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Molecular mechanism of chaperonin-assisted protein folding studied by single molecule imaging.

Research Project

Project/Area Number 14380320
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Biophysics
Research InstitutionThe University of Tokyo (2004)
Waseda University (2002-2003)

Principal Investigator

FUNATSU Takashi  The University of Tokyo, Graduate School of Pharmaceutical Sciences, Professor, 大学院・薬学系研究科, 教授 (00190124)

Co-Investigator(Kenkyū-buntansha) TAGUCHI Hideki  The University of Tokyo, Graduate School of Frontier Sciences, Associate Professor, 大学院・新領域創成科学研究科, 助教授 (40272710)
TADAKUMA Hisashi  The University of Tokyo, Graduate School of Engineering, Assistant Professor, 大学院・工学系研究科, 助手 (10339707)
Project Period (FY) 2002 – 2004
Project Status Completed (Fiscal Year 2004)
Budget Amount *help
¥16,900,000 (Direct Cost: ¥16,900,000)
Fiscal Year 2004: ¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2003: ¥5,700,000 (Direct Cost: ¥5,700,000)
Fiscal Year 2002: ¥7,900,000 (Direct Cost: ¥7,900,000)
Keywordsmolecular chaperon / single molecule imaging / GroEL / シャペロニン / 1分子イメージング / タンパク質間相互作用
Research Abstract

Chaperonin GroEL mediates substrate protein folding in a repeated cycle of ATP-driven GroEL-GroES association-dissociation processes. We have succeeded in detecting the processes at the level of a single molecule using total internal reflection fluorescence microscopy (TIRFM). Our results indicated that this process consists of two timers approximately 3 sec and 5 sec of the rate-limiting steps. In order to gain understating of this two timer process, acid-denatured GFP was used as a substrate protein for GroEL and its folding was observed. The result from this observation demonstrated that the substrate folding was inhibited in the first 3 sec period, and revealed a cis ADP*-complex, that is a new complex existed in the 5 sec rate-limiting step.
Furthermore, the existence of a so called football-shaped intermediate complex, consisting of a GroEL bound both sides with GroES, was examined using a new single molecule fluorescence imaging method. In this imaging, nano-holes with 100 nm diameter were fabricated in a metal film deposited on a slide-glass. This was used to localize evanescent field, reducing the fluorescence background, enabling us to image a single fluorescent molecule in the presence of a high concentration of fluorescence molecules. Using this method, processes of Gro-GroES association-dissociation was revealed at the level of a single molecule in the presence of fluorescently labeled 1 μM of GroES.

Report

(4 results)
  • 2004 Annual Research Report   Final Research Report Summary
  • 2003 Annual Research Report
  • 2002 Annual Research Report
  • Research Products

    (51 results)

All 2005 2004 2003 2002 Other

All Journal Article (37 results) Book (3 results) Publications (11 results)

  • [Journal Article] Micropatterning oligonucletides on single crystal diamond surface by photolithography2005

    • Author(s)
      Zhang, G., H.Umezawa, H.Hata, T.Zako, T.Funatsu, I.Ohdomari, H.Kawarada
    • Journal Title

      Jpn.J.Appl.Phys. 44

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] High-performance liquid chromatographic assay of N^G-monomethly-L-arginine, N^G, N^G-dimethyl-L-arginine, and N^G, N-<G'>-dimethyl-L-arginine using 4-fluoro-7-nitro-2,1,3-benzoxadiazole as a fluorescent reagent2005

    • Author(s)
      Nonaka, S., M.Tsunoda, K.Imai, T.Funatsu
    • Journal Title

      J.Chromatogr. A 1066

      Pages: 41-45

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Rapid assay for catechol-O-methyltransferase activity by high-performance liquid chromatography-fluorescence detection.2005

    • Author(s)
      Hirano, Y., M.Tsunoda, T.Funatsu, K.Imai
    • Journal Title

      J.Chromatogr. B 819

      Pages: 41-46

    • NAID

      10016981128

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Micropatterning oligonucletides on single crystal diamond surface by photolithography.2005

    • Author(s)
      Zhang, G.H.Umezawa, H.Hata, T.Zako, T.Funatsu, I.Ohdomari, H.Kawarada
    • Journal Title

