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

• 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
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11480185/
• 关键词: Quick-freeze electron microscopy; Three-demensional erconstruction; Dynein; Microtubule; Actomyosin; Processive movement; コンフォメーション変化; ホットスポット; ディープエッチ・レプリカ法; コンピュータ・シミュレーション; 酢酸ウラニル固定; フリーズ・ディープエッチレプリカ法; 1分子生理学; 構造生物学

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

本研究的主要目的是研究细胞质动力素/微管复合物的三维结构及其在滑动运动中的变化。由于很难获得非常新鲜的高活性材料，我们将材料转换为海胆精子鞭毛，并研究轴丝dyncin亚种的结构/功能关系。我们还研究了滑动肌动球蛋白复合物的三维结构，使用的重建方法，我们最近开发的“单分子生理学”的概念。在此基础上，我们讨论了在动力蛋白/微管滑动系统中起关键作用的进行性运动的分子机制。许多非常规肌球蛋白引起肌动蛋白丝的进行性运动。肌球蛋白-V是最有趣的，因为它的长臂部分和它的“步行”运动，沿着肌动蛋白丝的36 nm的步骤。肌球蛋白V的这种特征性行为 ...更多信息 被认为是支持“倾斜摆臂机构”有效性的最有力证据之一。我们研究了具有截短的α-臂的肌球蛋白-V突变体和天然具有短α-臂部分的肌球蛋白-VI的行为。有趣的是，这两个人都表现出与原始myosin-V相同的肌动蛋白进行性运动，步长为1.36 nm，具有长臂。我们通过电子显微镜检查了这些肌球蛋白头与ADP-Pi状态非常稳定的dystyosteoporosis肌球蛋白突变体一起与肌动蛋白丝的结合方式。令人惊讶的是，我们发现所有这些肌球蛋白头似乎都以大约36 nm的规则间隔与肌动蛋白结合，并且只与细丝的一侧结合。所有这些现象都很难用“倾斜长臂假说”来解释，并可能暗示一个被激发的肌球蛋白头与肌动蛋白的结合可能引起特殊的高亲和力位点根据我们的复制图像，功能性肌球蛋白头部的结构大致分为三类，通过上50 kD结构域与肌动蛋白结合的，通过下50 kD结构域与肌动蛋白结合的，以及通过两个50 kD结构域结合的。如果用不同结构的颗粒来代表肌球蛋白结构变化的时间进程，我们可以假定肌球蛋白头是由肌动蛋白丝进行的，并伴有一定的“摇摆运动”，海胆轴丝的B带成分是一个dyncin亚种。少

项目成果

Konishi,M.: "Structure and enzymatic properties of genetically truncated forms of the water-insoluble glycan-synthesizing glycotransferase from Strepto coccus sobrinus"Journal of Biochemistry. 126(2). 287-295 (1999)

Konishi,M.：“来自链球菌的水不溶性聚糖合成糖转移酶的基因截短形式的结构和酶学特性”生物化学杂志。

Tamano,K.: "Supramolecular structure of Shigella type III secretion machinery : the needle part is changeable in length and essential for delivery of effectors"EMBO Journal. 19(15). 3876-3887 (2000)

Tamano,K.：“志贺氏菌 III 型分泌机制的超分子结构：针头部分的长度是可变的，对于效应物的传递至关重要”EMBO 杂志。

Nishikawa S: "Class VI myosin moves processively along actin filaments backward with large steps"Biochemical and Biophysical Research Communications. 290(1). 311-317 (2002)

Nishikawa S：“VI 类肌球蛋白沿着肌动蛋白丝向后大步地持续移动”《生物化学和生物物理研究通讯》。

Konishi N: "Structure and enzymatic properties of genetically truncated forms of the water-insoluble glucan-synthesizing glucosyltransferase from streptococcus sobrinus"Journal of Biochemistry. 126(2). 287-295 (1999)

Konishi N：“来自远缘链球菌的水不溶性葡聚糖合成葡萄糖基转移酶的基因截短形式的结构和酶学特性”生物化学杂志。

Murayama,T Oba,T. Katayama,E (equal contribution): "Further Characterization of the type 3 ryanodine receptor (PyR3) purified from rabbit diaphragm"Journal of Biological Chemistry. 274(24). 17297-17308 (1999)

村山，T奥巴，T。