Time-lapse Electron Microscopy with Caged Compounds

笼状化合物的延时电子显微镜

• 批准号: 04558034
• 负责人: TSUKITA Shoichiro
• 金额: $ 4.99万
• 依托单位: National Institute for Physiological Sciences
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04558034/
• 关键词: muscle; caged compounds; rapid freezing; liquid helium; platelet; electron microscopy; IP3; temporal resolution; caged化合物; ケージドATP; 急速凍結法; アクチン; ミオシン; 紫外線; ミリ秒

The interaction between myosin subfragment 1 (S1) and actin filaments after the photolysis of P^3-1-(2-nitrophenyl)ethyl ester of ATP (caged ATP) was analyzed with a newly-developed freezing system using liquid helium. Actin and S1 (100muM each) formed a rope-like double helix characteristic of rigor in the presence of 5 mM caged ATP at room temperature. At 15 ms after photolysis, the rope-like double helix was partially disintegrated. The number of S1 attached to actin filaments gradually decreased up to 35 ms after photolysis, and no more changes were detected from 35 to 200 ms. After depletion of ATP, the rope-like double helix was reformed. Taking recent analyzes of actomyosin kinetics into consideration, we concluded that most S1 observed on actin filaments at 25-200 ms are so called "weakly-bound S1" (S1.ATP or S1.ADP.Pi) and that the weakly-bound S1 under a rapid association-dissociation equilibrium with actin filaments can be captured by electron microscopy by means of our newly-developed freezing system.This enabled us to directly compare the conformation of weakly- and strongly-bound S1. Within the resolution of deep-etch replica technique, there were no significatn conformational differences between weakly- and strongly-bound S1, and neither types of S1 showed any positive cooperativity in their binding to actin filaments. Close comparison revealed that the weakly- and strongly-bound S1 have different angles of attachment. As compared to strongly-bound S1, weakly-bound S1 showed broad distribution of attachment angle and a decreased tilt from the perpendicular to the filaments. These results are discussed with special reference to the molecular mechanism of acto-myosin interaction in the presence of ATP.

使用新开发的液氦冷冻系统分析 ATP（笼状 ATP）光解后肌球蛋白亚片段 1 (S1) 和肌动蛋白丝之间的相互作用。肌动蛋白和 S1（各 100μM）在室温下，在 5 mM 笼状 ATP 存在下形成具有严格特征的绳状双螺旋。光解后15毫秒，绳状双螺旋部分解体。光解后35毫秒内，附着在肌动蛋白丝上的S1数量逐渐减少，从35到200毫秒没有检测到更多变化。 ATP耗尽后，绳状双螺旋被重新形成。考虑到最近对肌动球蛋白动力学的分析，我们得出的结论是，在 25-200 ms 时在肌动蛋白丝上观察到的大多数 S1 都是所谓的“弱结合 S1”（S1.ATP 或 S1.ADP.Pi），并且与肌动蛋白丝快速缔合-解离平衡下的弱结合 S1 可以通过我们新开发的冷冻技术通过电子显微镜捕获 这使我们能够直接比较弱结合和强结合 S1 的构象。在深蚀刻复制技术的分辨率内，弱结合和强结合的 S1 之间没有显着的构象差异，并且两种类型的 S1 在与肌动蛋白丝的结合方面都没有表现出任何正协同性。仔细比较发现，弱结合和强结合的 S1 具有不同的附着角度。与强结合的 S1 相比，弱结合的 S1 显示出广泛的附着角分布和与细丝垂直方向的倾斜度减小。这些结果的讨论特别涉及 ATP 存在下肌动球蛋白相互作用的分子机制。

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