An understanding of mechanism in biological systems at the molecular level is based both on static structure and on changes in structure. The dynamic aspects of changes in structure during processes such as enzyme catalysis, photocycling in light-sensitive systems, ligand binding and release, and protein unfolding and refolding are critical. However, the rates of interconversion of structures under physiological conditions are large, and the lifetimes of transient structures correspondingly small. The observation of short-lived, transient structures by x-ray crystallographic techniques has therefore depended on the development and application of new techniques in several areas; 1) the rapid acquisition in a time-dependent manner of accurate x-ray data generated by intense synchrotron x-ray sources; 2) the uniform, synchronous, initiation of a structural reaction in the molecules in a crystal; 3) the ability to simultaneously monitor an optical property of the crystal such as absorbance, thus yielding both an optical and an x-ray measure of progress along the reaction coordinate; and 4) the ability to process the x-ray data to yield both the time-dependent difference in structure between that at a particular instant on the reaction coordinate and that of the reactants, and (under favorable circumstances) the time-independent structures through which the system progressively evolves. in turns out that it is substantially easier to study these slow structural reactions in crystals which occur on non- physiological time scales of tens of seconds or minutes. We concentrate here on much faster structural reactions which occur on sub-second time scales, down to times as short as 100 picoseconds when the new Advanced Photon Source comes into operation at Argonne National Laboratory. These factor structural reactions occur in the time scale where heat transfer processes are important. Great care has to be taken in the experimental design to minimize the photochemical and thermal gradients which may be introduced during laser initiation of the reaction, or through irradiation with an intense, polychromatic x-ray beam. Systems to be studied include photoactive yellow protein, which undergoes a photocycle and appears to be a simple, soluble bacterial photosensor; the carbon monoxide complexes of heme proteins such as myoglobin and hemoglobin; and other proteins which are either naturally photosensitive or can be made so by employing molecules such as photoactivatable substrates. Examples of the former include rhodopsin, and of the latter, restrictocin.
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