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

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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.