With this article, we will review and outline steps that are required to routinely determine the energetics of reactions in biomolecules in crystal and solution with newest X-ray sources. Kinetic data from time-resolved experiments on short time-scales must be interpreted in terms of chemical kinetics and tied to existing time-resolved experiments on longer time-scales. With the advent of fourth generation X-ray sources, new opportunities emerge to investigate structure and dynamics of biological macromolecules in real time in crystals and potentially from single molecules in random orientation in solution. ![]() With recent technological advances at synchrotrons, it is feasible to rapidly collect time-resolved crystallographic data at multiple temperature settings, from which barriers of activation can be extracted.
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