Analysis of a ADP-bound non-muscle myosin molecule

已有报道，当ADP加入肌动球蛋白复合物中时，平滑肌肌球蛋白和刷状缘肌球蛋白的轻链部分发生构象变化。而光镊实验揭示了力的产生是分两步进行的，从而提出了第二步力是由ADP解离产生的假说。然而，没有观察到加入ADP对平滑肌的力的降低。因此，ADP在Fight链中观察到的构象变化被认为是应变依赖性ADP解离，而不是动力冲程的一部分。本研究的目的是阐明应变依赖的ADP解离。遗憾的是，由于在实验装置和方法的开发上花费了大量的时间，因此没有得到关于ADP解离的应变依赖性的结论。然而，我们已经制作了一个很好的装置，可以测量单个肌球蛋白分子从刷状缘产生的力。我们描述了该装置的开发过程，刷状缘肌球蛋白的制备，以及珠状样品的制备，并显示了单个肌球蛋白分子产生力的结果。

It has been reported that conformational changes occurred at the light chain portion of smooth muscle myosin and brush border myosin when ADP was added to the actomyosin complex. And the optical tweezer experiment has revealed that the foece generation occurred by two steps, leading the hypothesis that the second step of force is generated by the ADP dissociation. However, the decrease in the force of smooth muscle by adding ADP was not observed. So, the conformational changes seen in the fight chain by ADP is thought to be the strain-dependent ADP dissociation, not a portion of a power stroke. The purpose of this study was to elucidate the strain-dependent ADP dissocation. Unfortunately, a lot of time was spent to develope the experimental apparatus and the method, so the conclusion on the strain-dependent ADP dissociation was not obtained. However, we have made a fine apparatus that measure the force generation of a single myosin molecule from brush border.We describe the process of developing the apparatus, the preparation of the brush border myosin, and the preparation of the beads sample, and show the result of the force generation of single myosin molecule.