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Molecular mechanism of protein motors revealed by the combination of genetic engineering and single-molecule analysis

Research Project

Project/Area Number 06404081
Research Category

Grant-in-Aid for Scientific Research (A)

Allocation TypeSingle-year Grants
Section一般
Research Field Biophysics
Research InstitutionThe University of Tokyo

Principal Investigator

SUTOH Kazuo  The University Graduate of Tokyo Graduate School of Arts and Sciences, 大学院・総合文化研究科, 教授 (20111453)

Project Period (FY) 1994 – 1996
Project Status Completed (Fiscal Year 1996)
Budget Amount *help
¥29,400,000 (Direct Cost: ¥29,400,000)
Fiscal Year 1996: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1995: ¥9,700,000 (Direct Cost: ¥9,700,000)
Fiscal Year 1994: ¥16,700,000 (Direct Cost: ¥16,700,000)
Keywordsgenetic engineering / single-molecule analysis / myosin / actin / motor protein / Dictyostelium disoideum / 遺伝子工学 / 分子モーター / X線結晶解析 / 蛋白質モーター / 組み換えミオシン / 結晶構造 / 蛋白質機械 / 分子機械
Research Abstract

We have constructed a myosin motor domain consisted of the heavy chain alone(760 residues), which retained motor functions. By using this construct, we succeeded to crystallize the myosin motor domain, and solve its structure at the atomic resolution (2.7A). By using this structural information, we carried out carried out site-directed mutagenesis of functionally important regions of myosin, especially the switch I and switch II loops. A loop comprising residues 454-459 of Dictyostelium myosin II is structurally and functionally equivalent to the switch II loop of the G-protein family. The consensus sequence of the switch II loop of the myosin family is DIXGFE.In order to determine the functions of each of the conserved residues, alanine scanning mutagenesis was carried out on the dictyostelium myosin II heavy chain gene. Examination of in vivo and in vitro motor functions of the mutant myosins revealed that the I455A and S456A mutants retained those functions while the D454A,G457A,F458A and E459A mutants lost them. Biochemical analysis of the latter myosins showed that the G457A and E459A mutants lost the basal ATPase activity by blocking of the isomerization and hydrolysis speps of the ATPase cycle, respectively. The F458A mutant, however, lost the actin-activated ATPase activity without loss of the basal ATPase activity. These results are discussed in terms of the crystal structure of the Dictyostelium myosin motor domain.

Report

(4 results)
  • 1996 Annual Research Report   Final Research Report Summary
  • 1995 Annual Research Report
  • 1994 Annual Research Report
  • Research Products

    (24 results)

All Other

All Publications (24 results)

