High-Resolution Structural Aanlyases of Dynein Molecules During Sliding By Quick-Freeze Deep-Etoh Replica Electron Microscopy

通过快速冷冻深乙醇复制电子显微镜对滑动过程中的动力蛋白分子进行高分辨率结构分析

基本信息

批准号：
11480185
负责人：
KATAYAMA Eisaku
金额：
$ 9.47万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

This project originally aimed to determine three-dimensional (3-D) structure of cytoplasmic dyncin/microtubule complex and its change upon sliding movement. Because of difficulty in obtaining very fresh materials with high activity, we switched the material to sea-urchin sperm flagella and studied the structure/function relationship of axonemal dyncin subspecies. We also investigated the 3-D structure of sliding actomyosin complex, using a reconstruction method which we recently developed according to the concept of "single molecule physiology". With the results of such studies as a basis, we discussed the molecular mechanism of processive motor activity which often seems to play crucial roles in dynein/microotuble sliding systems.Many members of unconventional myosins evoke processive movement of actin filament. Myosin-V is among the most intriguing for its long lever-arm moiety and its "walking" movement with 36 nm steps, along actin filament. Such characteristic behavior of myosin-V … More has been attributed as one of the strongest evidences to support the validity of "tilting lever-arm mechanisms". We examined the behavior of myosin-V mutant with truncated lever-arm and myosin-VI which naturally has short lever-arm moiety. Interestingly, both of them showed processive movement of actin with 〜36 nm step size that is identical to original myosin-V with long lever-arm. We checked the mode of binding of those myosin heads together with dyctyostelium myosin mutant whose ADP-Pi state is extraordinarily stabilized, to actin filament by electron microscopy. Surprisingly, we found that all of those myosin heads seem to bind to actin with 〜36 nm regular intervals, and only to one side of the filament. All of these phenomena are quite difficult to explain by "tilting lever-arm hypothesis" and might suggest the possibility that the binding of one energized myosin head to actin might evoke the special high affinity site(s) (hot-spot(s)) 〜36 nm away from the original site.According to our replica images, the structure of functioning myosin heads was roughly classified into three categories,' those bound to actin through upper 50kD domain, those bound through lower 50kD domain and those bound through both 50kD domains. If the particles with different structures are postulated to represent the time-course of myosin's structural change, we might assume that myosin head proceeds actin filament involving certain "rocking motion".The B-band component of sea urchin axoneme turned out to be a dyncin subspecies. Less

该项目最初旨在确定细胞质Dyncin/微管复合物的三维（3-D）结构及其在滑动运动时的变化。由于难以获得具有高活性的非常新鲜的材料，因此我们将材料切换到Sea-Curchin精子鞭毛，并研究了轴突Dyncin亚种的结构/功能关系。我们还使用了我们最近根据“单分子生理学”的概念开发的重建方法研究了滑动肌球蛋白复合物的3-D结构。根据此类研究的结果，我们讨论了过程运动活动的分子机制，这些运动活动通常在动力蛋白/微观滑动系统中起着至关重要的作用。非常常规肌球蛋白的成员引起了肌动蛋白细丝的过程。肌球蛋白V是其长杆臂部分及其“行走”运动最吸引人的，沿肌动蛋白丝沿36 nm步骤。肌球蛋白V的这种特征行为……更多被归因于支持“倾斜杠杆机制”的有效性的有力证据之一。我们使用截短的杠杆臂和肌球蛋白-VI检查了肌球蛋白V突变体的行为，这些杆自然具有短杠杆部分。有趣的是，它们俩都表现出肌动蛋白的进程运动，其步骤约为36 nm，与原始肌球蛋白V和长杆臂相同。我们检查了这些肌球蛋白头与二十叶骨肌球蛋白突变体的结合方式，其ADP-PI态非常稳定，以通过电子显微镜稳定在肌动蛋白丝中。令人惊讶的是，我们发现所有这些肌球蛋白头似乎都以〜36 nm的常规间隔与肌动蛋白结合，并且仅与细丝的一侧结合。所有这些现象都很难通过“倾斜杠杆假说”来解释，并且可能表明，一个能量的肌球蛋白与肌动蛋白的结合可能会引起特殊的高亲和力（s）（s）〜36 nm的特殊高亲和力（S）〜36 nm距离原始位点。与我们的复制型图像分类为50个类别，分类为50个差异，该结构分类为效应的肌无力的效果，这些结构是易于进行的，这些结构是毫无疑问的毛刺，这些结构是易于进行的，这些结构是在效果上划分的，这些结构是粗糙的，这些结构是粗糙的，这些结构是粗糙的，这些结构是粗糙的，这些结构是粗糙的，这些结构是粗糙的，该结构是毫无用处域，通过较低的50KD域和通过两个50KD域结合的域。如果张贴具有不同结构的颗粒以表示肌球蛋白结构变化的时间顺序，我们可能会假设肌球蛋白头会继续进行肌动蛋白丝，涉及某些“摇摆运动”。肌蛋白轴突的B波段成分被证明是Dyncin subspecies。较少的