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Control of thermal fluctuation of proteins by evanessent field trapping.

Research Project

Project/Area Number 09359004
Research Category

Grant-in-Aid for Scientific Research (A)

Allocation TypeSingle-year Grants
Section展開研究
Research Field 広領域
Research InstitutionOsaka University

Principal Investigator

YANAGIDA Toshio  Graduate School of Medicine, Osaka University Professor, 医学系研究科, 教授 (30089883)

Co-Investigator(Kenkyū-buntansha) SUZUKI Makoto  Department of Technology, Tohoku University, Professor, 工学部, 教授 (60282109)
KITAMURA Yuri  Graduate School of Medicine, Osaka University Assistant Professor, 医学系研究科, 助手 (90294074)
SAKO Yasushi  Graduate School of Medicine, Osaka University Associate Professor, 医学系研究科, 助教授 (20215700)
Project Period (FY) 1997 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥32,600,000 (Direct Cost: ¥32,600,000)
Fiscal Year 1999: ¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1998: ¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1997: ¥23,500,000 (Direct Cost: ¥23,500,000)
Keywordsmyosin / actin / surface plasmon / total internal reflection / chemi-mechano couppling / single molecule analysis / アクチンフィラメント / 光ピンセット / ガラスニードル / 回転拡散 / 全反射顕微鏡 / エバネッセント場 / 熱揺らぎ / 分子モーター / 科学 / 力学共役
Research Abstract

The aims of this study were development of new techniques to control thermal fluctuation of proteins and to apply these techniques to actin-myosin system toward the final goal that is to understand roles of thermal fluctuation in chemi-mechano couppling of actin-myosin system. During the period of this grant, we obtained the following achievments to the final goal.
(1)Single molecule imaging of fluorescently-labelled proteins on metal by surface plasmons in aqueous solution : We made an optical microscope using surface plasmon resonance at the meniscus between thin metal layer and water. By using this technique, evanessent field was enhanced 2-10 times compered to that by total internal reflection between glass and water. Active movement of single molecules of the fluorescently labelled motor proteins was observed on the surface of gold and alminium.
(2)Development of a technique that allows mechanical and ligand-binding events in a single myosin molecules to be monitored simultaneously … More : Using this technique, we found that the force generation of single myosin molecule does not always coincide with the release of bound nucleotide, presumably ADP. Instead the myosin head produces force several handreds of milliseconds after ADP is released.
(3)Development of a new instrument to capture and manipulate individual myosin molecules using a scanning probe : We found that single myosin head moves along an actin filament with regular steps of 。ォ5.5nm. Groups of two to five rapid steps in succession often produce displacement of 11 to 30nm. This multiple stepping is produced during just one biochemical cycle of ATP.
(4)Measurement of the torsional diffusion of actinfilaments : A single actin filament with bead attached to both ends was suspended in solution by optical tweezers. Torsional diffusion of the filament was observed by rotational movements of a bead in the optical tweezers in the presence of myosin with or without ATP. Amplitude of the rotation was increased in the presence of ATP, though torsional regidity of the filament was not changed. Less

Report

(4 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • 1997 Annual Research Report
  • Research Products

    (47 results)

All Other

All Publications (47 results)

