Protein-Bath Coupling of an Internal Reaction Coordinate at Intermediate Time Scales.

Thermally activated barrier-crossing processes are central to protein reaction kinetics. A determining factor for such kinetics is the extent to which the protein's motions are coupled to the surrounding bath. It is understood that slow large-scale conformational motions are strongly coupled to the environment, while fast librational motions are uncoupled. However, less is known about protein-bath coupling of reaction coordinates located on the interior of a protein and with dynamics on intermediate time scales. In this work, we use single molecule 2D fluorescence lifetime correlation spectroscopy to study the microsecond chemical reaction occurring in the chromophore pocket of eGFP. The equilibrium reaction involves a dihedral rotation of a glutamic acid residue and a rearrangement of the local hydrogen-bonding network surrounding the endogenous chromophore, with no accompanying large-scale conformational changes. We observe that the internal chemical reaction is coupled to the solvent viscosity, though the scaling deviates from Kramers' behavior. We attribute this deviation to the internal friction of the protein, which weakens the protein-solvent coupling at high viscosity and intermediate time scales.