Structure and Function of Molecular Motors

描述(由申请人提供)： 我们将继续研究肌球蛋白和动蛋白家族的马达将化学能转化为机械能的机制。运动蛋白马达的方法将建立在我们之前的工作的基础上，我们之前的工作确定了几个基于微管的多种构象的马达的结构，并使用光谱方法来确定这些结构在循环中是如何变化的。我们将获得Kinesin家族新成员的结构。我们将通过使用荧光和顺磁光谱探针获得动态信息来研究这些结构中发生的构象变化。我们将扩大我们对传统Kinesin的颈缩机制的了解。我们将启动对NCD的研究，这可能通过与激动素非常不同的机制发挥作用。肌球蛋白VI是一种前进的马达，步长很长，但杠杆臂很短，将使用光谱探测器来研究这一不寻常的马达。此外，我们还将研究另一个运动家族成员Dynein的运动区和货物结合区的结构和功能。这些结构和功能数据将得到发动机力学测量的补充。肌动蛋白细丝及其与肌球蛋白片段的复合体将形成低聚体，并确定其性质和结构。 我们将为我们的计划启动一个新的方向，并调查马达蛋白质的货运结构域，并确定它们的蛋白质伙伴，这些蛋白质伙伴共同将马达连接到它们的负载。货运域的特征不如马达域好，它们的众多合作伙伴中只有几个是已知的。我们将使用化学、结构、生化和遗传技术来鉴定与货物识别有关的蛋白质，确定选定的货物结合结构域及其伙伴的结构，并表征它们的功能。 总之，我们理解生物马达机制的目标需要：a)确定它们的原子分辨结构，b)了解这些结构在特定状态下是如何变化的，以及c)测量与这些状态相关的力学和能量学。在这个项目中，我们聚集了一组独特的研究人员，他们可以扩展我们在这些领域的每个领域的知识，从而更好地了解这两个产生力和运动的发动机的分子机制。

DESCRIPTION (provided by applicant): We will extend our studies of the mechanism by which chemical energy is transformed into mechanical energy by motors from the myosin and kinesin families. The approach for the kinesin motors will build on our previous work which determined the structures of several of the microtubule based motors in multiple conformations, and which used spectroscopic methods to determine how these structures changed during the cycle. We will obtain structures for new members of the kinesin family. We will investigate the conformational changes that occur in these structures by obtaining dynamic information using spectroscopic probes, both fluorescent and paramagnetic. We will expand our knowledge of the neckdocking mechanism of conventional kinesin. We will initiate studies of NCD, which may work through a mechanism very different from kinesin. Myosin VI is a processive motor with a long step, but a short lever arm, and spectroscopic probes will be used to investigate this unusual motor. In addition we will investigate the structure and function of both the motor region and the cargo-binding region of a member of another motor family, dynein. These structural and functional data will be complemented by measurements of motor mechanics. Oligomers of the actin filament and their complexes with myosin fragments will be formed, and their properties and structures determined. We will initiate a new direction for our program, and investigate the cargo domains of the motor proteins and identify their protein partners that together connect the motors to their loads. The cargo domains are less well characterized than are the motor domains, and only a few of their many partners are known. We will employ chemical, structural, biochemical, and genetic techniques to identify the proteins involved in cargo recognition, determine structures for selected cargo binding domains and their partners, and characterize their function. In summary, our goal of understanding the mechanism of biological motors requires: a) determination of their atomic resolution structures, b) knowledge of how these structures change in specific states, and c) measurements of the mechanics and energetics associated with these states. In this program we have brought together a unique group of investigators that can extend our knowledge in each of these areas leading to a better picture of the molecular mechanism of these two motors that produce force and motion.