  • [Publications] Y, Ishii et al.: Proceedings of the Australian Physiological and Pharmacological Society.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Y, Ishii et al.: "Imaging and nano-manipulation of single actomyosin motors at work"Chemical Physics. 30(1). 24-39 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Ide et al.: "An artificial lipid bilayer formed on an agarose-coated glass for simultaneous electrical and optical measurement of single Ion-channels"Biochem. Biophys. Res. Commun.. 265. 595-599 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Y, Ishii et al.: "Imaging and Nano-Manipulation of Single Actomyosin Motors at Work"Clinical and Experimental Pharmacology and Physiology. (In press).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Y, Ishii et al.: "Direct Inhibition of Microtubule-based Kinesin Motility by Local Anesthetics"Biophys. J.. (In press).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Y, Ishii et al.: "Molecular Interactions of Actin : Myosin interaction, motility assays and Ca-Regulatory proteins"D .D. Thomas and C. G. dos Remedios eds. Springer Verlag(Heidelberg) (In press).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K. Saito, M. Tokunaga, A. H. Iwane and T. Yanagida.: "Dual-colour microscopy of single fluorophores bound to myosin interacting with fluorescently labelled actin using anti-stoke fluorescence."J. Microscopy.. 183. 255 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Y. Inoue, Y. Yano-Toyoshima, A. Hikikoshi-Iwane, S. Morimoto, H. Higuchi and T. Yanagida.: "Movements of truncated kinesin fragments with a short or an artificial flexible neck."Proc. Natl. Acad. Sci. USA.. 94. 7275-7280 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] M. Tokunaga, K. Kitamura, K. Saito, A. Hikikoshi Iwane and T. Yanagida.: "Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy."Biochem. Biophys. Res. Commun.. 235. 47-53 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] A. Hikikoshi Iwane, T. Funatsu, Y. Harada, M. Tokunaga, O. Ohara, S.: "Morimoto and T. Yanagida. Single molecular assay of individual ATP turnovers by a myosin-GFP fusion protein expressed in vitro."FEBS Letters.. 407. 235-238 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] A. Hikikoshi Iwane, K. Kitamura, M. Tokunaga and T. Yanagida: "Myosin subfragment-1 is fully equipped with factors essential for motor function."Biochem. Biophys. Res Commun.. 230. 76-80 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Aoki, M. Hiroshima, K. Kitamura, M. Tokunaga and T. Yanagida: "Noncontract scanning probe microscopy with sub-piconewton force sensitivity."Ultramicroscopy.. 70. 45-55 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] H. Kojima, E. Muto, H. Higuchi and T. Yanagida: "Mechanics of single kinesin molecules measured by optical trapping nanometry."Biophys. J.. 73. 2012-2022 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] H. Higuchi,E. Mutoh, Y. Inoue and T. Yanagida: "Kinetics of force generation by single kinesin molecules activated by laser photolysis of caged ATP."Proc. Natl. Acad. Sci. USA. 94. 4395-4400 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] M. Tokunaga, T. Aoki, M. Hiroshima, K. Kitamura and T. Yanagida.: "Subpiconewton intermolecular force microscopy."Biochem. Biophys. Res. Commun.. 231. 566-569 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] C. Shingyoji, H. Higuchi, M. Yoshimura, E. Katayama and T. Yanagida.: "Dynein arms are oscillating force generators."Nature. 393. 711-714 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] H. Yokota, K. Saito and T. Yanagida: "Single molecule imaging of fluorescently-labeled proteins on metal by surface plasmons in aqueous solution."Phys. Rev. Letter.. 80(20). 4606-4609 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] A. Ishijima, H. Kojima, T. Funatsu, M. Tokunaga, H. Higuchi, H. Tanaka and T. Yanagida: "Simultaneous observation of individual ATPase and mechanical events by a single myosin molecule during interation with actin."Cell.. 92. 161-171 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] H. Tanaka, A. Ishijima, M. Honda, K. Saito and T. Yanagida: "Orientation dependence of displacements by a fingle one-headed myosin relative to the actin filament."Biophys. J.. 75. 1886-1894 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K. Kitamura, M. Tokunaga, A. H. Iwane and T. Yanagida.: "A single myosin head moves along an actin filament with regular steps of 。ォ5.3nm."Nature.. 397. 129-134 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Y. Harada, T. Funatsu, K. Murakami, Y. Nonoyama, A. Ishihama and T. Yanagida.: "Single molecule imaging of RNA polymerase-DNA interactions in real time."Biophys. J.. 76. 709-715 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Y. Ishii, T. Yoshida, T. Funatsu, T. Wazawa, and T. Yanagida.: "Fluorescence resonance energy transfer between single fluorophores attached to a coiled-coil protein in aqueous solution."Chemical Physics.. 247. 163-173 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] S. Iwatani, A. H. Iwane, H. Higuchi, Y. Ishii, and T. Yanagida.: "Mechanical and Chemical Properties of Cysteine-Modified Kinesin Molecules."Biochemistry.. 38. 10318-10323 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Ide and T. Yanagida.: "An artificial lipid bilayer formed on an agarose-coated glass for simultaneous electrical and optical measurement of single ion-channels."Biochem. Biophys. Res. Commun.. 265. 595-599 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Y. Sambongi, Y. Iko, M. Tanabe, H. Omote, A. Iwamoto-Kihara, I. Ueda, T. Yanagida, Y. Wada and M. Futai.: "Mechanical rotation of the c subunit oligomer in the ATP synthase (FoF1) : direct observation."Science.. 286. 1722-1724 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] R. Yamasaki, M. Hoshino, T. Wazawa, Y. Ishii, T. Yanagida, Y. Kawata, T. Higurashi, K. Sasaki, J. Nagai, and Y. Goto.: "Single molecular observation of the interaction of GroEL with substrate proteins."J. Mol. Biol.. (in press). (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Y. Miyamoto, E. Muto, T. Mashimo, A. H. Iwane, I. Yoshiya and T. Yanagida.: "Direct Inhibition of Microtubule-based Kinesin Motility by Local Anesthetics."Biophys. J.. 78 (in press.). (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Wazawa, Y. Ishii, T. Funatsu and T. Yanagida.: "Spectral fluctuation of a single fluorophore conjugated to a protein molecule."Biophys. J.. (in press.). (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Y. Ishii et al: "Imaging and nano-manipulation of single actomyosin motors at work"Proceedings of the Australian Physiological and Pharmacological Society. 30(1). 24-39 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Y. Ishil et al: "Fluorescence resonance energy transfer between single fluorophores attached to a coiled-coil protein in aqueous solution"Chemical Physics. 247. 163-173 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] T. Ide et al: "An artificial lipid bilayer formed on an agarose-coated glass for simultaneous electrical and optical measurement of single ion-channels"Biochem. Biophys. Res. Commun.. 265. 595-599 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Y. Ishii et al: "Imaging and Nano-manipulation of Single Actomyosin Motors at Work"Clinical and Experimental Pharmacology and Physiology. (in press).

