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

肌肉收缩是由肌动蛋白和肌凝蛋白丝的相对滑动运动引起的。滑动力是由肌凝蛋白头（运动蛋白）的结构变化产生的，肌凝蛋白头（运动蛋白）从肌凝蛋白丝中挤出，利用ATP水解的化学能与肌动蛋白丝相互作用。然而，肌凝蛋白马达产生力并在肌动蛋白和肌凝蛋白界面传递的分子机制尚不清楚。因此，本研究项目在单分子水平上对肌凝蛋白马达的力产生过程进行了详细的研究。我们采用了两种实验方法来解决这个问题；即(1)肌凝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)肌凝蛋白马达是否能沿肌动蛋白丝直线滑动。第一种方法，我们使用基因技术制备的重组肌凝蛋白马达。原子力显微镜悬臂尖在肌动蛋白结合的肌病环上进行了转化。因此，基于AFM悬臂的挠度，可以同时测量肌凝蛋白马达产生的力和引起的肌凝蛋白头部的结构变化。结果表明，肌凝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，制备了涂有肌凝蛋白丝的小微珠并检查了第二种方法，制备了涂有肌凝蛋白丝的小微珠并检查了这些微珠是如何沿着肌动蛋白丝滑动的，它们是笔直地沿着肌动蛋白丝滑动的。当肌凝蛋白丝的数量被肌凝蛋白丝包裹时，它们沿着肌动蛋白丝直线滑动。随着涂覆在微珠上的肌凝蛋白丝数量的减少，滑动运动变得非线性和不规则。这些结果表明，这种力是由肌凝蛋白马达本身的结构变化产生的，并将产生的力传递给滑动运动，而滑动运动的模式很大程度上取决于肌凝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