A serious limitation of the x-ray crystallographic approach to an understanding of macromolecular function has been its inability to reveal changes in structure on a biochemical time scale. Conventional crystallographic studies have revealed essentially static structures, a space average over all molecules in the crystal and a time average over the hours or tens of hours required for data collection. Thus, the structures of functionally crucial, transient intermediates in ligand binding, catalysis and conformational change have had to be inferred from a series of static structures, presumed to be similar to the crystallographically inaccessible intermediates. Accurate inferences have been hard to draw; mechanism is less readily understood than structure. The availability of very intense, polychromatic synchrotron x-ray sources such as CHESS and recent advances in x-ray optics have greatly diminished the time necessary to record an x-ray diffraction pattern from a macromolecular crystal. This time stands at well under one second on film now, with the immediate prospect of a further reduction to roughly 25 msec with focussing optics. We propose to apply our polychromatic, Laue diffraction technique to the development of time-resolved macromolecular crystallography. That is, we will initiate a structural reaction in the crystal, monitor the time-dependent changes in structure factors, and analyze the results in terms of time-dependent structures. Systems to be developed include the thermal unfolding of ribonuclease A, hen egg white and phage T4 lysozymes in response to a rapid temperature jump, at temperatures below and approaching the melting temperature; denaturant-induced unfolding, where a concentration jump is applied in a flow cell; the flash photolysis of carboxymyoglobin; and the synthesis and development of """"""""caged"""""""" substrates, effectors and chelators, modelled on """"""""caged ATP"""""""", and their application to enzymes where a phosphate group is in some way involved, and to calcium binding proteins.
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