Non-Equilibrium Dynamics of a Pump Protein Measured with Multidimensional Single-Molecule Spectroscopy

Many biological processes, from intracellular transport to trans-membrane ion pumping, rely on non-equilibrium conformation dynamics to drive directional processes. Here, we study the dynamics of bacteriorhodopsin during its light-activated, multi-step catalytic cycle that results in vectorial H+ transport across a membrane. Applying single-molecule 2D fluorescence lifetime correlation spectroscopy (sm-2D-FLCS), we are able to monitor the dynamics of several intermediates of the reaction cycle from microseconds to seconds. The nature of the experiment allows forward and reverse transitions to be measured separately, which is crucial in determining the efficiency of protein motor action. Wild-type bacteriorhodopsin shows forward transitions are more favorable by several orders of magnitude, indicating efficient directional motion and successful ion transport. In contrast, common protein mutants that destabilize the H+ transfer chain are shown to have near identical forward and reverse dynamics, consistent with bidirectional H+ transport and no motor efficiency.