Mechanisms of Processivity in Molecular Motors

描述（由申请人提供）：在上一个资助期，我发现肌球蛋白V，肌球蛋白VI，驱动蛋白1和CENP-E-进化上不同但功能相似的分子马达-每个都以相同的方式协调其两个催化结构域（“头部”）的酶学：通过门控核苷酸结合到前导头部。在这个更新申请中，我现在希望进一步探索变构通信的这个问题，这次通过研究来自同一家族但具有不同功能的两个马达如何通信-在一个头部内（分子内）以及头部之间（分子间）。驱动蛋白1作为单个马达运输货物，在其微管（MT）轨道上进行超过100步而不解离。Eg 5在合奏中滑动反平行主轴MT，对抗来自ncd和动力蛋白的持续相反力;并且它平均每次进行运行仅需要8步。这些功能差异反映在不同的酶学中。与驱动蛋白1不同，ATP与Eg 5的结合是缓慢的，并且与颈接头对接紧密偶联。我将重点介绍三种结构 这两种马达之间的差异很大，我认为它在介导分子内和分子间的通讯中起着关键作用。它们是环L5、颈连接体和颈卷曲螺旋。在目标1中，我将研究L5环如何调节核苷酸结合和偶联到机械元件-颈连接器的运动的时间。在这个目标的实验将利用国家的最先进的瞬态动力学和光谱方法。在目标2中，我将研究颈连接体和颈卷曲螺旋的多态性如何导致运动持续合成能力的差异。这项工作将结合联合收割机的国家的最先进的方法，在目标1与单分子力学研究。总的来说，目标1和2应该导致一个全面的模型的发展，如何驱动蛋白电机“微调”他们的分子生理学通过调整离散数量的结构。驱动蛋白1功能障碍与许多神经退行性疾病和多种恶性肿瘤中的化疗抗性有关，并且Eg 5已被深入研究作为开发用于治疗癌症的新抗有丝分裂剂的靶标。因此，很可能一个分子水平的模型，如何电机功能不仅会影响我们的理解病理生理学，但也指出了新的治疗方法。 公共卫生相关性：分子马达驱动多种形式的细胞生理学。在这个应用程序中，我建议调查如何两个成员的驱动蛋白家族的分子马达调整其结构，以适应他们的酶的细胞对他们的具体要求。我预计，这项工作将阐明设计原则，以了解现有的电机如何工作，以及产生的见解，如何新的电机可以定制工程，以服务于特定的功能。

DESCRIPTION (provided by applicant): In the last funding period, I found that myosin V, myosin VI, kinesin 1, and CENP-E--evolutionarily diverse but functionally similar molecular motors--each coordinate the enzymology of their two catalytic domains ("heads") in the same way: by gating nucleotide binding to the leading head. In this renewal application, I now wish to explore this issue of allosteric communication further, this time by investigating how two motors that come from the same family but serve different functions communicate--both within one head (intra- molecularly) as well as between heads (inter-molecularly). Kinesin 1 transports cargoes as a single motor, taking greater than 100 steps on its microtubule (MT) track without dissociating. Eg5 slides anti-parallel spindle MTs in ensembles, working against sustained opposing forces from ncd and dynein; and it only takes on average 8 steps per processive run. These functional differences are reflected in different enzymologies. Unlike kinesin 1, ATP binding to Eg5 is slow and tightly coupled to neck linker docking. I will focus on three structures that vary considerably between these two motors and which I propose play key roles in mediating both intra- and inter-molecular communication. These are loop L5, the neck linker, and the neck coiled coil. In Aim 1, I will examine how loop L5 regulates the timing of nucleotide binding and coupling to movements of the mechanical element--the neck linker. Experiments in this aim will utilize state- of-the-art transient kinetic and spectroscopic methodologies. In Aim 2 I will examine how polymorphisms in the neck linker and the neck-coiled coil contribute to differences in motor processivity. This work will combine the state-of-the-art methodologies developed in Aim 1 with single molecule mechanical studies. Taken together, Aims 1 and 2 should lead to the development of a comprehensive model of how kinesin motors "fine tune" their molecular physiology by adjusting a discrete number of structures. Kinesin 1 dysfunction has been linked to a number of neuro-degenerative diseases and to chemotherapy resistance in a variety of malignancies, and Eg5 has been intensively investigated as a target for the development of new anti-mitotics for the treatment of cancer. It is therefore likely that a molecular level model of how motors function will not only impact our understanding of pathophysiology but also point to new therapeutic approaches. PUBLIC HEALTH RELEVANCE: Molecular motors drive diverse forms of cell physiology. In this application, I propose to investigate how two members of the kinesin family of molecular motors adjust their structures in order to adapt their enzymologies to the specific demands placed on them by the cell. I anticipate that this work will illuminate design principles for understanding how existing motors work as well as generate insights into how new motors could be custom engineered to serve specific functions.