1989 Fiscal Year Final Research Report Summary
Studies on mechanism of muscle contraction by cryoelectron microscopy with site-specific molecular and heavy metal labelings
| Project/Area Number |
61065001
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| Research Category |
Grant-in-Aid for Specially Promoted Research
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| Allocation Type | Single-year Grants |
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| Research Institution | University of Tokyo |
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Principal Investigator |
WAKABAYASHI Takeyuki University of Tokyo, Physics Dept., Professor, 理学部, 教授 (90011717)
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| Co-Investigator(Kenkyū-buntansha) |
SUTOH Kazuo University of Tokyo, College of Arts & Sciences, Associate Professor, 教養学部, 助教授 (20111453)
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| Project Period (FY) |
1986 – 1989
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| Keywords | Cryoelectron Microscopy / Actin / Myosin / Undecagold Cluster / Site-Specific Lebel / Avidin-Biotin / Site-Directed Antibodies / Three-Dimensional Reconstitution |
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| 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.
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