Studies Of Myosin V

哺乳动物中有三个肌球蛋白V基因，分别为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。在体外，野生型肌球蛋白V分子沿着附着在盖子表面的肌动蛋白连续移动。平均游程长度约为1微米。许多研究人员一直对使用肌球蛋白V作为纳米机器的马达，沿着肌动蛋白细丝的工程轨迹移动感兴趣。为了增加肌球蛋白V的游程长度，我们克隆了驱动卷曲表达的cDNA构建物，这些卷曲形成的三聚体、四聚体和五聚体结构与肌球蛋白V的杠杆臂和运动区融合在一起。初步数据表明，我们成功地制造了一个三头肌球蛋白，该分子比两头野生型分子具有更长的游程长度。

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.