      Jpn.J.Appl.Phys. 44

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] High-performance liquid chromatographic assay of N^G-monomethyl-L-arginine, N^G,N^G-dimethyl-L-arginine, and N^G,N^G-dimethyl-L-arginine using 4-fluoro-7-nitro-2,1,3-benzoxadiazole as a fluorescent reagent2005

    • Author(s)
      Nonaka, S., M.Tsunoda, K.Imai, T.Funatsu.
    • Journal Title

      J Chromatogr.A. 1066

      Pages: 41-45

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Rapid assay for catechol-O-methyltransferase activity by high-performance liquid chromatography-fluorescence detection.2005

    • Author(s)
      Hirano, Y., M.Tsunoda, T.Funatsu, K.Imai
    • Journal Title

      J Chromatogr.B. 819

      Pages: 41-46

    • NAID

      10016981128

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] The immobilization of DNA on microstructured patterns fabricated by maskless lithography.2004

    • Author(s)
      Zhang, G.-J., T.Tanii, T.Funatsu, I.Ohdomari
    • Journal Title

      Sensors and Actuators B 97

      Pages: 243-248

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Patterning of DNA nanostructures on silicon surface by electron beam lithography of self-assembled monolayer.2004

    • Author(s)
      Zhang, G.-J., T.Tanii, T.Funatsu, I.Ohdomari
    • Journal Title

      Chem Comm.

      Pages: 786-787

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] GroEL mediates protein folding with a two successive timer mechanism2004

    • Author(s)
      Ueno T., H.Taguchi, H.Tadakuma, M.Yoshida, T.Funatsu
    • Journal Title

      Molecular Cell 14

      Pages: 423-434

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Kinetics and binding sites for interaction of prefoldin with group II chaperonin : contiguous non-native substrate and chaperonin binding sites in archaeal prefoldin2004

    • Author(s)
      Okochi, M., T.Nomura, T.Zako, R.Iizuka, H.Ueda, T.Funatsu, M.Leroux, M.Yohda
    • Journal Title

      J.Biol. Chem 279

      Pages: 31788-31795

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Preferential immobilization of biomolecules on silicon microstructure array by means of electron beam lithography on organosilane self-assembled monolayer resist2004

    • Author(s)
      Tanii, T., T.Hosaka, T.Miyake, G.Zhang, T.Zako, T.Funatsu, I.Ohdomari
    • Journal Title

      Appl.Surf.Sci. 234

      Pages: 102-106

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Kinetic analysis of interactions between archaeal prefoldin and chaperonin.2004

    • Author(s)
      Zako T., T.Funatsu, M.Yohda
    • Journal Title

      Recent Res.Develop. Biophys 3

      Pages: 475-483

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Specific flow control systems using IR laser indeced sol-gel tranfer of hydrogel2004

    • Author(s)
      Tanaka, J.-I., Y.Shirasaki, M.Tatsuoka, T.Funatsu, S.Watanabe, S.Shoji, T.Edura, J.Mizuno, K.Tsutsui, Y.Wada
    • Journal Title

      NSTI Nanotechnology Conference and Trade Show-NSTI Nanotech 2004 1

      Pages: 296-299

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] The immobilization of DNA on microstructured patterns fabricated by mask less lithography.2004

    • Author(s)
      Zhang, G., T.Tanii, T.Zako, T.Funatsu, I.Ohdomari
    • Journal Title

      Sensors and Actuators B. 97

      Pages: 243-248

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Patterning of DNA nanostructures on silicon surface by electron beam lithography of self-assembled monolayer.2004

    • Author(s)
      Zhang, G.-J., T.Tanii, T.Funatsu, I.Ohdomari
    • Journal Title

      Chem Commun.

      Pages: 786-787

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] GroEL mediates protein folding with a two successive timer mechanism.2004

    • Author(s)
      Ueno T., H.Taguchi, H.Tadakuma, M.Yoshida, T.Funatsu
    • Journal Title

      Molecular Cell. 14

      Pages: 423-434

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Kinetics and binding sites for interaction of prefoldin with group II chaperonin : contiguous non-native substrate and chaperon in binding sites in archaeal prefoldin.2004

    • Author(s)
      Okochi, M., T.Nomura, T.Zako, R.Iizuka, H.Ueda, T.Funatsu, M.Leroux, M.Yohda
    • Journal Title

      J.Biol.Chem. 279

      Pages: 31788-31795

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Kinetic analysis of interactions between archaeal prefoldin and chaperonin.2004

