CAN LAUE CATCH MAXWELL - OBSERVATION OF SHORT-LIVED SPECIES BY LAUE X-RAY CRYSTALLOGRAPHY

Now that the Laue method has been established as a tool for protein crystallography, the main problem involved in any prospective time-resolved X-ray diffraction study is one of chemistry. The reaction or process in question must be initiated on a timescale that is fast compared with its kinetics. For most biochemical events photochemistry is the most suitable trigger. but not all substrates can be caged for photochemical release. This problem can be solved by the novel strategy of caging the enzyme with a photoreversible covalent inhibitor. The logic of this method will be discussed and its application to a time-resolved study of the reaction of a suicide substrate with the protease gamma chymotrypsin shown. The question of real-time crystallographic 'movies' of enzymatic reactions can now be considered. It seems likely that following a reaction in real time in a single experiment will be very difficult if not impossible in most cases, in part because even a synchronized process will rapidly become asynchronous in a protein crystal, and also because it will be very difficult to know exactly what species one is observing at any instant unless one has extremely high resolution. It seems that the best use of the Laue technique will be to study unstable species that can be accumulated in the crystal under defined conditions for short periods of time. An entire reaction sequence can then be obtained as a series of individual steps, each of which is obtained from a separate experiment.