    • Related Report
      1999 Annual Research Report
  • [Publications] Y. Miyamoto et al: "Direct Inhibition of Microtubule-based Kinesin Motility by Local Anesthetics"Biophys. J.. (in press).

    • Related Report
      1999 Annual Research Report
  • [Publications] Y. Ishii et al: "Molecular Interactions of Actin : Myosin interaction, motility assays and Ca- Regulatory proteins"D. D. Thomas and C. G. dos Remedios eds. Springer Verlag(Heidelberg) (in press).

    • Related Report
      1999 Annual Research Report
  • [Publications] A,Ishijima et al: "Simultaneous observation of individual ATPase and mechanical events by a single myosin molecule during interation with actin." Cell. 92. 161-171 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] C.Shingyoji et al: "Dynein arms are oscillating force generators." Nature. 393. 711-714 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] H.Yokota et al: "Single molecule imaging of fluorescently-labeled proteins on metal by surface plasmons in aqueous solution." Phys.Rev.Letter.80(20). 4606-4609 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] H.Tanaka et al: "Orientation dependence of displacements by a fingle one-headed myosin relative to the actin filament." Biophys.J.75. 1886-1894 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] X. Liu et al: "Filament structure as an essential factor for regulation of Dictyostelium myosin by regulatory light chain phosphorylation." Proc.Natl.Acad.Sci.USA.95. 14124-14129 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] K.Kitamura et al: "A single myosin head moves along an actin filament with regular steps of〜5.3nm." Nature. 397. 129-134 (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] A.H.Iwane: "Myosin Subfragment-1 is Fully Equipped with Factors Essential for Motor Function" Biochemical and Biophysical Research Communication. 230. 76-80 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] M.Tokunaga: "Subpiconewton Intermolecular Force Microscopy" Biochem.and Biophys.Res.Commun.231. 556-569 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] M.Tokunaga: "Single Molecule Imaging of Fluorophores and Enzymatic Reactions Achieved by Objective-Type Total Internal Reflection Fluorescence Microscopy" Biochem.and Biophys.Res.Commun.235. 47-53 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] H.Kojima: "Mechanics of Single Kinesin Molecules Measured by Optical Trapping Nanometry" Biophys.J.73. 2012-2022 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Y.Harada: "Single Molecule Imaging and Nano-manipulation of Biomolecules" Method in Cell Biology. 55. 117-128 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] T.Aoki: "Non-contact acanning probe microscopy with oub-pioonewton force sensitivity" Ultramicroscopy. 70. 45-55 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 柳田敏雄: "生物物理から見た生命像3" 吉岡書店, 164 (1997)

    • Related Report
      1997 Annual Research Report

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Published: 1997-04-01   Modified: 2016-04-21  

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