    • Author(s)
      Zako T., T.Funatsu, M.Yohda
    • Journal Title

      Recent Res.Develop.Biophys. 3

      Pages: 475-483

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Single molecule imaging of chaperon in functions2004

    • Author(s)
      Ueno, T., Tadakuma H., T.Funatsu
    • Journal Title

      Tanpakusitsu Kakusan Koso 49

      Pages: 855-856

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] GroEL Mediates Protein Folding with a Two Successive Timer Mechanism2004

    • Author(s)
      Ueno, T., Taguchi, H., Tadakuma, H., Yoshida, M., Funatsu, T.
    • Journal Title

      Molecular Cell 14

      Pages: 423-434

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Patterning of DNA nanostructures on silicon surface by electron beam lithography of self-assembled monolayer.2004

    • Author(s)
      Zhang, G., Tanii, T., Funatsu, T., Ohdomari, I.
    • Journal Title

      Chem Comm.

      Pages: 786-787

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Kinetics and binding sites for interaction of prefoldin with group II chaperonin : contiguous non-native substrate and chaperonin binding sites in archaeal prefoldin.2004

    • Author(s)
      Okochi, M., et al.
    • Journal Title

      J.Biol.Chem. 279

      Pages: 31788-31795

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Kinetic analysis of interactions between archaeal prefoldin and chaperonin.2004

    • Author(s)
      Zako T., Funatsu, T., Yohda, M.
    • Journal Title

      Recent Res.Develop.Biophys. 3

      Pages: 475-483

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Preferential immobilization of biomolecules on silicon microstructure array by means of electron beam lithography on organosilane self-assembled monolayer resist.2004

    • Author(s)
      Tanii, T., et al.
    • Journal Title

      Appl.Surf.Sci. 234

      Pages: 102-106

    • Related Report
      2004 Annual Research Report
  • [Journal Article] 1分子イメージングによるシャペロン研究2004

    • Author(s)
      上野太郎, 多田隈尚史, 船津高志
    • Journal Title

      蛋白質・核酸・酵素 49(7 sup.)

      Pages: 855-856

    • Related Report
      2004 Annual Research Report
  • [Journal Article] PDMS microfluidics devices with PTFE passivated channels.2003

    • Author(s)
      Kanai, M., D.Uchida, S.Sugiura, Y.Shirasaki, J.S.Go, H.Nakanishi, T.Funatsu, S.Shoji
    • Journal Title

      Micro Total Analysis System 2003

      Pages: 429-432

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] mRNA核外輸送の可視化2003

    • Author(s)
      多田隈尚史, 船津高志, 谷時雄
    • Journal Title

      蛋白質核酸酵素 48

      Pages: 421-429

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] PDMS microfluidics devices with PTFE passivated channels.2003

    • Author(s)
      Kanai, M., D.Uchida, S.Sugiura, Y.Shirasaki, J.S.Go, H.Nakanishi, T.Funatsu, S.Shoji.
    • Journal Title

      Micro Total Analysis System 2003.

      Pages: 429-432

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Imaging of mRNA nuclear export in a living cell2003

    • Author(s)
      H.Tadakuma, T.Funatsu, T.Tani
    • Journal Title

      Tanpakusitu Kakusan Koso 48

      Pages: 421-429

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Rapid and Sensitive Detection Method of A Bacterium Using GFP Reporter Phage.2002

    • Author(s)
      Funatsu, T., T.Taniyama, T.Tajima, H.Tadakuma, H.Namiki
    • Journal Title

      Microbiol. Immuno 46

      Pages: 365-369

    • NAID

      50000571049

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Microscopicanalysis of polymerization dynamics with individual actin filaments2002

    • Author(s)
      Fujiwara, I., S.Takahashi, H.Tadakuma, T.Funatsu, S.Ishiwata
    • Journal Title

      Nature Cell Biol. 4

      Pages: 666-673

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] A Novel Biomolecule Sorter Using Thermosensitve Hydrogel in Micro Flow System2002

    • Author(s)
      Shirasaki, Y., H.Makazu, K.Tashiro, S.Ikeda, T.Sekiguchi, S.Shoji, S.Tsukita, T.Funatsu
    • Journal Title

      Micro Total Analysis System 2002

      Pages: 925-927

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Rapid and Sensitive Detection Method of A Bacterium Using GFP Reporter Phage.2002

    • Author(s)
      Funatsu, T., T.Taniyama, T.Tajima, H.Tadakuma, H.Namiki
    • Journal Title

