W0101

Proteins are engaged in a myriad of tasks that are essential to life. To understand in mechanistic detail how proteins function, it is crucial to know the time ordering of events that give rise to their designed (or modified) function. Myoglobin (Mb), a ligand-binding heme protein, has long served as a model system for investigating ligand transport and binding in proteins. The dynamics of ligand escape from various mutants of MbCO have been investigated using femtosecond time-resolved polarized IR spectroscopy. The CO residence time in the primary docking site was found to be more than 1000 times shorter in L29F (~140 ps) than in wild-type MbCO. The static structure of this mutant provided few clues regarding the ultrafast ligand escape dynamics. To probe structural dynamics in proteins on ultrafast time scales, we have developed the method of picosecond time-resolved macromolecular crystallography on the ID09B beamline at the European Synchrotron and Radiation Facility. Recent improvements in the methodology have extended the time resolution to 150 ps and the spatial resolution to less than 2 Å. Time-resolved diffraction images from single crystals of photolyzed L29F MbCO were acquired under near ambient conditions at time delays ranging from 100 ps to 3 microseconds. The frame-by-frame structural evolution allows us to literally “watch” the protein as it functions. Conformational changes on the picosecond time scale are far more dramatic than the structural differences between the static carboxy and deoxy states. The correlated motion of CO and several side chains provides a structural explanation for the rapid expulsion of this toxic ligand from the primary ligand docking site. This expulsion triggers ligand migration to numerous internal cavities, suggesting a circuitous path for ligand escape into the surrounding solvent.