Control of thermal fluctuation of proteins by evanessent field trapping.

The aims of this study were development of new techniques to control thermal fluctuation of proteins and to apply these techniques to actin-myosin system toward the final goal that is to understand roles of thermal fluctuation in chemi-mechano couppling of actin-myosin system。在这一赠款期内，我们获得了后续的成就，达到了最终目标。（1）金属表面等离子体上荧光标记的蛋白质的单分子成像：我们在薄金属层和水中之间制造光学显微镜的表面等离子体共振。由于使用了这项技术，最好的领域已经增强了2-10次，达到了玻璃和水之间的全部内部反射。荧光标记的马达蛋白的单分子的活性运动被观察到黄金和铝的表面。（2）允许机械和配体结合事件在单个肌红蛋白分子中的技术的发展，以便完全监测 ... More 使用这一技术，我们发现单一肌红蛋白分子的原力一代并不总是与绑定核肽的释放结合在一起，预先确定ADP。在ADP发布后，Myosin头部生产了几个百万分的手工艺品。（3）利用扫描探针捕捉和操纵个人肌氨酸分子的新仪器的发展：我们发现了一个单一的肌氨酸头部在正常步骤上沿着活性膜移动的行为。5.5纳米。两个到五个快速步骤在成功中经常产生的11 - 30纳米置换。这几个步骤只产生了ATP的一个生物化学周期。（4）对活性成分的局部差异的测量：一种带有珠子附着的单活性成分薄膜，两个结果都在光学调味器的解决方案中暂停。薄膜的损伤差异被观察到在光学tweezer中存在肌红蛋白或无ATP肌红蛋白的旋转运动。旋转的幅度在ATP的存在中增加了，三个不同的菲律宾地区也没有改变。Less（低）

The aims of this study were development of new techniques to control thermal fluctuation of proteins and to apply these techniques to actin-myosin system toward the final goal that is to understand roles of thermal fluctuation in chemi-mechano couppling of actin-myosin system. During the period of this grant, we obtained the following achievments to the final goal.(1)Single molecule imaging of fluorescently-labelled proteins on metal by surface plasmons in aqueous solution : We made an optical microscope using surface plasmon resonance at the meniscus between thin metal layer and water. By using this technique, evanessent field was enhanced 2-10 times compered to that by total internal reflection between glass and water. Active movement of single molecules of the fluorescently labelled motor proteins was observed on the surface of gold and alminium.(2)Development of a technique that allows mechanical and ligand-binding events in a single myosin molecules to be monitored simultaneously … More : Using this technique, we found that the force generation of single myosin molecule does not always coincide with the release of bound nucleotide, presumably ADP. Instead the myosin head produces force several handreds of milliseconds after ADP is released.(3)Development of a new instrument to capture and manipulate individual myosin molecules using a scanning probe : We found that single myosin head moves along an actin filament with regular steps of 。ォ5.5nm. Groups of two to five rapid steps in succession often produce displacement of 11 to 30nm. This multiple stepping is produced during just one biochemical cycle of ATP.(4)Measurement of the torsional diffusion of actinfilaments : A single actin filament with bead attached to both ends was suspended in solution by optical tweezers. Torsional diffusion of the filament was observed by rotational movements of a bead in the optical tweezers in the presence of myosin with or without ATP. Amplitude of the rotation was increased in the presence of ATP, though torsional regidity of the filament was not changed. Less