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

肌肉收缩是通过肌动蛋白和肌球蛋白丝的相对滑动而发生的。滑动力是由利用ATP水解的化学能与肌动蛋白丝相互作用而从肌球蛋白丝挤出的肌球蛋白头（马达蛋白）的结构变化产生的。然而，肌球蛋白马达如何产生力以及力如何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，本研究在单分子水平上详细研究了肌球蛋白马达的力产生过程。我们提出了两种实验方法来解决这个问题;即，(1)肌球蛋白马达是否能在不与肌动蛋白丝相互作用的情况下产生力;（2）肌球蛋白马达是否能沿肌动蛋白丝直线沿着滑动。第一种方法是利用基因工程技术制备重组肌球蛋白马达，利用原子力显微镜的悬臂梁探针将其转移到肌动蛋白结合的肌病环上 ...更多信息 （AFM）。因此，肌球蛋白马达产生的力和肌球蛋白头部的结构变化可以同时测量基于AFM悬臂梁的偏转。结果表明，肌球蛋白马达可以在不与肌动蛋白丝相互作用的情况下产生力。在第二种方法中，制备了涂覆有肌球蛋白细丝的小微珠，并检查了这些第二种方法，涂覆有肌球蛋白细丝的小微珠是如何制备的，并检查了这些珠是如何沿着肌动蛋白细丝滑动的，它们是如何沿着沿着肌动蛋白细丝直线滑动的。由于肌球蛋白丝被肌球蛋白细丝包裹，它们沿着肌动蛋白丝直线滑动。随着微球表面肌球蛋白丝数量的减少，滑动运动变得非线性和不规则，这些结果表明，力是由肌球蛋白马达自身结构的变化产生的，并且所产生的力被传递到滑动运动中，滑动运动的模式强烈地依赖于肌球蛋白马达和肌动蛋白丝之间的相互作用。

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