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Studies on mechanism of muscle contraction by cryoelectron microscopy with site-specific molecular and heavy metal labelings

Research Project

Project/Area Number 61065001
Research Category

Grant-in-Aid for Specially Promoted Research

Allocation TypeSingle-year Grants
Research InstitutionUniversity of Tokyo

Principal Investigator

WAKABAYASHI Takeyuki  University of Tokyo, Physics Dept., Professor, 理学部, 教授 (90011717)

Co-Investigator(Kenkyū-buntansha) SUTOH Kazuo  University of Tokyo, College of Arts & Sciences, Associate Professor, 教養学部, 助教授 (20111453)
Project Period (FY) 1986 – 1989
Project Status Completed (Fiscal Year 1989)
Budget Amount *help
¥185,000,000 (Direct Cost: ¥185,000,000)
Fiscal Year 1989: ¥7,000,000 (Direct Cost: ¥7,000,000)
Fiscal Year 1988: ¥25,000,000 (Direct Cost: ¥25,000,000)
Fiscal Year 1987: ¥86,000,000 (Direct Cost: ¥86,000,000)
Fiscal Year 1986: ¥67,000,000 (Direct Cost: ¥67,000,000)
KeywordsCryoelectron Microscopy / Actin / Myosin / Undecagold Cluster / Site-Specific Lebel / Avidin-Biotin / Site-Directed Antibodies / Three-Dimensional Reconstitution / 電子顕微鏡法 / 筋肉収縮 / 抗体 / ビオチン / クライオ電子顕微鏡 / 重原子標識 / 分子標識法 / 重原子標識法 / 三次元像再構成法 / 急速凍結 / 収縮の分子的メカニズム / ケイジド化合物 / 生体機能の分子的機序 / 筋収縮 / 三次元像再構成 / 分子標識 / 三次元再構成 / 立体構造
Research Abstract

1)WE employed an avidin-biotin system as an electron microscopic probe to visualize specific sites on the myosin head. We determined three-dimensional locations of several functionally important sites on the head by the use of image reconstitution technique. One of these site is the ATP ase site of myosin. The other is the SH1 thiol, which is the most reactive cys residue on the head and supposed to be involved in ATPase and actin-binding in some way. The former is located at the middle part of the head and is on the opposite side to the actin-binding site. On the other hand, the SH1 thiol is on the same side to the actin-binding site and also at the middle of the head. The head bends at the middle. The ATPase site and the SH1 thiol are located at this bend-region.
2)We synthesized monomaleimide derivative of undecagoid-cluster. The diameter of the gold cluster is only 0.8nm. The gold cluster can be covalently attached to thiol groups through the maleimide group. We used to this heavy metal label to visualize cys 374 of actin molecule. We label actin with this gold cluster. By peptide mappings, we showed that 80% of cys 374 of actin are selectively labeled. We obtained electron microscopic image of gold-labeled actin filament by the use of cryoelectron microscopy. Three-dimensional location of the gold cluster was determined by the image reconstitution technique. Thus we determined three-dimensional location of a specific residue of actin molecule by cryoelectron microscopy.
3)WE constructed an apparatus for time-resolved cryoelectron microscopy. It is equipped with YAG laser for flash photolysis, which is driven by microcomputer. By using caged material (caged ATP etc), we can carry out time-resolved electron microscopy.

Report

(5 results)
  • 1989 Annual Research Report   Final Research Report Summary
  • 1988 Annual Research Report
  • 1987 Annual Research Report
  • 1986 Annual Research Report
  • Research Products

    (69 results)

All Other

All Publications (69 results)

