Enzyme transient state kinetics in crystal and solution from the perspective of a time-resolved crystallographer

With recent technological advances at synchrotrons [Graber et al., J. Synchrotron Radiat. 18, 658-670 (2011)], it is feasible to rapidly collect time-resolved crystallographic data at multiple temperature settings [Schmidt et al., Acta Crystallogr. D 69, 2534-2542 (2013)], from which barriers of activation can be extracted. With the advent of fourth generation X-ray sources, new opportunities emerge to investigate structure and dynamics of biological macromolecules in real time [M. Schmidt, Adv. Condens. Matter Phys. 2013, 1-10] in crystals and potentially from single molecules in random orientation in solution [Poon et al., Adv. Condens. Matter Phys. 2013, 750371]. Kinetic data from time-resolved experiments on short time-scales must be interpreted in terms of chemical kinetics [Steinfeld et al., Chemical Kinetics and Dynamics, 2nd ed. (Prentience Hall, 1985)] and tied to existing time-resolved experiments on longer time-scales [Schmidt et al., Acta Crystallogr. D 69, 2534-2542 (2013); Jung et al., Nat. Chem. 5, 212-220 (2013)]. 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. In eight sections, we aim to describe concepts and experimental details that may help to inspire new approaches to collect and interpret these data. (C) 2014 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 Unported License.