Functionally relevant protein dynamics monitored by time-resolved quasielastic neutron scattering

Conformational flexibility is often considered as a prerequisite for protein function; however, unambiguous evidence is so far lacking. Previously, dynamics-function correlations were inferred from coincidental secession of protein dynamics and function studied in independent experiments, e.g., upon cooling or dehydration. Using the novel experimental technique of time-resolved quasielastic neutron scattering with in situ light excitation, we are now able to directly examine the modulation of picosecond protein dynamics during the functional cycle of the prototypical membrane protein bacteriorhodopsin (BR) in real time. These experiments are performed using native purple membranes hosting this light-driven vectorial proton pump equilibrated at specific hydration levels provided by D₂O vapors. We find that actinic light energy initiating protein function is converted into excess fluctuations of the protein within ∼150 μs, which subsequently decay with a time constant of 250 ± 50 μs. The latter decay time is very similar to the rise time of 280 μs observed for the buildup of the M2 intermediate, a late substate of the M photocycle intermediate characterized by deprotonation of the retinal Schiff base, as observed by time-resolved absorption difference spectroscopy. The M intermediate can be subdivided into M1, which precedes, and M2, which follows a major global structural change of BR. We thus conclude that the excess fluctuations of the protein decay along with a global structural change between the M1 and the M2 intermediates of BR. This reveals that light-modulated protein dynamics are directly involved in functionally relevant processes by promoting the global structural changes in BR. Thus, our findings challenge the simple model of picosecond fluctuations acting as passive "lubricating grease" for structural changes, but suggest a more active role as an integral part of the energy conversion mechanisms during the photocycle of BR as well as for the function of enzymes in general.