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Studies on muscle contraction Mechanism based on molecular structure of mutant motor proteins

Research Project

Project/Area Number 02102009
Research Category

Grant-in-Aid for Specially Promoted Research

Allocation TypeSingle-year Grants
Research InstitutionThe University of Tokyo

Principal Investigator

WAKABAYASHI Takeyuki  Univ.of Tokyo, School of Science, Professor, 大学院・理学系研究科, 教授 (90011717)

Co-Investigator(Kenkyū-buntansha) SUTOH Kazuo  Univ.of Tokyo, College of Arts and Science, Associate Professor, 教養学部, 助教授 (20111453)
Project Period (FY) 1990 – 1993
Project Status Completed (Fiscal Year 1993)
Budget Amount *help
¥201,900,000 (Direct Cost: ¥201,900,000)
Fiscal Year 1993: ¥13,000,000 (Direct Cost: ¥13,000,000)
Fiscal Year 1992: ¥20,900,000 (Direct Cost: ¥20,900,000)
Fiscal Year 1991: ¥58,000,000 (Direct Cost: ¥58,000,000)
Fiscal Year 1990: ¥110,000,000 (Direct Cost: ¥110,000,000)
KeywordsMuscle / Myosin / Actin / Tubulin / Kinesin / Electron microscopy / X-ray scattering / Site-directed mutagenesis / 収縮 / 三次元像再構成 / 遺伝子改変 / 滑り運動 / 筋収縮機構 / 筋収縮蛋白 / 生物物理学 / 三次元像再開成 / 超微細構造 / 蛋白質工学 / クライオ電子顕微鏡 / 重原子標識 / 筋肉収縮 / 三次元像再構成法 / 部位特異的標識
Research Abstract

Our goal is to understand the molecular mechanism of motor proteins on the basis of atomic structure of proteins.
First we developed new techniques for electron microscopy. Using one of new techniques we developed, that is, the site-directed heavy atom labelling electron microscopy (SHALEM), we decided the position of the 5^<th> amino acid of myosin only by electron microscopy. Also, we reconstructed three-dimensional structure of muscle during isometric contraction.
Second, we detected the structural change of myosin during ATP hyrdrolysis by electron microscopy, small angle X-ray scattering, and crystallography using X-ray diffraction. This is the first observation of detection of myosin structural change during ATP hydrolysis.
Third, we observed the structural change of thin filaments in the addition of Ca ions by cryo-electron microscopy.
Last, we found the myosin-actin interaction sites on the subdomain-1 of actin using protein engineering techniques. Also we found the motor domain of myosin. This part is essential for sliding movement of actin and production of force. Furthermore, we use protein engineering to produce the mutant actins, which activate myosin ATPase in a highly cooperative manner. We found that the replacement of only 3 amino acid residues cause the high cooperativity in Ca^<2+>-induced activation of ATPase. This region is also one of the binding sites of tropomyosin in the presence of Ca.

Report

(5 results)
  • 1993 Annual Research Report   Final Research Report Summary
  • 1992 Annual Research Report
  • 1991 Annual Research Report
  • 1990 Annual Research Report
  • Research Products

    (53 results)

All Other

All Publications (53 results)

