Studies Of Myosin V

哺乳动物有三种肌球蛋白V基因，分别称为Va、Vb和Vc。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 -肌动蛋白运输囊泡和细胞器，每次扩散相遇时，它们交替进行几个36纳米的步骤。我们证明了肌凝蛋白的atp酶活性需要钙才能达到最大活性，并且表明，在缺乏钙的情况下，肌凝蛋白V采用折叠的、无活性的结构。我们用阴性染色和低温电镜观察了肌动蛋白上行走的肌球蛋白V的结构。这些图像提供了两个头部附着在肌动蛋白上的肌球蛋白V分子的清晰图像，并将使我们能够观察杠杆臂的位置和刚度。我们正在研究肌动蛋白结合的肌球蛋白V突变体的结构，其中IQ基序之间的间距被改变，并将通过体外运动测定和光学捕获来补充这些研究。我们还研究了突变型肌球蛋白V分子的结构，其中颈部区域通过改变IQ motif的数量而改变。这些研究表明，肌凝蛋白V的颈部已经进化到允许它沿着肌动蛋白走36纳米的步伐，这与螺旋重复相匹配。我们突变了肌凝蛋白V （S217A）的开关-1区域，发现该突变改变了占空比并影响了马达的加工能力。Switch-2突变体影响易位速度和ADP释放率。光学捕获研究表明，在一定的应变水平下，肌凝蛋白V附着在肌动蛋白上的动力行程可以逆转，这可能有助于在失速期间维持肌凝蛋白的附着。我们已经开始研究肌凝蛋白Vc的力学特性和肌凝蛋白Vb的调控特性。

There are three myosin V genes in mammals, termed Va, Vb and Vc. 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 are examining the structure of actin-bound myosin V mutants in which the spacing between IQ motifs were altered and will complement these studies with in vitro motility assays and optical trapping. We also examined the structure of mutant myosin V molecules in which the neck region was altered by changing the number of IQ motilfs. 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. 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. We have begun to study the mechanical properties of myosin Vc and the regulatory properties of myosin Vb.