GFP chromophore photophysics: ultrafast dynamics and hot ground state cooling in the neutral form

The neutral GFP chromophore is the photoactive form in many photoconvertible and photoswitchable fluorescent proteins, yet experimental characterisation of its ultrafast dynamics requires clear assignment of the associated transient signals. Defining these dynamics is important for understanding how protonation state tunes the intrinsic photophysics of GFP chromophores. Using complementary ultrafast electronic and vibrational spectroscopies supported by explicit-solvent calculations and spectral simulations, we show that the neutral chromophore (called the protonated state) and a pre-twisted derivative relax barrierlessly on a sub-500 fs timescale via a Z-E isomerisation pathway. This relaxation proceeds with minimal involvement of the phenyl-ring torsion coordinate that is central to the photophysics of the deprotonated, anionic chromophore. Although the internal conversion pathway passes through a twisted charge-transfer region, there is no distinct intermediate charge-transfer state in solution; instead, the dominant picosecond transients arise from cooling of a hot ground-state product. Explicit solvation calculations reveal that solvent stabilisation brings the crossing region into close proximity with the twisted coordinate, bypassing a metastable twisted charge-transfer intermediate. The combined ultrafast electronic and vibrational spectroscopy and computational modelling strategy provides a practical framework for distinguishing hot ground state cooling from excited-state intermediates when interpreting picosecond signals in fluorescent protein photoswitches and other photoisomerisable molecules.