  • [Publications] Saeki,K., Sutoh,K.,& Wakabayashi,T.: "Tropomyosin-binding sites on the Dictyostelium actin surface as identified by site-directed mutagenesis." Biochemistry. 35. 14465-14472 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Aizawa,H., Sutoh,K.,& Yahara,I.: "Overexpression of cofilin stimulates bundling of actin filaments,membrane ruffling and cell movement in Dictyostelium discoideum cells." J.Cell Biol. 132. 335-344 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Fisher,AJ., Smith,C.A, Thoden,J., Smith,R., Sutoh,K.,et al.: "Structures of Beryllium and Aluminium metallofluoride-Mg-ADP complexes of the Dictyostelium discoideum myosin motor domain." Biochemistry. 34. 8960-8972 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Fisher,A.J., Smith,C.A., Sutoh,K., Holden,H.M,& Rayment,I.: "The preliminary structure of the ATP-bound state of myosin." Biophys.J. 68. 19S-28S (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Aizawa,H,Sutoh,K, Tsubaki,S., Kawashima,S., Ishii,A & Yahara,I.: "Identification,characterization,and intracellular distribution of cofilin in Dictyostelium discoideum." J.Biol.Chem.270. 10923-10932 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Morio,T., Adachi,H., Sutoh,K., Yanagisawa,K.,& Tanaka,Y.: "Bsr-REMI : An improved Method for gene tagging using a new vector in Dictyostelium." J.Plant Res.108. 111-114 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Suzuki, Y., Yasunaga, T., Ohkura, R., T.Wakabayashi, and Sutoh, K.: "Swing of leve-arm of myosin motor at the isomerization and Pi-release steps." submitted. (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Sasaki, N.and Sutoh, K.: "Structure-Mutation Analysis of the ATPase Site of Dictyoselium discoideum Myosin II" Adv.Biophys.(in press). (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Sasaki, N., Shimada, T., and Sutoh, K.: "Mutational analysis of teh switch II loop of Dictyostelium myosin II." J.Biol.Chem.(in press). (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Sutoh, K.: "The use of recombinant molecules." Current Methods in Muscle Physiology (ed.Sugi, H.). 163-171 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Nagasaki, A., Sutoh, K., Adachi, H., and Sutoh, K.: "A novel Dictyostelium discoideum gene required for cAMP-dependent cell aggregation." Biochem.Biophys.Res.Commun.244. 505-513 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Adachi, H., Takahashi, Y., Hasebe, T., Shirouzu, M., Yokoyama, S., Sutoh, K.: "Dictyostelium IQ-GAP-related protein specifically involved in the completion of cytokinesis." J.Cell Biol.137. 891-898 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Shimada, T., Sasaki, N.Ohukura, R., & Sutoh, K.: "Alanine scanning mutagenesis of switch I region of the ATPase site of Dictyostelium discoideum myosin II." Biochemistry. 36. 14037-14043 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Bobkov, A.A., Sutoh, K., & Reisler, E.: "Nucleotide and actin binding properties of the isolated motor domain from Dictyostelium discoideum myosin" J.Muscel Res.Cell Motil.18. 563-571 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Fujita, H., Sugiura, S., Momomura, S., Omata, M., Sugi, H., & Sutoh, K.: "Characterization of mutant myosins equivalent to human familial hypertophic cardiomyopathy mutants." J.Clinc.Invest.99. 1010-1015 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Suzuki, Y., Ohkura, R., Sugiura, S., Yasuda, R., Kinoshita, K., Tanokura, M., & Sutoh, K.: "Modulation of actin filament sliding by mutations of the SH2 cysteine in Dictyostelium myosin II." Biochem.Biophys.Res.Commun.234. 701-706 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Sugi, H., Akimoto, T., Sutoh, K., Chaen, S., Oishi, N., and Suzuki, S.: "Dynamic electron microscopy of ATP-induced myosin head movement in living muscle thick filaments." Proc.Natl.Acad.Sci.USA94. 4378-4382 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Saeki, K., Sutoh, K., & Wakabayashi, T.: "Tropomyosin-binding sites on the Dictyostelium actin surface as identified 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
      1996 Final Research Report Summary
  • [Publications] Aizawa, H., Sutoh, K., & Yahara, I.: "Overexpression of cofilin stimulates bundling of actin filaments, membrane ruffling and cell movement in Dictyostelium discoideum cells." J.Cell Biol.132. 335-344 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Fisher, A.J., Smith, C.A., Thoden, J., Smith, R., Sutoh, K., Holden, H.M., & Rayment, I.: "Structures of Beryllium and Aluminium metallofluoride-Mg-ADP complexes of the Dictyostelium discoideum myosin motor domain." Biochemistry. 34. 8960-8972 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Fisher, A.J., Smith, C.A., Sutoh, K., Holden, H.M., & Rayment, I.: "The preliminary structure of The ATP-bound state of myosin." Biophys.J.68. 19s-28s (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Aizawa, H., Sutoh, K., Tsubaki, S., Kawashima, S., Ishii, A., & Yahara, I.: "Identification, Characterization, and intracellular distribution of cofilin in Dictyostelium discoideum." J.Biol.Chem.270. 10923-10932 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Morio, T.Adachi, H., Sutoh, K., Yanagisawa, K., & Tanaka, Y.: "Bsr-REMI An improved Method for gene tagging using a new vector in Dictyosteliunm." J.Plant Res.108. 111-114 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] A.J.Fisher: "Xray structaro of mgyos in Motor Domain of Dictyosteliun a:scoiueum" Biochemistry. 34. 8960-8972 (1995)

    • Related Report
      1995 Annual Research Report

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Published: 1994-04-01   Modified: 2016-04-21  

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