Mechanism of energy transduction in motor proteins - Single molecule mechanics and the ATPase reaction -

我们已经改进了全内反射荧光显微术（TIRFM），以全视频速率可视化水溶液中的单个荧光染料分子（Funatsu等人，Nature，374，555 '95）。我们将这种方法扩展到测量个人ATP周转率。通过直接观察荧光ATP类似物的缔合-（水解）-解离来检测单个驱动蛋白分子的单个ATP周转事件，其中Cy 3连接到核糖。采用光阱纳米测量技术测量了单个驱动蛋白分子的基本力学事件。如先前报道的，清楚地观察到8-nm台阶（Svoboda等人，Nature，365，721，92）。为了同时测量单电机力学和ATP营业额，我们将单分子成像的光学器件与单电机力学的光学捕获纳米测量相结合。首先，我们检测单个驱动蛋白分子连接到一个珠子，其位置是由一个光学陷阱控制的ATP营业额。当驱动蛋白分子在溶液中时，除了玻璃表面上的微管外，ATP周转率非常小，为-0.2s^1<-1>。而当它与微管接触时，速率大大提高到-10s<-1>。因此，现在可以测量受控负载下单个电机的ATP周转率。我们试图同时测量单个驱动蛋白分子的8 nm位移步骤和ATP周转率。ATP周转事件似乎大多与8 nm步长相关，但有时不相关。然而，由于Cy 3 ATP对驱动蛋白的低亲和力，8 nm的基本步骤不够清楚。为了获得基本机械事件和ATP失误之间的耦合，需要对装置进行改进。不会花很长时间的。由于Cy 3 ATP作为肌动球蛋白马达的正常ATP，本方法可能更有希望用于肌动球蛋白马达。该项目也在进行中。

We have refined total internal reflection fluorescence microscopy (TIRFM) to visualize single fluorescent dye molecules in aqueous solution at a full video rate (Funatsu, et al., Nature, 374,555 '95). We extened this method to measurements of individual ATP turnovers. Individual ATP turnover events by single kinesin molecules were detected by directly observing association-(hydrolysis)-dissociation of fluorescent ATP analogue, in which Cy3 was attached to ribose. Elementary mechanical events of single kinesin molecules were measured by optical trapping nanometry. 8-nm steps were clearly observed as reported previously (Svoboda, et al. Nature, 365, 721 '92). For simultaneous measurements of single motor mechanics and the ATP turnover, we combined the optics for single molecule imaging with optical trapping nanometry for single motor mechanics. First, we detected individual ATP turnovers by single kinesin molecules attached to a bead, of which position was controlled by an optical trap. When the kinesin molecule was in solution, apart from a microtubule on a glass surface, the ATP turnover rate was very small, -0.2s^<-1>. While, when it was brought into contact with a microtubule, the rate was greatly enhanced to be -10s^<-1>. Thus, it is now possible to measure the ATP turnover rates of single motors under controlled loads. We have tried to measure 8-nm displacement steps and ATP turnovers by single kinesin molecules simultaneously. ATP turnover events appeared to mostly correlated to 8-nm steps but sometimes do not. Because of low affinity of Cy3 ATP for kinesin, however, 8-nm elementary steps were not sufficiently clear. In order to gain the coupling between elementary mechanical events and ATP turnovers, improvement of the apparatus is necessary. It will not take long time. Since Cy3 ATP functions as normal ATP for actomyosin motors, the present method may be more hopeful for actomyosin motors. This project is also in progress.