  • [Publications] Hitaka,K.: "Effect of phosphoryation by protein kinase C on the conformation of chicken gizzaed myosin" J.Biol.Chem.567. 1234-1238 (1986)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sutoh,K.: "Electron microscopic visualization of the ATPase site of myosin by the use of a biotinylated photo-affioity ADP analog." Proc.Natl.Acad.Sci.USA. 83. 212-216 (1986)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Maruyama,K.: "Connectin causes aggregation of myosin rods but not myosin heads." Biomedical Reseaech. 6. 423-427 (1986)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sutoh,K.: "Actin-fragmin interaction as revealed by chemical crosslinking." Biochemistry. 25. 435-440 (1986)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sutoh,K.: "Improved method for mapping the binding site of an actin-binding protien." Biochemistry. 25. 6168-6192 (1986)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Tokunaga,M.: "Position of the amino-acid treminus of myosin light chain-1 and light chain-2 determined by electron microscopy using monoclnal antibody." J.Mol.Biol.194. 245-255 (1987)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sutoh,K.: "Electron microscopic visullization of the N-terminus of the myosin heavy chain by the use of a site-directed antibody." J.Mol.Biol.195. 953-956 (1987)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Tokunaga,M.: "Location of the ATase site of myosin determined by threedimensional electron microscopy." Nature. 329. 635-638 (1987)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sutoh,K.: "Identification of two segments of myosin subfragmentl that can be crosslinked to the SH1 thiol." Biochemistry. 26. 4511-4516 (1987)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sutoh,K.: "A short hydrophobic segment next to Trp130 is close to the ribose ring of ADP bound to the ATPase site." Biochemistry. 26. 7648-7654 (1987)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Suzuki,M.: "Monoclonal antibodies which recognize trimethylated N-terminus± of histone H2B." Jpn.Acad.63. 385-388 (1987)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Hirose,K.: "Thin filaments of rabbit skeletal muscle are in helical register." J.Mol.Biol.204. 797-801 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sutoh,K.: "Spatial proximity of the glycine-rich loop and the SH2 thiol in myosin subfragment 1." Biochemistry. 27. 2964-2969 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Suzuki,M.: "Packing of DNA in cricket sperm." J.Mol.Biol.204. 653-661 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sutoh,K.: "End-label fingerprintings show that an N-terinal segment of depactin participate in interaction with actin." Biochemistry. 28. 102-106 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sutoh,K.: "Electron microscopic mappings of myosin head with site-directed antibodies." J.Mol.Biol.206. 357-363 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sutoh,K.: "End-label fingerprinting show that the N- and C-termini of actin are in the contact site with gelsolin." Biochemistry. 28. 5269-5275 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Saeki,K.: "New method to prepare single-headed heavy meromyosin with high purity and a high yield." J.Biochemistry. 106. 606-611 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Hitaka, K.: "Effect of phosphorylation by protein kinase C on the conformation of chicken gizzard myosin" J.Biol. Chem.567. 1234-1238 (1986)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sutoh, K.: "Electron microscopic visualization of the ATPase site of myosin by the use of a biotinylated photo-affinity ADP analog." Proc.Natl.Acad.Sci.USA. 83. 212-216 (1986)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Maruyama, K.: "Connection causes aggregation of myosin rods but not myosin heads." Biomedical Research. 6. 423-427 (1986)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sutoh, K.: "Actin-fragmin interaction as revealed by chemical crosslinking." Biochemistry. 25. 435-440 (1986)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sutoh, K.: "Improved method for mapping the binding site of an actin-binding protein." Biochemistry. 25. 6168-6192 (1986)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Tokunaga, M.: "Position of the amino-acid terminus of myosin light chain-1 and light chain-2 determined by electron microscopy using monoclonal antibody." J.Mol.Biol.194. 245-255 (1987)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sutoh, K.: "Electron microscopic visualization of the N-terminus of the myosin heavy chain by the use of a site-directed antibody." J.Mol.Biol.195. 953-956 (1987)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Tokunaga, M.: "Location of the ATPase site of myosin determined by three-dimensional electron microscopy." Nature. 329. 635-638 (1987)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sutoh, K.: "Identification of two segments of myosin subfragmentl that can be crosslinked to the SH1 thiol." Biochemistry. 26. 4511-4516 (1987)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sutoh, K.: "A short hydrophobic segment next to Trp130 is close to the ribose ring of ADP bound to the ATPase site." Biochemistry. 26. 7648-7654 (1987)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Suzuki, M.: "Monoclonal antibodies which recognize trimethylated N-terminus<plus-minus> of histone H2 " Jpn.Acad.63. 385-388 (1987)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Hirose, K.: "Thin filaments of rabbit skeletal muscle are in helical register." J.Mol.Biol.204. 797-801 (1988)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sutoh, K.: "Spatial proximity of the glycine-rich loop and the SH2 thiol in myosin subfragment 1." Biochemistry. 27. 2964-2969 (1988)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Suzuki, M.: "Packing of DNA in cricket sperm." J.Mol.Biol.204. 653-661 (1988)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sutoh, K.: "End-label fingerprintings show that an N-terinal segment of depactir participate in interaction with actin." Biochemistry. 28. 102-106 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sutoh, K.: "Electron microscopic mappings of myosin head with site-directed antibodies" J.Mol.Biol.206. 357-363 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sutoh, K.: "End-label fingerprintings show that the N- and C-termini of actin are in the contact site with gelsolin." Biochemistry. 28. 5269-5275 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Saeki, K.: "New method to prepare single-headed heavy meromyosin with high purity and a high yield." J.Biochemistry. 106. 606-611 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] K.Sutoh: "Electron microscopic visualization of the ATPase site of myosin by photoaffinity labeling with a biotinylated photoreactive ADP analog." Proc.Natl.Acad.Sci.USA. 83. 212-216 (1986)

