Time‐resolved X‐ray crystallography: uncovering reaction intermediates in biochemical processes

Abstract

X-ray crystallography plays a fundamental role in modern biochemistry and molecular biology, providing the structural basis for understanding the functionality of biological macromolecules. Indeed, biochemistry and molecular biology textbooks greatly benefit from the use of three-dimensional structures to address general concepts and to illustrate specific aspects of the biochemical processes. Structural analyses of model systems such as serine proteases are widely used to illustrate the molecular bases of key biochemical events, including protein-protein recognition, catalysis and enzyme regulation [ 1,2]. Moreover, the comparative analysis of the three-dimensional structures of deoxygenated and oxygenated hemoglobin, in the absence and presence of modulators, has provided a structure-based framework for a rational approach to complex phenomena such as allostery [3-51. Remarkably, about five new three-dimensional structures are deposited every day in the Protein Data Bank (http://www.rcsb.org/pdb), underlining the potentiality and the educational role of crystallography in biochemistry and molecular biology teaching. The biological activity of macromolecules is generally characterized by rapid (4 1 s) structural changes. However, due to the long time average represented by X-ray crystal structures (X-ray diffraction data are collected over intervals of several minutes to hours), the use of this technique in describing transient biochemical processes was, until recently, very limited. From this point of view, the development of time-resolved multiwavelength Laue crystallography was a major breakthrough, since it allows one to follow the time course of biochemical processes, through the related three-dimensional struc-