ANALYSIS OF MOTOR PROTEIN FUNCTION

描述：(逐字摘自申请者的摘要)的基本目标 这项提议是为了从以下方面获得生化、生物物理和结构数据 运动蛋白家族和肌球蛋白家族马达蛋白都可以更好地定义 产生力和功的蛋白质构象的变化。水晶 这两个家族的运动域的结构似乎是合理的 涉及构象配位的作用力假设 马达的核苷酸位置、转换区和颈部的变化。 然而，检验这些假说将需要将目前的 在生化/机械反应中具有特定中间体的已知结构 循环，并确定是否存在其他当前未解析的结构。 电子顺磁共振(EPR)波谱将用于监测 核苷酸位置和转化子的构象和取向的变化 在从弱国向强国过渡的过程中，信息至关重要 以了解力量是如何产生的。中的相应区域 Kinesin家族的马达也将被用自旋标签探测。定向 来自EPR探测器的信息将使用我开发的协议获得 使微管流动对准。对非传染性疾病的研究建立在之前的数据基础上， 确定了探针的内源性特定位置和具有交联性的状态 半胱氨酸，可能对应于在 肌球蛋白。我还将探讨以前未曾预料到的领域移动 S1-PI管的开口。结构比较表明， 互补结构域运动发生在运动蛋白家族中--关闭 圆周率管的。一组相关的实验将检查圆周率的运动 动蛋白家族马达的管子。PI管的打开和关闭将 通过测量其上的距离并使用胶水将其粘合进行监控 交联剂。这一地区的运动可能起到重要作用 在测定肌球蛋白和肌球蛋白的核苷酸结合和释放速率时 Kinesin家族马达。总而言之，以上获得的数据将把结构 具有动力学和能量数据的信息，提供了更完整的画面 马达蛋白质是如何产生力和位移的。一项基本原则 这一提议的动机是对晶体结构的认识 肌球蛋白和MT的X射线结构的异同 马达可以显著提高实验设计水平。虽然这种方法 将显示为双行道，显然是最显著的优势 归因于研究较少的微管运动家族。

Description: (Verbatim from the applicant's abstract) The fundamental goal of this proposal is to obtain biochemical, biophysical, and structural data from both kinesin-family and myosin-family motor proteins to better define the changes in protein conformation that produce force and work. The crystal structures of motor domains from both families have suggested plausible hypotheses for force production involving the co-ordination of conformational changes at the nucleotide site, the converter region, and neck of the motor. Testing these hypotheses, however, will require both correlating the presently known structures with specific intermediates in the biochemical/mechanical cycle, and determining whether other, currently unresolved structures exist. Electron paramagnetic resonance (EPR) spectroscopy will be used to monitor changes in conformation and orientation at the nucleotide site and converter region during the transition from weak to strong states, information crucial for understanding how force is produced. The corresponding regions in kinesin-family motors will also be probed with spin labels. Orientation information from EPR probes will be obtained using protocols I have developed to flow-align microtubules. The study of ncd builds on previous data that identified an endogenous specific site for probes and a state with cross-linked cysteines, possibly corresponding to the well-studied cross-linked state in myosin. I will also explore a previously unanticipated domain movement in S1-the opening of the Pi-tube. A structural comparison suggests that the complementary domain movement occurs in the kinesin-family of motors-a closing of the Pi-tube. A related set of experiments will examine motions of the Pi tube of the kinesin-family motor. Opening and closing of the Pi-tube will be monitored by measuring distances across it and by gluing it shut using cross-linking reagents. The movement of this region may play an important role in determining rates of nucleotide binding and release in both myosin and kinesin family motors. Together, the data obtained above will link structural information with kinetic and energetic data, providing a more complete picture of how motor proteins function to produce force and displacement. A fundamental motivation for this proposal is that recognition of crystal structure differences and similarities between the X-ray structures of myosin and MT motors can significantly enhance experimental design. Although this approach will be shown to be a two-way street, the most significant advantage clearly accrues to the much less studied microtubule motor family.