Time-resolved 3-dimensional Structural Study of Biological Reactions by Flash Photolysis and Frozen-hydrated Electron Microscopy

本研究的目的是开发所需的技术可视化的构象变化的生物大分子在反应中，使用闪光光解的笼状化合物和冷冻水合电子显微镜。在这个系统中，生物反应是由笼状化合物的闪光光解开始的，并通过在液态乙烷中快速冷冻到融化温度而停止。这项研究是基于首席研究员在英国进行的一系列试点实验。从这些实验中已经认识到，最难解决的问题是由于闪光光解引起的样品的温度升高。这个问题在冷冻水合电子显微镜中最为明显，因为样品是在支撑膜上的非常薄的溶液层（小于0.5 μ m，优选约0.1 μ m），该支撑膜对用于光解的紫外光不透明。这意味着，除非我们能够使支撑膜对光完全反射，否则无法消除温度升高。在这项工作的过程中，我们已经意识到，这个问题比预期的要复杂，因为脉冲激光器（钕-UAG）的闪光持续时间很短，以至于从支撑膜到电子显微镜网格几乎不发生热传导。尽管如此，我们已经成功地进行了实验，其中超过10%的笼状ATP转化为ATP，保持膜蛋白（来自肌浆网的钙ATP酶）分子的规则排列。最有效的是使用银作为支撑膜和激光束对支撑膜的大入射角。因此，似乎有可能以毫秒级的时间分辨率捕获反应中生物大分子的三维结构。

The purpose of this research is to develop the technology required for visualizing the conformation change of biological macromolecules in reactions, using flash photolysis of caged compounds and frozen-hydrated electron microscopy. In this system, biological reactions are started by flash photolysis of caged compounds and stopped by rapid freezing in liquid ethane cooked down to the milting temperature. This research is based on a series of pilot experiments conducted in England by the principal investigator. It has been realized from these experiments that the most difficult problem to be solved is the temperature rise of the specimen due to flash photolysis. This problem is most pronounced with frozen-hydrated electron microscopy, because the specimen is a very thin layer (less than 0.5 mum, preferably about 0.1 mum) of solution on a supporting film that is not transparent to the ultraviolet light used for photolysis. This means that the temperature rise cannot be eliminated unless we can make the supporting film perfectly reflective to the light. During the course of this work, we have realized that this problem is severer than expected because the duration of the flash from the pulse laser(Neodymium-UAG) is so short that heat conductance hardly takes place from the supporting film to the electron microscope grid. Nevertheless, we have succeeded in conducting the experiments in which better than 10% of caged ATP is converted into ATP keeping the regular arrangements of molecules of a membrane protein (calcium ATPase from sarcoplasmic reticulum). Most effective are the use of silver for the supporting film and a large incident angle of the laser beam to the supporting film. Thus it seems possible to capture three-dimensional structures of biological macromolecules in reaction with a millisecond time resolution.