Studies Of Myosin V

最近的研究提供了强有力的证据表明，单个V类肌球蛋白分子沿着F-肌动蛋白连续运输囊泡和细胞器，每次扩散相遇都要走几个36 nm的步骤。我们证明，肌球蛋白的ATPase活性需要钙才能达到最大活性，并表明在没有钙的情况下，肌球蛋白V采用折叠的非活性结构。我们用负染和冷冻电子显微镜研究了行走在肌动蛋白上的肌球蛋白V的结构。这些图像给出了肌球蛋白V分子的清晰图像，两个头都附着在肌动蛋白上，这将使我们能够观察到杠杆臂的位置和僵硬。我们已经研究了肌动蛋白结合肌球蛋白V突变体的结构，在这些突变体中，颈部长度发生了变化。这些研究表明，肌球蛋白V的颈部已经进化到允许它沿着与螺旋重复序列匹配的肌动蛋白迈出36纳米的步伐。 用原子力显微镜研究了果蝇和小鼠肌球蛋白V尾部的延展性。在这两种情况下，这些肌球蛋白尾巴的力-伸展曲线与兔骨骼肌肌球蛋白的显著不同，因为实现伸展所需的力更少。进行性小鼠肌球蛋白V和非进行性果蝇肌球蛋白V的尾巴行为也有差异。模拟研究表明，可伸展的肌球蛋白V尾巴对于粘性环境中的进行性运动非常有用。 我们突变了肌球蛋白V(S217A)的Switch-1区域，并发现该突变改变了占空比和影响马达的加工性。Switch-2突变体影响转位速度和ADP释放率。 光学捕捉研究表明，在一定的应变水平下，肌球蛋白V与肌动蛋白结合的PowerStroke可以被逆转，这可能有助于在失速过程中保持肌球蛋白的附着。

Recent studies provide strong evidence that single class V myosin molecules transport vesicles and organelles processively along F-actin, taking several 36-nm steps, hand over hand, for each diffusional encounter. We demonstrated that the ATPase activity of myosin required calcium for maximal activity and showed that, in the absence of calcium, myosin V adopted a folded, inactive structure. We have used negative staining and cryo-electron microscopy to examine the structure of myosin V that is walking on actin. These images give clear pictures of myosin V molecules with both heads attached to actin and will allow us to make observations about lever arm position and stiffness. We have examined the structure of actin-bound myosin V mutants in which the neck length was altered. These studies demonstrate that the neck of myosin V has evolved to allow it to take 36 nm strides along actin which matches the helical repeat. Atomic force microscopy was used to study the extensibility of the tails of myosin V from Drosophila and mouse. In both cases, the force-extension curve of these myosin tails differed dramatically from that of rabbit skeletal muscle myosin in that less force is needed to bring about extension. There were also differences in the behavior of the tail from the processive mouse myosin V and the nonprocessive Drosophila myosin V. Modeling studies have shown that an extensible myosin V tail would be very useful for processive movement in a viscous environment. We have mutated the switch-1 region of myosin V (S217A) and found that this mutant alters the duty ratio and effects processivity of the motor. Switch-2 mutants affect the speed of translocation and the ADP release rate. Optical trapping studies show that under certain levels of strain, the powerstroke of myosin V attached to actin can be reversed which may aid in maintaining myosin attachment during stall.