ANALYSIS OF MOTOR PROTEIN FUNCTION

描述：（申请人摘要中的逐字逐句） 这项建议是从以下方面获得生物化学、生物物理和结构数据： 驱动蛋白家族和肌球蛋白家族马达蛋白，以更好地定义 产生力和功的蛋白质构象变化。晶体 来自两个家族的运动域的结构表明可能是 涉及构象协调的力产生假说 在核苷酸位点、转换区和马达颈部发生变化。 然而，测试这些假设需要将目前的 已知的结构与特定的中间体在生化/机械 周期，并确定是否存在其他目前尚未解决的结构。 电子顺磁共振（EPR）光谱将用于监测 核苷酸位点和转换器的构象和方向变化 在从弱国向强国过渡期间，信息至关重要 来理解力是如何产生的中的对应区域 还将用自旋标记探测驱动蛋白家族马达。取向 从EPR探测器获得的信息将使用我开发的协议 来排列微管非传染性疾病的研究建立在以前的数据基础上， 确定了探针的内源性特异性位点和交联状态， 半胱氨酸，可能对应于充分研究的交联状态， 肌球蛋白我还将探讨一个以前没有预料到的领域运动， S1-Pi管的开口。结构比较表明， 互补结构域运动发生在驱动蛋白家族的马达-一个关闭 关于Pi-tube一组相关的实验将检查圆周率的运动 驱动蛋白家族马达的管。打开和关闭PI管将 通过测量它的距离和用胶水把它粘上来监测， 交联试剂。这一区域的运动可能起到重要作用 在测定肌球蛋白和肌球蛋白中核苷酸结合和释放的速率时， 驱动蛋白家族发动机总之，上面获得的数据将连接结构 信息与动力学和能量数据，提供更完整的图片 马达蛋白是如何产生力和位移的一项基本 该提议的动机是认识到晶体结构 肌球蛋白和MT的X射线结构的异同 电动机可以显著增强实验设计。虽然这种方法 将被证明是一个双向的街道，最显着的优势明显 属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.