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Time-lapse Electron Microscopy with Caged Compounds

笼状化合物的延时电子显微镜

基本信息

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

项目摘要

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.

采用新研制的液氦冷冻系统，分析了P^3-1-（2-硝基苯基）ATP乙酯（笼式ATP）光解后肌凝蛋白亚片段1 （S1）与肌动蛋白丝的相互作用。肌动蛋白和S1（各100muM）在室温下，在5 mM笼状ATP存在下形成绳状双螺旋结构，具有刚性特征。光解15ms后，绳状双螺旋部分解体。在光解后35 ms内，附着在肌动蛋白丝上的S1数量逐渐减少，在35 ~ 200 ms内没有变化。ATP耗尽后，绳状双螺旋重组。考虑到最近对肌动球蛋白动力学的分析，我们得出结论，在25-200 ms时在肌动蛋白丝上观察到的大多数S1是所谓的“弱结合S1”（S1）。ATP或s1， adp。Pi)，并且利用我们新开发的冷冻系统可以在电子显微镜下捕获与肌动蛋白丝快速结合-解离平衡的弱结合S1。这使我们能够直接比较弱键和强键S1的构象。在深蚀刻复制技术的分辨率范围内，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中都没有表现出任何积极的协同性。通过比较发现，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的附着角分布较宽，从垂直于细丝的角度倾斜较小。这些结果讨论了在ATP存在下肌动蛋白-肌球蛋白相互作用的分子机制。