  • [Publications] 若林他: "X線小角散乱によるMgATP" Science. 258. 443-447 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] 須藤他: "Site-directed mutations of Dictyostelium a ctin : Disruption of a nagatire charge dustev at the A" Proc.Natl.Acad.Sci.U.S.A.88. 7711-7714 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Strynadka他: "Mdecular structure of the acyl-enzyme intermidiate in beta lactom hydrolysis at 1.7A nosolution" Nature. 359. 700-705 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] 吉川他: "Three-dimcnsional structure of tubulin-kinesin complex." Nature. 376. 274-277 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] 孟他: "Determination of the protein components of natiue thin fitaments isolated from natural actonyosin" J.Biochem.118. 422-427 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] 佐伯他: "Tropomyosiu binding sitels)on the Dictyosterium Actins surface as identify by site-dirocted mutagenesis" Biodiemistry. 35. 14465-14472 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Max, N., Hayashi, N., Wakabayashi, T.: "Computer simulation and animation of muscle crossbrige motion" J.Visual.And Comp.Annim.1. 9-14 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Fujiyama, Y., Yokoyama, K.Yoshida, M., Wakabayashi, T.: "Electron microscopy of the reconstituted complexes of the F1-ATPase with various subnit constitution revealed the location of the gamma-subunit in the central cavity of the molecule" FEBS. 271. 111-115 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Konishi, J., Denda, K., Oshima, T., Wakagi, Uchida, E., Ohsumi, Y., Anraku, Y., Matsumoto, T.Wakabayashi, T., Mukohata, Y., Ihara, K., Inatomi, K., Kato, K., Ohta, T., Allison, W.S., Yoshida, M.: "Archaebacterial ATPases : Relationship to Other Ion-translocating ATPase Families examined in terms of immunological cross-reactivity" J.Biochem.108. 554-559 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Tokunaga, M., Sutoh, K., Wakabayashi, T.: "Structure and Structural Change of the Myosin Head." Advances in Biophysics. 27. 157-167 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Sutoh, K., Ando, M., Sutoh, K., & Toyoshima, Y.Y.: "Site-directed mutations of Dictyostelium actin : Disruption of a negative charge cluster at the N-terminus." Proc.Natl.Acad.Sci.USA.88. 7711-7714 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Tomioka, A.Ribi, H.O., Tokunaga, M., Furuno, T., Sasabe, H., Miyano K., and Wakabayashi, T.: "Three-dimensional Structure of Polymeric Actin" Advances in Biophysics. 27. 169-183 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Hirose, K., and Wakabayashi, T.: "Conformations of Crossbridges in Contracting Skeletal Muscle" Advances in Biophysics. 27. 197-203 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Wakabayashi, K., Tokunaga, M., Kohno, L,Sugimoto, Y., Hamanaka, T., Takazawa, Y., Wakabayashi, T., Amemiya, Y.: "Shape Change of the Myosin Head induced by MgATP,sudied by small-angle synchrotron X-ray scattering in solution" J.Muscle Res.Cell Motil., absract.13. 481 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Wakabayashi, K., Tokunaga, M., Kohno, I., Sugimoto, Y., Hamanaka, T., Takazawa, Y., Wakabayashi, T., Amemiya, Y.: "Small-angle synchrotron X-ray scattering reveals distinct shape changes of the myosin head during hydrolysis of ATP" Science. 258. 443-447 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Strynadka, N.C.J., Adachi, H., Jensen, S.E., Johns, K., Seilecki, Betzel, C., Sutoh, K., & James.M.N.G.: "Molecular structure of the acyl-enzyme intermidiate in beta-lactom hydrolysis at 1.7 A resolution" Nature. 359,6397. 700-705 (1992)

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

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Sutoh., K.: "A transformation vector for Dictyostelium discoideum with a new selectable marker bsr" Plasmid. 30. 150-154 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Hirose, K.and Wakabayashi T.: "Structural Change of crossbridges of rabbit skeleta muscle during isometric contraction." J.Muscle Res.Cell Motil.14. 432-445 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Yamada, A.and Wakabayashi, T.: "Movement of actin away from center of reconstrituted rabbit skeletal muscle myosin filament is slower than that in the opposite direction" Biophys.J.64. 565-569 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Itakura, S.Yamakawa, H., Toyoshima, Y.Y., Ishijima, A., Kojima, T., Harada, Y., Yanagida, T., Wakabayashi, T., and Sutoh, K.: "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
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Ishikawa, T.& Wakabayashi T.: "Calcium induced change in three-dimensional structure of thin filaments of rabbit skeletal muscle as revealed by cryo-electron microscopy" Biochem.Biophys.Res.Commun.203. 951-958 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kikkawa, M., Ishikawa, T., Miyazawa, A., Fujiyoshi, Y., Wakabayashi, T.& Hirokawa, N.: "Direct visualization of the microtubule lattics seam both in vitro and in vivo" J Cell Biol.127. 1965-1971 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Fisher, A., Smith, C.A., Thoden, J., Smith, R., Sutoh, K., Holden, H.M., & Rayment, I.: "Structural studies of myosin : nucleotide complexes : a revised model for the molecular basis of muscle contraction" Biophys.J.68. 19s-28s (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Fisher, A.J., Smith, C.A., Thoden, J.B., Smith, R., Sutoh, K., Holden, H.M., & Rayment, I.: "X-ray structures of myosin motor domain of Dictyostelium discoideum complexed with MgADPBeF-and MgADPAIF" Biochemistry. 34. 8960-8972 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Ishikawa, T., Wakabayashi, T.: "Proposal of Alignment-Independent Classification of Electron Microscopic Images with Helical Symmetry and its Applicaiton to Reconstrituted Thin Filaments of Skeleral Muscle" Ultramicroscopy. 57. 91-101 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Kikkawa, M., Ishikawa, T., Miyazawa, A., Fujiyoshi, Y., Wakabayashi, T.& Hirokawa, N.: "Three-dimensional structure of tubulin-kinesin complex" Nature. 376. 274-277 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Meng, Y., Yasuanga, T., & Wakabayashi, T.: "Determination of the protein components of native thin filaments isolated from natural actomyosin : nebulin and alpha-actinin are associated with actin filaments" J.Biochem. 118. 422-427 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Saeki, K., Sutoh, K.& Wakabayashi, T.: "Tropomyosin-binding site(s) on the Dictyosterium Actin surface as identify by site-directed mutagenesis" Biochemistry. 35. 14465-14472 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Yasunaga, T., Saeki, K., & Wakabayashi, T.: "Facilitationg effect of ADP and phosphate on the sliding movement of actin filaments on the myosin-coated surface" J.Mus.Res.Cell Motil.Abstract. 17. 282 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Yasunga, T., & Wakabayashi, T.: "Extensible and object-oriented system Eos suppllies a new environment for image analysis of electron micrographs of macromolecules" J.Struct.Biol.116. 155-160 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] A.Yamada & T.Wakabayashi: "Movement of actin away from the center of reconstituted rabbit myosin filament is slower than in the opposite direction" Biophysical J.64. 565-569 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] M.Johara et al.: "Charge-reversion mutagenesis of Dictyostelium actin to map the surface recognized by myosin during ATP-driven sliding motion" Proc.Natl.Acad.Sci.U.S.A.90. 2127-2131 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] K.Sutoh: "A Transformation Vector for Dictyostelium discoideum with a New Selectable Marker bsr" Plasmid. 30. 150-154 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] K.Sutoh: "Identification of actin surface interacting with myosin during the actin-myosin sliding" Adv.Exp.Med.Biol.332. 241-245 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] K.Hirose & T.Wakabayashi: "Structural change of crossbridges of rabbit skeletal muscle during isometric contraction" J.Muscle Res.Cell Motil.14. 432-445 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] S.Itakura et al.: "Force-Generating Domain of Myosin Motor" Biochem.Biophys.Res.Commun.196. 1504-1510 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] K.Wakabayashi et al.: "Small-angle Synchrotron X-ray Scattering Reveals Distinct Shape Change of the Myosin Head During Hydrolysis of ATP" Science. 258. 443-447 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] A.Yamada & Y.Wakabayashi: "Movement of actin away from the center of reconstituted rabbit myosin filament is slower than in the opposite direction" Biophysical J.(1993)