      Microbiol.Immuno. 46

      Pages: 365-369

    • NAID

      50000571049

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Microscopicanalysis of polymerization dynamics with individual actin filaments.2002

    • Author(s)
      Fujiwara, I., S.Takahashi, H.Tadakuma, T.Funatsu, S.Ishiwata.
    • Journal Title

      Nature Cell Biol. 4

      Pages: 666-673

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] A Novel Biomolecule Sorter Using Thermosensitve Hydrogel in Micro Flow System2002

    • Author(s)
      Shirasaki, Y., H.Makazu, K.Tashiro, S.Ikeda, T.Sekiguchi, S.Shoji, S.Tsukita, T.Funatsu
    • Journal Title

      MicroTotal Analysis System 2002.

      Pages: 925-927

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Synthesis of benzofurazan derivatization regents for carboxylic and acids and its application to analysis of fatty acids in rat plasma by high-performance liquid chromatography/electrospray ionization mass spectrometry.

    • Author(s)
      Tsukamoto Y., T.Santa, H.Saimaru, K.Imai, T.Funatsu
    • Journal Title

      Biomed.Chromatogy. (In press)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Synthesis of benzofurazan derivatization regents for carboxylic and acids and its application to analysis of fatty acids in rat plasma by high-performance liquid chromatography/electrospray ionization mass spectrometry.

    • Author(s)
      Tsukamoto Y., T.Santa, H.Saimaru, K.Imai, T.Funatsu
    • Journal Title

      Biomed.Chromatogy. (In press)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Book] 「1分子イメージングによるシャペロン研究」細胞における蛋白質の一生(蛋白質核酸酵素 5月号増刊)pp.855-856(小椋光、遠藤斗志也、森正敬、吉田賢右 編)2004

    • Author(s)
      上野太郎, 多田隈尚史, 船津高志
    • Total Pages
      1152
    • Publisher
      共立出版
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Book] Particles and molecules handling in micro channels" Lab-on-a-chip. Miniaturized System for (Bio) Chemical Analysis and Synthesis (R.E.Oosterbroek and A van den Berg eds.) pp. 205-2142003

    • Author(s)
      Shoji, S., K.Tashiro, T.Sekiguchi, H.Sato, N.Honda, T.Funatsu
    • Total Pages
      394
    • Publisher
      Elsevier B.V.
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Book] Particles and molecules handling in micro channels Lab-on-a-chip. Miniaturized System for (Bio)Chemical Analysis and Synthesis (R.E.Oosterbroek and A van den Berg eds.)2003

    • Author(s)
      Shoji, S., K.Tashiro, T.Sekiguchi, H.Sato, N.Honda, T.Funatsu
    • Publisher
      Elsevier B.V.
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Publications] G.Zhang: "The immobilization of DNA on microstructured patterns fabricated by maskless lithography"Sensors and Actuators B. 97. 243-248 (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] G.Zhang: "Patterning of DNA nanostructures on silicon surface by electron beam lithography of self-assembled monolayer."Chem Comm.. (in press). (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] M.Kanai: "PDMS microfluidics devices with PTFE passivated channels"Micro Total Analysis System 2003. 429-432 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] 船津高志: "単一分子計測とバイオフォトニクス"応用物理. 72. 727-730 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] S.Shoji: "Particles and molecules handling in micro channels"Lab-on-a-Chip. Miniaturized Systems for (Bio)Chemical Analysis and Synthesis. 205-214 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] 多田限尚史: "mRNA核外輪送の可視化"蛋白質核酸酵素. 48. 421-429 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Funatsu T., et al.: "Rapid and sensitive detection method of a bacterium using GFP reporter phage"Microbiol Immuno. 46. 365-369 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Fujiwara I., et al.: "Microscopic analysis of polymerization dynamics with individual actin filaments"Nature Cell Biol.. 4. 666-673 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Shirasaki Y., et al.: "A novel biomolecule sorter using thermosensitive hydrogel in micro flow system"Micro Total Analysis System 2002. 925-927 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 船津高志: "1分子蛍光イメージング技術による生命現象の解析"可視化情報学会誌. 22. 18-21 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 座古保, 船津高志: "生体機能を1分子レベルで探る"分光研究. 51. 3-14 (2002)

    • Related Report
      2002 Annual Research Report

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Published: 2002-04-01   Modified: 2016-04-21  

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