STRUCTURAL STUDIES ON MICROTUBULE MOTORS

肌动蛋白机械酶超家族的成员在执行其在细胞中的许多关键功能的过程中沿着微管移动。现在可以得到正端定向和负端定向的动蛋白的X射线结构，以及微管蛋白原丝的高分辨电子晶体结构。尽管付出了很多努力，但在制备用于结晶学分析的轨道发动机复合体晶体方面仍未取得成功。将冷冻电子显微镜获得的低分辨率(20-30A)3D图与高分辨率的组件结构相结合，是了解组件在相互作用周期不同阶段的详细工作原理的唯一途径。研究将集中在Kinesin超家族的三个成员上：传统的二聚体正端定向运动(脑Kinesin)，二聚体负端定向运动(NCD)和单体正端定向运动(Unc104)。金簇标记蛋白质的冷冻-EM和图像分析，再加上差异图谱和建模，将被用于：-了解动蛋白和微管蛋白的近原子相互作用，-可视化核心运动区域内的运动，-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。这些数据将为Kinesin超家族的三个成员如何产生定向运动提供详细的结构理解。这项工作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础性和根本性的相关性。

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.