Dynamic Stokes shift in green fluorescent protein variants

Solvent reorganization around the excited state of a chromophore leads to an emission shift to longer wavelengths during the excited-state lifetime. This solvation response is absent in wildtype green fluorescent protein, and this has been attributed to rigidity in the chromophore's environment necessary to exclude nonradiative transitions to the ground state. The fluorescent protein mPlum was developed via directed evolution by selection for red emission, and we use time-resolved fluorescence to study the dynamic Stokes shift through its evolutionary history. The far-red emission of mPlum is attributed to a picosecond solvation response that is observed at all temperatures above the glass transition. This time-dependent shift in emission is not observed in its evolutionary ancestors, suggesting that selective pressure has produced a chromophore environment that allows solvent reorganization. The evolutionary pathway and structures of related fluorescent proteins suggest the role of a single residue in close proximity to the chromophore as the primary cause of the solvation response.