    • Related Report
      1989 Annual Research Report
  • [Publications] K.Sutoh: "Actin-fragmin interactions as revealed by chemical crosslinking." Biochemistry. 25. 435-440 (1986)

    • Related Report
      1989 Annual Research Report
  • [Publications] K.Sutoh: "Improved method for mapping the binding site of an actin binding protein in the actin sequence." Biochemistry. 25. 6186-6192 (1986)

    • Related Report
      1989 Annual Research Report
  • [Publications] K.Sutoh: "Identification of two segments of myosin subfragment 1 heavy chain that can be crosslinked to the SH1 thiol." Biochemistry. 26. 4511-4516 (1987)

    • Related Report
      1989 Annual Research Report
  • [Publications] K.Sutoh: "A short hydrophobic segment next to Trp130 in myosin heavy chain is close to the ribose ring of ADP bound in the ATPase site." Biochemistry. 26. 7648-7654 (1987)

    • Related Report
      1989 Annual Research Report
  • [Publications] M.Suzuki: "Monoclonal Antibody which Recognizes Trimethylated N-Terminus of Histone H2B." Proc.Japan Acad.63. 385-388 (1987)

    • Related Report
      1989 Annual Research Report
  • [Publications] M.Tokunaga: "Position of the Amino-terminus of Myosin Light Chain-1 and Light Chain-2 determined by Electron Microscopy Using Monoclonal Antibody." J.Mol.Biol.194. 245-255 (1987)

    • Related Report
      1989 Annual Research Report
  • [Publications] K.Sutoh: "Electron Microscopic Visualization of the N-Terminus of the Myosin Heavy Chain using a Site-directed Antibody." J.Mol.Biol.195. 953-956 (1987)

    • Related Report
      1989 Annual Research Report
  • [Publications] M.Tokunaga: "Location of the ATPase site of myosin determined by three-dimensional electron microscopy." Nature. 329. 635-638 (1987)

    • Related Report
      1989 Annual Research Report
  • [Publications] K.Sutoh: "Spatial proximity of the glycine-rich loop and the SH2 thiol in myosin subfragment 1." Biochemistry. 27. 2964-2969 (1988)

    • Related Report
      1989 Annual Research Report
  • [Publications] M.Suzuki: "Packing of DNA in Cricket Sperm.A Compact Mode of DNA Packaging." J.Mol.Biol.204. 653-661 (1988)

