Nonequilibrium Dynamics of Directional H+ Trans-Membrane Transport

The connection between protein function and equilibrium/nonequilibrium conformation dynamics remains an outstanding problem in biophysics. In the case of motor proteins, nonequilibrium fluctuations are accessed through energy input, either through ATP hydrolysis or photon absorption, to achieve directional transport. Bacteriorhodopsin is a model light-driven motor protein which performs a rudimentary form of photosynthesis through directional H+ transport across a membrane. In this work, we use single-molecule 2-dimensional fluorescence spectroscopy to study the dynamics of bacteriorhodopsin during its multistep catalytic cycle. Using the endogenous retinal chromophore as a probe, we monitor the forward and reverse transitions between several intermediates of the cycle over 5 decades in time, allowing the direct measurement of efficiency and directionality of the proton transport. We show that most conformational transitions are reversible, though directionality of the catalytic cycle is determined by a select few irreversible switch steps. Disruption of the switch steps, either through site-specific mutation or changes in pH, hinders forward transition rates in the catalytic cycle that results in loss of pump directionality.