Time-lapse Electron Microscopy with Caged Compounds

利用新研制的液氦冷冻系统，研究了笼状ATP的P^3-1-(2-硝基苯基)乙酯光解后肌球蛋白亚段1(S1)与肌动蛋白细丝之间的相互作用。在室温下，肌动蛋白和S1(各100微米)在5 mm笼养的ATP存在下形成具有僵硬特征的绳状双螺旋。光解15ms后，绳状双螺旋部分解体。光解后至35ms，附着在肌动蛋白细丝上的S1数目逐渐减少，从35ms到200ms，无更多变化。耗尽三磷酸腺苷后，绳状双螺旋重新形成。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25-200ms观察到的肌动蛋白细丝上的大多数S1都是所谓的弱结合S1(S1.ATP或S1.ADP.PI)，利用我们新开发的冷冻系统，可以用电子显微镜捕捉到与肌动蛋白细丝处于快速结合-解离平衡下的弱结合S1，这使我们能够直接比较弱结合S1和强结合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之间没有显著的构象差异，并且两种类型的S1在与肌动蛋白细丝的结合方面都没有表现出任何正的协同性。近距离比较发现，弱束缚和强束缚的S1具有不同的附着角度。与强结合的S1相比，弱结合的S1具有较宽的附着角分布和较小的垂直于纤维的倾斜度。这些结果的讨论特别参考了在ATP存在的情况下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-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.