Studies Of Myosin V

在哺乳动物中存在三种肌球蛋白V基因，称为Va、Vb和Vc。最近的研究提供了强有力的证据表明，单一的V类肌球蛋白分子运输囊泡和细胞器procedescent沿着F-肌动蛋白，采取几个36纳米的步骤，手在手，为每个扩散遇到。我们证明，肌球蛋白的ATP酶活性需要钙的最大活性，并表明，在钙的情况下，肌球蛋白V通过折叠，无活性的结构。我们已经使用负染色和冷冻电子显微镜检查肌球蛋白V的结构，是肌动蛋白上行走。这些图像给出了清晰的肌球蛋白V分子的图像，其两个头部都连接到肌动蛋白上，并将使我们能够观察杠杆臂的位置和刚度。在体外，野生型肌球蛋白V分子沿着附着在盖玻片表面的肌动蛋白向前移动。 平均游程长度为约1 μ m。 许多研究人员一直有兴趣使用肌球蛋白V作为纳米机器中的马达，以沿着肌动蛋白丝的工程轨道移动。为了增加肌球蛋白V的运行长度，我们克隆了cDNA构建体，其驱动卷曲螺旋的表达，形成三聚体，四聚体和五聚体结构融合到肌球蛋白V的杠杆臂和马达结构域。初步数据显示，我们已经成功地制造了三头肌球蛋白，并且该分子具有比双头野生型分子更长的运行长度。

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.