Single-molecule analysis of recombinant motor protein of skeletal muscle by the use of atomic force microscope

原子力显微镜对骨骼肌重组运动蛋白的单分子分析

• 批准号: 12680660
• 负责人: YAMADA Takenori
• 金额: $ 2.24万
• 依托单位: Tokyo University of Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680660/
• 关键词: myosin; atomic force microscope; single molecule manipulation; single molecule analysis; motor protein; molecular motor; 単一分子計測・操作; 一分子解析

Muscle contraction takes place by relative sliding motions of actin and myosin filaments. The sliding force is produced by the structural changes of myosin heads (motor protein) extruded from myosin filaments by interacting with actin filaments utilizing the chemical energy of the ATP hydrolysis. However the molecular mechanism of how the force is produced in myosin motor and transmitted at the actin and myosin interface is still unclearIn the present research project, therefore, we investigated in detail the force generating processes of myosin motor at single molecule level. We made two experimental approaches to attack this problem; i.e., (1) whether or not myosin motor can generate force without the interaction with actin filaments and (2) whether or not myosin motor slides straightly along actin filamentsIn the first approach, we used recombinant myosin motors prepared by the gene technology.Transfored at actin binding myopathy loop by the cantilever tip of atomic force microscope … More (AFM). Thus the force to be generated by myosin motor and the structural changes to be induced in myosin head could simultaneously be measured based on the deflections of AFM cantilever. The results obtained suggested that myosin motor could generate force without the interaction with actin filaments. In the second approach, small micro-beads coated with myosin filaments were prepared and examined how these second approach, small micro-beads coated with myosin filaments were prepared and examined how these beads slid along actin filaments, they straightly slid along actin filaments. As the number of myosin filaments coated with myosin flaments, they straightly slid along actin filaments. As the number of myosin filaments coated to micro-beads was decreased, sliding motions became non-linear and irregularThese results suggest that the force is produced by the structural changes of myosin motor it self and the produced force is transmitted to the sliding motion, the pattern of which strongly depends on the interaction between myosin motor and actin filaments Less

肌肉收缩是通过肌动蛋白和肌球蛋白细丝的相对滑动运动发生的。滑动力是由肌球蛋白细丝通过与肌动蛋白细丝相互作用，利用三磷酸腺苷水解的化学能，产生肌球蛋白头部(马达蛋白)的结构变化而产生的。然而，肌球蛋白马达如何产生力并在肌动蛋白和肌球蛋白界面传递的分子机制尚不清楚，因此，我们在单分子水平上对肌球蛋白马达的力产生过程进行了详细的研究。为了解决这个问题，我们提出了两种实验方法：(1)肌球蛋白马达是否可以在不与肌动蛋白细丝相互作用的情况下产生力；(2)肌球蛋白马达是否可以沿着肌动蛋白细丝直线滑动。在第一种方法中，我们使用了基因技术制备的重组肌球蛋白马达，通过原子力显微镜…的悬臂尖端在肌动蛋白结合肌病环上进行转导更多(AFM)。因此，基于AFM悬臂梁的挠度可以同时测量肌球蛋白马达所产生的力和肌球蛋白头部所引起的结构变化。这些结果表明，肌球蛋白马达可以在不与肌动蛋白细丝相互作用的情况下产生力。在第二种方法中，制备了包裹着肌球蛋白细丝的小球，并研究了第二种方法，包裹着肌球蛋白细丝的小球是如何制备的，并研究了这些小球是如何沿着肌动蛋白细丝滑动的，它们是沿着肌动蛋白细丝直线滑动的。随着肌球蛋白细丝包裹肌球蛋白纤维的数量增加，它们沿着肌动蛋白细丝直线滑动。随着包裹在微珠上的肌球蛋白细丝数量的减少，滑动运动变得非线性和不规则。这些结果表明，这种力是由肌球蛋白马达本身的结构变化产生的，所产生的力传递到滑动运动中，其模式强烈依赖于肌球蛋白马达和肌动蛋白细丝之间的相互作用

项目成果

Kunioka Y., Sasaki N., Wakayama J., Aimi M., Sutoh K., Yamada T: "Simultaneous detection of the ATP hydrolysis and the motions in recombinant myosin heads"4th International Conference on Biophysics. 14. (2001)

Kunioka Y.、Sasaki N.、Wakayama J.、Aimi M.、Sutoh K.、Yamada T：“同时检测重组肌球蛋白头中的 ATP 水解和运动”第四届国际生物物理学会议。

C.Yagi,M.Shohara,Y.Kunioka,T.Yamada: "Does a Myosin Filament Rotate as It Slides along Actin Filaments?"J.Musc.Res.Cell Motil. 21. 195-196 (2000)

C.Yagi、M.Shohara、Y.Kunioka、T.Yamada：“肌球蛋白丝沿着肌动蛋白丝滑动时会旋转吗？”J.Musc.Res.Cell Motil。

国岡由紀, 佐々木直哉, 若山純一, 野口裕允, 須藤和夫, 山田武範: "AFM measurements of ATP-induced movements in myosin heads"J Muscle Res Cell Motil. 23. 176-176 (2002)

Yuki Kunioka、Naoya Sasaki、Junichi Wakayama、Hiromichi Noguchi、Kazuo Sudo、Takenori Yamada：“肌球蛋白头中 ATP 诱导运动的 AFM 测量”J Muscle Res Cell Motil 23. 176-176 (2002)。

国岡由紀,若山純一,佐々木直哉,大岩和弘,須藤和夫,山田武範: "ミオシンの化学-力学過程を原子間力顕微鏡で調べる"日本生物物理学会第38回年会講演予稿集. 40. S200-S200 (2000)

Yuki Kunioka、Junichi Wakayama、Naoya Sasaki、Kazuhiro Oiwa、Kazuo Sudo、Takenori Yamada：“使用原子力显微镜研究肌球蛋白的化学机械过程”第 38 届日本生物物理学会年会论文集。 S200 (2000)

国岡由紀,若山純一,佐々木直哉,大岩和弘,須藤和夫,山田武範: "AFM-TIRFM同時観察による生体モーター分子の研究"第48回応用物理学関係連合講演会講演予稿集. (発表予定). (2001)

Yuki Kunioka、Junichi Wakayama、Naoya Sasaki、Kazuhiro Oiwa、Kazuo Sudo、Takenori Yamada：“通过同时 AFM-TIRFM 观察研究生物运动分子”第 48 届应用物理学会会议记录（即将发表）。