Structure and function of Myosin VI

描述（由申请人提供）：肌球蛋白VI可能是非常规肌球蛋白中最非常规的。它使用了许多尚未被很好理解的独特机制来完成与肌球蛋白V类似步长但方向相反的进行性运动，以在细胞内锚定和运输。肌球蛋白VI可以作为单体或二聚体存在，其细胞意义知之甚少。该项目将利用体外表达和功能测定，结构测定和体内测定，试图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。这些目标将通过实现以下具体目标来实现：肌球蛋白VI转换器中产生大的可变（但本质上是加端定向的）步长的适应性是什么？假设：虽然肌球蛋白VI转化器末端的独特插入物（插入物2）仅负责肌球蛋白VI的方向性逆转，但肌球蛋白VI转化器具有额外的适应性，可实现独特的动力冲程前状态并产生较大且可变的步长。目标2.不寻常的肌球蛋白VI杠杆臂+延伸的性质和目的是什么？假设：肌球蛋白VI的大步长部分是由于其转换器的不寻常运动和构象，这产生了比肌球蛋白V更大的摆动（动力冲程）。由于这种更大的摆动，较短的杠杆臂可以提供与肌球蛋白V杠杆臂相同的步长。目的3.进一步探索持续合成能力过程中头部门控的机制假设：肌球蛋白VI的独特插入片段（插入片段1）负责缓慢的ATP结合，随着反向应变，ATP结合变得更慢，而ADP可以进入核苷酸结合口袋。这允许门控二聚体的引线头，以及锚定二聚体。目标4。肌球蛋白VI是如何二聚化的？它在细胞中是二聚体还是单体？（与Karen Avraham合作）假设：肌球蛋白VI二聚化受到货物相互作用的调节。我们建议，受管制的二聚体消除了需要调节运动活动。

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.