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。野生型肌凝蛋白V分子沿着附着在盖盖表面的肌动蛋白运动。平均运行长度约为1微米。许多研究人员对利用肌凝蛋白V作为纳米机器的马达，使其沿着肌动蛋白丝的工程轨迹运动很感兴趣。为了增加肌凝蛋白V的运行长度，我们克隆了驱动线圈表达的cDNA结构，形成三聚体、四聚体和五聚体结构，融合到肌凝蛋白V的杠杆臂和运动域。初步数据表明，我们成功地制造了一个三头肌凝蛋白，并且该分子比双头野生型分子具有更长的运行长度。

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. Wild type myosin V molecules move processively along actin attached to coverslip surfaces in vitro. The average run length is about 1 um in length. Many investigators have been interested in using myosin V as a motor in nanomachines to move along engineered tracks of actin filaments. In order to increase the run lengths of myosin V, we have cloned cDNA constructs that drive the expression of coiled-coils that form trimeric, tetrameric and pentameric structures fused to the lever arm and motor domain of myosin V. Preliminary data show that we have successfully made a three-headed myosin and that this molecule has a longer run length than seen with the two-headed wild type molecule.