Structural Biology Group

Structure determination of unstable reaction intermediates

p21_caged_reaction.jpgMany (bio)chemical reactions are complex, often involving several reaction intermediates. For a thorough understanding of the reaction mechanism it is necessary to know all steps occurring along the reaction coordinate the kinetics of their interconversion, their equilibrium constants, and the three-dimensional structures of the intermediates. Crystallography, NMR, and electron microscopy are excellent tools for determining three-dimensional structures to atomic resolution, but are generally regarded as static methods, averaging over space and data collection time.

p21_reaction_flow.jpgBecause data acquisition is generally lengthy and intermediates are usually short-lived, their structures cannot normally be determined by conventional approaches. Thus, transition state or substrate analogues, inhibitors and mutants are traditionally used to obtain mechanistic information. In the last two decades, however, crystallographic structure determination of species that are short-lived has become feasible either on ultra-fast time scales ("time-resolved crystallography") or by slowing reactions via manipulation of, for example, temperature, pH, or slow substrates ("kinetic crystallography", "trapping"). Both approaches require rapid and efficient initiation of the reaction in the crystal. We have developed protocols for handling caged compounds in crystallographic studies which opened up investigations of enzymatic systems operating on phosphate-containing substances.