1993 Fiscal Year Final Research Report Summary
Mechanisms of molecular motors analyzed with optical tweezers
| Project/Area Number |
04452331
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
分子遺伝学・分子生理学
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| Research Institution | Keio University |
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Principal Investigator |
KINOSITA Kazuhiko Keio University, Faculty of Science and Technology, Professor, 理工学部, 教授 (30124366)
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| Co-Investigator(Kenkyū-buntansha) |
SUZUKI Naoya Nagoya University, Faculty of Science, Instructor, 理学部, 助手 (50222063)
MIYATA Hidetake Keio University, Faculty of Science and Technology, Assistant Professor, 理工学部, 専任講師 (90229865)
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| Project Period (FY) |
1992 – 1993
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| Keywords | Myosin / Actin / Plastic beads / Protein conformational changes / Fluorescence polarization imaging / Rigor bond / Unbinding force / Molecular individualism |
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| Research Abstract |
To elucidate the mechanisms of motility and force production by molecular motors, we have analyzed the dynamic properties of an in vitro motility system in which actin filaments slide over myosin molecules distributed on a glass surface.
1. We attached a plastic bead selectively at the tail (barbed) end of an actin filament. The bead was held in an optical trap, and movement of the bead due to the tug-of-war between myosin molecules and the optical trap was analyzed at a nanometer precision. At low myosin densities and low ATP concentrations, unitary steps of myosin were resolved, which distributed around 8 nm. The steps may represent the size of a conformational change (s) in myosin : real-time detection of conformational changes in a single protein molecule is possible by attaching to the protein a huge probe (plastic bead) through a flexible string of actin.
2. The orientation of actin monomers in an actin filament was assessed continuously in real time by attaching a fluorophore rigidly to each actin monomer and detecting the fluorophore orientation by fluorescence polarization imaging. With up to a few fluorophores in each filament, the axial rotation of sliding actin filaments has been demonstrated.
3.Unbinding force between a single myosin molecule and actin, in the absence of ATP,was measured with optical tweezers. Under an applied force of 10 pN,the bond broke in about 1s. Lower force resulted in slower unbinding. The unbinding force was independent of the direction of pull, indicating that myosin is a flexible molecule. Repeated measurement on the same myosin molecules revealed molecular individualism, in that some myosin always required a high tension for unbinding whereas others were consistently weaker.
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