A New Rapid Freezing Apparatus for Electron Microscopy

用一种新研制的液氦冷冻系统研究了P^3-1-（2-硝基苯）乙基ATP酯（笼状ATP）光解后肌球蛋白亚片段1（S1）与肌动蛋白丝的相互作用。肌动蛋白和S1（各100 μ M）在室温下存在5 mM笼状ATP时形成具有僵直特征的绳状双螺旋。在光解后15 ms，绳状双螺旋部分解体。附着在肌动蛋白丝的S1的数量逐渐减少到35毫秒后光解，并没有更多的变化被检测到从35至200毫秒。耗尽ATP后。重新形成绳状双螺旋。考虑到最近对肌动球蛋白动力学的分析，我们的结论是，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），并且在与肌动蛋白丝的快速缔合-解离平衡下弱结合的S1可以通过电子显微镜通过我们新的-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之间没有显著的构象差异，并且两种类型的S1在与肌动蛋白丝的结合中均未表现出任何正协同性。通过比较发现，弱结合和强结合的S1具有不同的连接角度。与强结合的S1相比，弱结合的S1显示出较宽的附着角分布，并且从垂直于细丝的方向的倾斜度减小。这些结果进行了讨论，特别是参考在ATP的存在下的肌动蛋白-肌球蛋白相互作用的分子机制。

The interaction between myosin subfragment 1 (S1) and actin filaments after the photolysis of P^3-1-(2-nitropheny1)ethy1 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 analyses 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 significant 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 discussed with special reference to the molecular mechanism of acto-myosin interaction in the presence of ATP.