Cryophotolysis of ortho-nitrobenzyl derivatives of enzyme ligands for the potential kinetic crystallography of macromolecules.

Abstract

Photolabile precursors of biologically active molecules (caged compounds) have been developed to allow temporally and spatially controlled release of bioactive compounds by rapid photolysis. [1] Caged compounds can be used to photochemically trigger an enzymatic reaction within an enzyme crystal and are thus valuable tools for studying protein dynamics at the atomic level by time-resolved X-ray crystallography. [2] Real-time-resolved X-ray crystallographic studies of fast biological processes have been applied mainly to a series of naturally light-sensitive proteins by using rapid data collection techniques (Laue diffraction) combined with high-intensity synchrotron X-ray beams. [3] However, caged ligands can also be used as photochemical triggers to capture transient structural species within enzyme crystals, provided photofragmentation reactions are fast, synchronous, and homogeneous throughout the crystals. The few successful examples which have been described so far concern enzymes whose reaction rates in the crystal were slow enough to allow both completion of the photolytic reaction and generally of the Laue data collection. [4] Alternatively, enzyme mutants may be produced to further increase the reaction intermediates’ lifetime. [5] Unfortunately, a series of technical pitfalls are associated with this method, including difficulties with the Laue method (essentially sensitivity to crystal disorder) [6]