Single molecule research of molecular interactions using scanning probe microscopy

为了解决肌动球蛋白产生力的单个机械事件，我们开发了一种新的仪器，使用扫描探针捕获和直接操作单个肌动蛋白亚片段-1 （S1）。用荧光标记显示单个S1分子，并通过测量探针的位移来确定与吸收在玻璃表面的肌动蛋白束相互作用时的位移。这种迁移不是突然发生的，而是逐步发展的。位移上升阶段的步骤是有规则的（尺寸约为5.3 nm），在单个位移事件中观察到一到五个步骤。步长是恒定的，与[ATP]（0.1和1）和温度（20和27℃）无关。步骤之间的平均停留时间与[ATP]无关（在0.1 μM和1 μM时分别为3.2和4.8 ms），但总体位移持续时间与[ATP]有很大关系（在0.1 μM和1 μM时分别为2和0.2秒）。当ATP浓度为1 μm时，上升相的平均滑动速度分别为1.3 μm/s和3.1 μm/s，与ATP浓度为1 μm时的平均滑动速度接近，但比ATP浓度为1 μm时的平均滑动速度大100倍。这些结果表明，单个肌凝蛋白头沿肌动蛋白丝以5.3 nm的规则步骤移动，并经历5个步骤，每次ATP水解产生约30 nm的最大位移。

To resolve the individual mechanical events of force generation by actomyosin, we have developed a new instrument to capture and directly manipulate individual myosin subfragment-1 (S1) using a scanning probe. The single S1 molecules are visualized with a fluorescent label and the displacements during interaction with an actin bundle absorbed on a glass surface were determined by measuring the displacements of the probe. The displacements did not take place abruptly but, instead, developed in a stepwise fashion. The steps in the rising phase of displacements were regular (approximately 5.3 nm in size) and one to five steps were observed during single displacement events. The step size was constant, independent of [ATP] (0.1 and 1 ) and the temperature (20 and27℃). The mean dwell times between the steps were independent of [ATP] (3.2 and 4.8 ms at 0.1 and 1 μM, respectively), although the duration of overall displacements was greatly dependent (2 and 0.2 sec at 0.1 and 1 μM, respectively). The mean sliding velocities at the rising phase were 1.3 and 3.1 μm/s at 20 and 27℃, respectively, at 1 μM ATP, which are close to those at >1 μM ATP in a myosin-coated surface assay but >l00-fold greater than those at 1 μM ATP. These results show that a single myosin head moves along an actin filament with regular 5.3 nm steps and undergoes five steps to produce maximum displacement of approximately 30 nm for each ATP hydrolysis.