Turning on the Fluorescence from Isolated GFP Chromophore Anions at Cryogenic Temperatures

The chromophore anion derived from the green fluorescent protein is one of the best-studied chromophores in the gas phase, but attempts to measure fluorescence have failed at room temperature. Here we unequivocally show that the chromophore exhibits fluorescence in the gas phase when cooled to low temperatures (<150 K), thereby validating previous hypotheses. The experimental confirmation is enabled by a unique mass-spectroscopy setup, allowing for fluorescence observation near or at the 0-0 transition without inducing heat in the ions upon photon absorption. The low-temperature conditions effectively simulate the restricted motion experienced within the protein, inhibiting internal conversion via a conical intersection along a twist motion coordinate. Fluorescence-excitation experiments at 100 K reveal an absorption-band maximum at 481.6±0.2 nm, while the dispersed fluorescence spectrum shows maximum emission at 483.6±0.5 nm. Remarkably, both values closely resemble those for proteins cooled to 77 K. We estimate that after excitation at the band maximum, radiation is the only pathway back to the ground state. Franck-Condon simulations at the ωB97XD/aug-cc-pVDZ level of theory nicely reproduce the experimental spectra and identify the fluorescent form to be planar, and that an in-plane scissoring mode (80 cm^{-1}) is active for both absorption and emission.