Structure and function of Myosin VI

描述（由申请人提供）：Myosin VI可能是非常规肌球蛋白中最非常规的一种。它使用许多独特的机制来完成与肌凝蛋白V相似的步长过程运动，但方向相反，目的是在细胞内锚定和运输。肌凝蛋白VI可以作为单体或二聚体存在，其细胞意义尚不清楚。该项目将利用体外表达和功能测定、结构测定和体内测定来进一步了解肌球蛋白VI的功能以及这些分子在细胞中的作用。这些目标将通过解决以下具体目标来实现：目标1。肌凝蛋白VI转换器中产生大且可变（但固有的正端定向）步长的适应性是什么？假设：虽然在肌凝蛋白VI转换器末端的独特插入，插入2，完全负责肌凝蛋白VI的方向性逆转，但肌凝蛋白VI转换器具有额外的适应性，允许独特的动力冲程前状态，并创建大而可变的步长。目标2。不寻常的肌球蛋白VI杠杆臂+伸展的性质和目的是什么？假设：肌凝蛋白VI的大步长部分是由于其转体的不同寻常的运动和构象，它比肌凝蛋白V产生更大的摆动（动力冲程）。随着这种更大的摆动，更短的杠杆臂可以提供与肌凝蛋白V相同的步长。目标3。假设：myosin VI独特的插入物insert 1负责ATP的缓慢结合，当反向应变时，ATP的结合速度会变得更慢，而ADP可以进入核苷酸结合袋。这允许二聚体的导联头的门控，以及二聚体的锚定。目标4。肌凝蛋白VI是如何二聚的，它在细胞中作为二聚体或单体起作用吗？（与Karen abraham合作）假设：肌球蛋白VI二聚化受货物相互作用调节。我们认为调节二聚化消除了调节运动活动的需要。

DESCRIPTION (provided by applicant): Myosin VI is perhaps the most unconventional of unconventional myosins. It uses a number of unique mechanisms that are not well understood to accomplish processive movements of similar step sizes to myosin V, but of opposite directionality, for the purposes of anchoring and transporting within cells. Myosin VI can exist as a monomer or a dimer, the cellular significance of which is poorly understood. This project will utilize in vitro expression and functional assays, structural determinations and in vivo assays to attempt to further our understanding of how myosin VI functions and what these molecules do in a cell. These goals will be realized by addressing the following specific aims: Aim 1. What are the adaptations in the myosin VI converter that create a large and variable (but inherently plus-end directed) step size? Hypothesis: While the unique insert at the end of the myosin VI converter, insert 2, is solely responsible for the reversal of directionality of myosin VI, the myosin VI converter has additional adaptations that allow a unique pre-powerstroke state and create a large and variable step size. Aim 2. What is the nature and purpose of the unusual myosin VI lever arm + extension? Hypothesis: The large step size of myosin VI is in part due to the unusual movements and conformations of its converter, which creates a larger swing (powerstroke) than for myosin V. With this larger swing, a shorter lever arm can provide the same step size as the myosin V lever arm. Aim 3. Further probe the mechanism of head gating during processivity Hypothesis: The unique insert, insert 1, of myosin VI is responsible for slow ATP binding, which becomes still slower with reverse strain, while ADP has access to the nucleotide binding pocket. This allows gating of the lead head of a dimer, as well as anchoring of a dimer. Aim 4. How does myosin VI dimerize and does it function as a dimer or monomer in cells? (collaboration with Karen Avraham) Hypothesis: Myosin VI dimerization is regulated by cargo interactions. We propose that the regulated dimerization obviates the need to regulate motor activity.