    • Related Report
      1992 Annual Research Report
  • [Publications] K.Wakabayashi et al.: "Shape Change of Myosin Head induced by MgATP,studied by small-angle Synchrotron X-ray scattering in solution" J.Mucsle Res.Cell Motil.13. 481-481 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] N.C.J.Strynadka et al.: "Molecular structure of the acyl-enzyme intermediate in β-lactam hydrolysis at 1.7 A resolution" Nature. 359. 700-705 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] M.Hirono et al.: "Expression of Tetrahymena actin gene and chimeric actin genes in Dictyosterium cells" J.Muscle Res.Cell Motil.13. 483-484 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] K.Sutoh: "Site-directed mutagenesis of Dictyosterium actin" J.Muscle Res.Cell Motil.13. 484-484 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] M.Johara et al.: "Charge-reversion mutagenesis of Dictyostelium actin to map the surface recognized by myosin during ATP-driven sliding motion" Proc.Natl.Acad.Sci.U.S.A.(in press).

    • Related Report
      1992 Annual Research Report
  • [Publications] M.Hirono et al.: "A chimeric actin carrying N-terninal portion of Tetrahymena actin does not find to DNaseI" Biochem.Biophys.Res.Commun.184. 1511-1516 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Tokunaga,M.: "Structure and Structural Change of the Myosin Head" Advances in Biophysics. 27. 157-167 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Tomioka,A.: "Three-dimensional Structure of Polymeric Actin" Advances in Biophysics. 27. 160-183 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Hirose,K.: "Conformations of Crossbridges in Contracting Skeletal Muscle" Advances in Biophysics. 27. 197-203 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Sutoh,K.: "Site-directed mutations of Dictyostelium actin:Disruption of a negative charge chuster at the N-terminus" Proc.Natl.Acad.Sci.USA. 88. 7711-7714 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] J.Konishi et al.: "Archaebacterial ATPases:Relationship to IonーTranslocating ATPase Families Examined in Terms of Immunological CrossーReactivity." J.Biochem.108. 554-559 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] M.Tokunaga,K.Sutoh & T.Wakabayashi: "Structure and Structural Change of the Myosin Head." Advances in Biophysics. (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] K.Hirose & T.Wakabayashi: "Conformations of Crossbridges in Contracting Skeletal Muscle." Advances in Biophysics. (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] T.Yasunaga,K.Saeki & T.Wakabayashi: "How subnanomolar ATP,far less than myosin molecules,can drive Fーactin smoothly?" Nature.

    • Related Report
      1990 Annual Research Report

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Published: 1990-04-01   Modified: 2016-04-21  

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