Femtosecond Time-resolved Laue Crystallography: Using and ERL to Watch Proteins Function on the Chemical Time Scale
Philip Anfinrud
Laboratory of Chemical Physics, NIH/NIDDK
A detailed mechanistic understanding of how proteins function requires knowledge not only of their static structures, but also how their structures evolve as they execute their designed function. We recently developed the technique of picosecond time-resolved Laue crystallography [1,2] and used this method to visualize, with near-atomic resolution, structural changes in myoglobin as it evolves from the carboxy to the deoxy state. This transition was triggered with picosecond laser pulses and probed with picosecond X-ray pulses. This “pump-probe” approach recovered time-resolved diffraction “snapshots” whose corresponding structures were stitched together into movies that unveiled protein structure changes and ligand migration in real time. The driving force for this structural transition resides in a photolysis-induced displacement of the heme iron, which moves approximately 0.30-0.36 Å in the proximal direction. Correlated displacements of the heme, the protein backbone, and other side chains are evident throughout the protein at 100 ps (see Fig. 1), the earliest time accessible to synchrotron X-ray pulses. To witness how this piston-like iron motion drives the structural changes observed in this snapshot would require significantly improved time resolution. Hard X-ray pulses generated by an ERL are expected to be about 1000 times shorter than those generated by synchrotrons. Such short pulses would provide an unprecedented opportunity to investigate protein function at near-atomic resolution on the chemical time scale. However, to effectively exploit X-ray pulses from an ERL, there are numerous issues that require careful consideration. These issues will be discussed.
Figure: Experimentally determined electron densities within a 6.5-Å thick slice through the myoglobin molecule before (magenta) and 100 ps after (green) photolysis. Where both densities overlap, they blend to white. The white stick model corresponds to the unphotolyzed structure and is included to guide the eye. The direction of molecular motion follows the magenta to green color gradient. Three large scale displacements near the CO-binding site (large arrows) are accompanied by more subtle correlated rearrangements throughout the entire protein (small arrows; not drawn to scale).
References:
1. F. Schotte, M. Lim, et al.; "Watching a Protein as it Functions with 150-ps Time-resolved X-Ray Crystallography"; Science 300(5627): 1944-7 (2003)
2. F. Schotte, J. Soman, et al.; "Picosecond Time-resolved X-Ray Crystallography: Probing Protein Function in Real Time"; J. Struct. Biol. 147(3): 235-46 (2004)
