Okinawa
• [Presentation] 高速AFMによる生体分子のダイナミクス解析 (2012)
  • Author(s): 内橋貴之
  • Organizer: KEK PF 第1回先進的観測技術研究会
  • Place of Presentation: つくば
  • Invited
• [Presentation] HS-AFM observations of conformational dynamics of ring-shaped ATPases: F1 and TClpB
  • Author(s): 内橋貴之, 飯野亮太, 渡辺洋平, 野地博行, 安藤敏夫
  • Organizer: 231st IMEG seminars & Minisymposium: ATP/GTP駆動分子マシナリーの高速AFMイメージングと分子機構解明の進展
  • Place of Presentation: 熊本大学発生医学研究所
  • Invited
• [Presentation] 高速原子間力顕微鏡による生体試料の動的構造解析 ~一分子から細胞まで~
  • Author(s): 内橋貴之
  • Organizer: 第１回新学術領域「植物環境感覚」「少数性生物学」ジョイントシンポジウム
  • Place of Presentation: 大阪大学中之島センター
  • Invited
• [Presentation] Application of high-speed atomic force miscroscopy –from proteins to Cells
  • Author(s): T. Uchihashi, N. Kodera and T. Ando
  • Organizer: 12th International Conference on Frontiers of Polymers and Advanced Materials
  • Place of Presentation: Auckland, New Zealand,
  • Invited
• [Presentation] High-Speed Atomic Force Microscopy for Imaging of Protein Dynamics
  • Author(s): T. Uchihashi and T. Ando
  • Organizer: 2013 MRS Fall Meeting, Symposium LL: Advances in Scanning Probe Microscopy
  • Place of Presentation: Boston, USA
  • Invited
• [Presentation] High-speed AFM for observing cell dynamics
  • Author(s): T. Uchihashi
  • Organizer: 4th Asia-Pacific Symposium on Nanobionics
  • Place of Presentation: Melbourne, Australia
  • Invited
• [Presentation] 高速原子間力顕微鏡 ~分子の動きを直視して理解する~
  • Author(s): 内橋貴之
  • Organizer: 生物物理若手の会 第53回夏の学校
  • Place of Presentation: 伊豆長岡温泉
  • Invited
• [Presentation] Single-Molecule Imaging of Proteins at Work with High-Speed Atomic Force Microscopy
  • Author(s): T. Uchihashi, N. Kodera and T. Ando
  • Organizer: IMS Workshop on "Hierarchical Molecular Dynamics: From Ultrafast Spectroscopy to Single Molecule Measurements"
  • Place of Presentation: 岡崎
  • Invited
• [Presentation] 高速AFMによるタンパク質機能動態の直接観察
  • Author(s): 内橋貴之
  • Organizer: 日本生化学会九州支部例会シンポジウム「タンパク質構造の動きと機能発現」
  • Place of Presentation: 佐賀大学
• [Book] “Development of high-speed AFM and its biological applications”, Chapter 8 in Atomic Force Microscopy in Nanobiology (2014)
  • Author(s): T. Uchihahsi, N. Kodera and T. Ando
  • Total Pages: 34
  • Publisher: Pan Stanford Publishing
• [Book] 膜タンパク質構造研究(岩田想編) (2013)
  • Author(s): 内橋貴之、安藤敏夫
  • Publisher: 化学同人
• [Book] Nanoscale Liquid Interfaces: Wetting, Patterning, and Force Microscopy at the Molecular Scale (edited by Thierry ONDARÇUHU, Jean-Pierre Aimé ) (2013)
  • Author(s): T. Ando, T. Uchihashi
  • Publisher: Pan Stanford Publishing
• [Book] Nano-visualization of proteins in action using high-speed AFM" in Single-molecule Studies of Proteins. Biophysics for the Life Sciences Vol. 2 (2013)
  • Author(s): T. Uchihashi, N. Kodera, T. Ando
  • Publisher: Springer
• [Book] Single-molecule Studies of Proteins. Biophysics for the Life Sciences (2013)
  • Author(s): Takayuki Uchihashi
  • Total Pages: 269
  • Publisher: Springer