STRUCTURAL STUDIES ON MICROTUBULE MOTORS

机械酶驱动蛋白超家族的成员在细胞中执行其许多关键功能的过程中沿着微管移动。正端定向和负端定向驱动蛋白的X射线结构现在都是可用的，微管蛋白原丝的高分辨率电子晶体结构也是如此。尽管做了很多努力，但在制备用于晶体学分析的轨道-运动复合物的晶体方面还没有成功。将组件的高分辨率结构与通过低温电子显微镜获得的低分辨率（20- 30 A）3D图相结合是了解组件在相互作用周期中各个阶段的详细工作的唯一方法。研究将集中在驱动蛋白超家族的三个成员：一个传统的二聚体，正端定向电机（脑驱动蛋白），二聚体负端定向电机（ncd），和单体正端定向电机（unc 104）。金簇标记蛋白的冷冻EM和图像分析，加上差异映射和建模将用于：-了解驱动蛋白和微管蛋白的近原子相互作用，-可视化核心运动域内的运动，-可视化颈部区域的部分运动，-了解二聚体分子的两个头部是如何协调的。这些数据将提供一个详细的结构了解如何驱动蛋白超家族的三个成员产生定向运动。这项工作福尔斯属于基础生物医学研究的范畴，对健康和疾病状态都具有基本和根本的相关性。

Members of the kinesin superfamily of mechanoenzymes move along microtubules in the course of carrying out their many critical functions in the cell. X-ray structures of both plus-end directed and minus-end directed kinesins are now available, as is the high resolution electron crystallographic structure of the tubulin protofilament. Despite much effort, there has been no success in preparing crystals of the track- motor complexes for crystallographic analyses. Combining the high resolution structures of the components with low resolution (20-30A) 3D maps obtained by cryo-electron microscopy is the only way to understand the detailed workings of the assemblies at various stages in the cycle of interaction. Studies will focus on three members of the kinesin superfamily: a conventional dimeric, plus-end directed motor (brain kinesin), a dimeric minus-end directed motor (ncd), and a monomeric plus-end directed motor (Unc104). Cryo-EM and image analysis of gold-cluster labeled proteins, coupled with difference mapping and modeling will be used to: - understand the near-atomic interactions of kinesins and tubulin, - visualize motions within the core motor domain, - visualize the movements of part of the neck region, - understand how the two heads of dimeric molecules are coordinated. These data will provide a detailed structural understanding of how three members of the kinesin superfamily produce directional movement. This work falls into the category of Basic Biomedical Research and has a basic and fundamental relevance for both the healthy and diseased states.