    • Related Report
      1989 Annual Research Report
  • [Publications] K.Hirose: "Thin filaments of Rabbit Skeletal Muscle are in Helical Register." J.Mol.Biol.204. 797-801 (1988)

    • Related Report
      1989 Annual Research Report
  • [Publications] K.Sutoh: "End-label fingerprintings show that an N-terminal segment of depactin participates in interaction with actin." Biochemistry. 28. 102-106 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] K.Sutoh: "End-labeling fingerprinting shows that the N- and C-termini of actin are in the contact site with gelsolin." Biochemistry. 28. 5269-5275 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] K.Sutoh: "Electron Microscopic Mapping of Myosin Head with Site-directed Antibodies." J.Mol.Biol.206. 357-363 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] K.Saeki: "New Method to Prepare single-Headed Heavy Meromyosin with High Purity and a High Yield." J.Biochem.106. 606-611 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] T.Wakabayashi: "Three-Dimensional Image Reconstruction from Electron Micrographs of Biological Macromolocules." J.Electron Microsc.38. S102-S104 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] M.Tokunaga;M.Suzuki;K.Saeki;T.Wakabayashi: J.Mol.Biol.194. 245-255 (1987)

    • Related Report
      1988 Annual Research Report
  • [Publications] K.Sutoh;M.Tokunaga;T.Wakabayashi: J.Mol.Biol.,. 195. 953-956 (1987)

    • Related Report
      1988 Annual Research Report
  • [Publications] M.Tokunaga;K.Sutoh;C.Toyoshima;T.Wakabayashi: Nature. 329. 635-638 (1987)

    • Related Report
      1988 Annual Research Report
  • [Publications] M.Suzuki;K.Saeki;M.Tokunaga;T.Wakabayashi: Proc.Japan Acad.,. 63,Ser.B,. 385-388 (1987)

    • Related Report
      1988 Annual Research Report
  • [Publications] K.Sutoh,: Biochemisty,. 26. 7648-7654 (1987)

    • Related Report
      1988 Annual Research Report
  • [Publications] K.Hirose;T.Wakabayashi: J.Mol.Biol.204. 797-801 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] SUTOH, Kazuo: Proc. Natl. Acad. Sci. USA. 83. 212-216 (1986)

    • Related Report
      1987 Annual Research Report
  • [Publications] TOKUNAGA, Makio: J. Mol. Biol.194. 245-255 (1987)

    • Related Report
      1987 Annual Research Report
  • [Publications] SUTOH, Kazuo: J. Mol. Biol.195. 953-956 (1987)

    • Related Report
      1987 Annual Research Report
  • [Publications] SUTHOH, Kazuo: Biochemistry. 26. 7648-7654 (1987)

    • Related Report
      1987 Annual Research Report
  • [Publications] TOKUNAGA, Makio: Nature. 329. 635-638 (1987)

    • Related Report
      1987 Annual Research Report
  • [Publications] SUZUKI, Masashi: Proc. Japan Acad. Ser B.63. 385-388 (1987)

    • Related Report
      1987 Annual Research Report
  • [Publications] K.Sutoh;K.Yamamoto;T.Wakabayashi: Proc.Natl.Acad.Sci.,U.S.A.83. 212-216 (1986)

    • Related Report
      1986 Annual Research Report
  • [Publications] K.Maruyama;S.Kimura;K.Yamamoto;T.Wakabayashi;T.Suzuki: Biomedical Research,. 6. 423-427 (1986)

    • Related Report
      1986 Annual Research Report
  • [Publications] K.Hitaka;T.Nakamura;E.Kaneko;H.Onishi;T.Wakabayashi: J.Biol.Chem.,. 567. 1234-1238 (1986)

    • Related Report
      1986 Annual Research Report
  • [Publications] M.Tokunaga;M.Suzuki;K.Saeki;T.Wakabayashi: J.Mol.Biol.193. (1987)

    • Related Report
      1986 Annual Research Report

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Published: 1987-03-31   Modified: 2016-04-21  

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