Kinetic crystallography

Structural intermediates along a catalytic pathway are separated by the requirement that an energy barrier must be crossed when proceeding from one intermediate state to the next. Within the field of x-ray crystallography there exist two distinct methodologies for determining the structural changes associated with the build up of a specific reaction intermediate. 

An appealing method is to perform time resolved experiments for which the reaction is initiated at room temperature & rapid X-ray diffraction data collection methodologies are employed so as to capture structural information from the desired intermediate. This approach is technically challenging & requires very well diffracting crystals with low mosaic spread. Nevertheless, impressive technological achievements have been reported using the 100 ps single bunch of the European Synchrotron Radiation Facility, where the groups of Keith Moffat & Michael Wulff have presented time-resolved difference electron density maps for the photodissociation of a myoglobin:carbon monoxide complex [Srajer et al., Science 274, 1726-1729 (1996)] & described light-driven structural changes in photoactive yellow protein [Perman et al.Science 279, 1946-1950 (1998)].

Example of 100 ps time-resolved X-ray diffraction data (figure courtesy of Thomas Ursby): Laue (broad spectrum X-ray beam) diffraction data were collected from a single crystal of myoglobin:carbon monoxide complex at room temperature following photoexcitation by a ns laser [Srajer et al., Science274, 1726-1729 (1996)].

An alternative approach is to use cryogenic temperatures so as to prolong the life-time of an intermediate state of interest to the point where standard monochromatic data collection strategies may be applied. This is possible since, at low temperature, the energy required to cross an energy barrier from one intermediate state to the next is "frozen out". This approach typically yields higher quality X-ray diffraction data than that recovered using the room-temperature time-resolved Laue-diffraction approach, as illustrated by closely related low-temperature studies on the myoglobin:carbon monoxide photodissociation reaction [Schlicting et al., Nature 371, 808-812 (1994)] & photoactive yellow protein [Genick et al., Nature 392, 206-209 (1998)]. The use of low-temperature so as to slow the kinetics & thereby trap a high population of the desired intermediate in 3D protein crystals has coined the phrase "kinetic crystallography".

Example of a kinetic crystallography experiment: a single crystal of bacteriorhodopsin is first frozen to 100 K on a cryo-loop & then illuminated with green light. A portion of the resulting difference electron density (or difference Fourier map) is overlaid on the photograph [Edman et al., Nature 401 822-826(1999)].

Kinetic crystallography comes in various shapes & forms: freeze-trap techniques describe methodology whereby a protein crystal is first frozen & the reaction is then initiated at low temperature; alternatively trap-freeze methodology corresponds to near room temperature (or low temperature) reaction initiation followed by quenching in liquid nitrogen. In both cases the crystal is subsequently mounted within an X-ray beam & monochromatic X-ray diffraction data are collected. Within our group we have applied these techniques to study structural rearrangements in bacteriorhodopsin, sensory rhodopsinIIphotosynthetic reaction centre & elastase.