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Control of thermal fluctuation of proteins by evanessent field trapping.

通过消失场捕获控制蛋白质的热波动。

基本信息

批准号：
09359004
负责人：
YANAGIDA Toshio
金额：
$ 20.86万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

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

本研究的目的是发展新的技术来控制蛋白质的热涨落，并将这些技术应用于肌动蛋白-肌球蛋白系统，最终目的是了解热涨落在肌动蛋白-肌球蛋白系统的化学-机械耦合中的作用。在此期间，我们取得了以下成果，最终目标。（1）在水溶液中通过表面等离子体激元对金属上的荧光标记蛋白质进行单分子成像：我们在薄金属层和水之间的弯月面处使用表面等离子体激元共振制作了光学显微镜。用这种技术，倏逝场比玻璃与水之间的全内反射增强2-10倍。在金和氧化铝表面上观察到荧光标记的马达蛋白的单个分子的主动运动。（2）开发能够同时监测单个肌球蛋白分子中的机械和配体结合事件的技术 ...更多信息利用这种技术，我们发现单个肌球蛋白分子的力产生并不总是与结合的核苷酸（可能是ADP）的释放一致。相反，肌球蛋白头在ADP释放后几毫秒内产生力。（3）研制了一种利用扫描探针捕获和操纵单个肌球蛋白分子的新仪器：我们发现单个肌球蛋白头沿着肌动蛋白丝以规则的步长运动。波长5.5nm。连续两到五个快速步骤的组通常产生11到30 nm的位移。这种多重步进产生于ATP的一个生化循环中。（4）肌动蛋白丝扭转扩散的测量：用光镊将一根两端带有珠子的肌动蛋白丝悬浮在溶液中。在有或没有ATP的肌球蛋白的存在下，通过在光镊中珠的旋转运动观察到细丝的扭转扩散。在ATP存在下，旋转的幅度增加，但丝的扭转刚度没有改变。少