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1994 Fiscal Year Final Research Report Summary

Mechanism of energy transduction in motor proteins - Single molecule mechanics and the ATPase reaction -

Research Project

Project/Area Number 04404094
Research Category

Grant-in-Aid for General Scientific Research (A)

Allocation TypeSingle-year Grants
Research Field 分子遺伝学・分子生理学
Research InstitutionOsaka University

Principal Investigator

YANAGIDA Toshio  Osaka Univ. Facuity of Engineering Science Professor, 基礎工学部, 教授 (30089883)

Co-Investigator(Kenkyū-buntansha) HARADA Yoshie  Exploratory Research for Advanced Technology (Yanagida Biomotron Project) Resear, 研究員 (10202269)
Project Period (FY) 1992 – 1994
Keywordsmolecular motor / actin filament / myosin filament / actomyosin
Research Abstract

We have refined total internal reflection fluorescence microscopy (TIRFM) to visualize single fluorescent dye molecules in aqueous solution at a full video rate (Funatsu, et al., Nature, 374,555 '95). We extened this method to measurements of individual ATP turnovers. Individual ATP turnover events by single kinesin molecules were detected by directly observing association-(hydrolysis)-dissociation of fluorescent ATP analogue, in which Cy3 was attached to ribose. Elementary mechanical events of single kinesin molecules were measured by optical trapping nanometry. 8-nm steps were clearly observed as reported previously (Svoboda, et al. Nature, 365, 721 '92). For simultaneous measurements of single motor mechanics and the ATP turnover, we combined the optics for single molecule imaging with optical trapping nanometry for single motor mechanics. First, we detected individual ATP turnovers by single kinesin molecules attached to a bead, of which position was controlled by an optical trap. When the kinesin molecule was in solution, apart from a microtubule on a glass surface, the ATP turnover rate was very small, -0.2s^<-1>. While, when it was brought into contact with a microtubule, the rate was greatly enhanced to be -10s^<-1>. Thus, it is now possible to measure the ATP turnover rates of single motors under controlled loads. We have tried to measure 8-nm displacement steps and ATP turnovers by single kinesin molecules simultaneously. ATP turnover events appeared to mostly correlated to 8-nm steps but sometimes do not. Because of low affinity of Cy3 ATP for kinesin, however, 8-nm elementary steps were not sufficiently clear. In order to gain the coupling between elementary mechanical events and ATP turnovers, improvement of the apparatus is necessary. It will not take long time. Since Cy3 ATP functions as normal ATP for actomyosin motors, the present method may be more hopeful for actomyosin motors. This project is also in progress.

  • Research Products

    (15 results)

All Other

All Publications (15 results)

  • [Publications] M.Jorara: "Charge reversion mutagenesis of Dictyostelium actin to map the surface recognized by myosin during ATP-driven sliding motion." Proc.Natl.Acad.Sci.90. 2127-2131 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Yanagida: "Coupling between ATPase and force-generating attachment-detachment cycles of actomyosin in vitro." Adv.Exp.Med.Biol.332. 339-349 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] E.Prochniewicz: "Cooperativity in F-actin : Chemical modifications of actin monomers affect the functional interactions of myosin with unmodified monomers in the same actin filament." Biophysical J.65. 113-123 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Yanagida: "Nano-manipulation of actomyosin molecular motors in vitro : a new working principle." Trends in Biochemical Sciences. 18. 319-324 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K.Saitoh.: "Movement of Single Myosin Filaments and Myosin Step Size on an Actin Filament Suspended in Solution by a Laser Trap" Biophys.J.66. 769-777 (1994)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Funatsu: "Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution." Nature. 866. 3364-3365 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] M.Johara, Y.Y.Toyoshima, A.Ishijima, H.Kojima, T.Yanagida and K.Sutoh: "Charge-reversion mutagenesis of dictyostelium actin to map the surface recognized by myosin during ATP-driven sliding motion." Proc. Natl. Acad. Sci. USA. 90. 2127-2131 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T.Yanagida, Y.Harada and A.Ishijima: "Nano-manipulation of actomyosin molecular motors in vitro : a new working principle." Trends in Biochemical Sciences.18. 319-324 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] E.Prochniewicz, E.Katayama, T.Yanagida and D.D.Thomas: "Cooperativity in F- actin : Chemical modifications of actin monomers affect the functional interactions of myosin with unmodified monomers in the same actin filament." Biophysical J.65. 113-123 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T.Yanagida, A.Ishijima, K.Saitoh and Y.Harada: "Coupling between ATPase and force-generation attachment-detachment cycles of actomyosin in vitro." "Mechanism of Myofilament Sliding in Muscle Contraction" (H.Sugi eds Plenum Publishing Co. (New York) Adv. Exp. Med. Biol.332. 339-349 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] S.Itakura, H.Yamakawa, Y.Toyoshima, A.Ishijima, T.Kojima, Y.Harada, T.Yanagida, T.Wakabayashi & K.Sutoh: "Force-generating domain of myosin motor." Biochem. Biophys. Res. Commun.196. 1504-1510 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] A.Ishijima, Y.Harada, H.Kojima, T.Funatsu, H.Higuchi & T.Yanagida: "Single-molecule analysis of the actomyosin motor using nano-manipulation." Biochem. Biophys. Res. Commun.199. 1057-1063 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K.Saitoh, T.Aoki, T.Aoki & T.Yanagida: "Movement of single myosin filaments and myosin step size on actin filament suspended in solution by a laser trap." Biophys. J.66. 769-777 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] H.Kojima, A.Ishijima & T.Yanagida: "Direct measurement of stiffness of single actin filaments with and without tropomyosin by in vitro nanomanipulation." Proc. Natl. Acad. Sci. USA. 91. 12962-12966 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T.Funatsu, Y.Harada, M.Tokunaga, K.Saito & T.Yanagida: "Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution." Nature. 374. 555-559 (1995)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1999-03-09  

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