Experimental studies of the photophysics of gas-phase fluorescent protein chromophores

To better understand the photophysics of photoactive proteins, the absorption bands of several gas-phase biomolecules have been studied recently at the electrostatic heavy-ion storage ring ELISA by a photofragmentation technique. In the present paper we discuss the involved photophysics and photochemistry for protonated and deprotonated model chromophores of the Green Fluorescent Protein (GFP) and the Red Fluorescent Protein (RFP). We show specifically that the delayed dissociation after photoabsorption can be understood in terms of a thermally activated process of the Arrhenius type. The rate of dissociation as a function of time after laser excitation is modeled in a calculation which is based on calculated heat capacities of the chromophores. Absorption of only one photon is enough to dissociate the deprotonated GFP chromophore on a time scale of milliseconds whereas absorption of two to three photons occurs for other chromophore ions. The difference is attributed to different activation energies, pre-exponential factors and locations of the absorption bands. We obtain activation energies for the dissociation that are of the order of 1-3 eV. Collision-induced dissociation experiments were performed to help identifying the fragmentation channels. Loss of methyl is found to be the dominant fragmentation channel for the deprotonated GFP chromophore and is also likely to be important for the protonated GFP chromophore at high temperatures. Other channels are open for the RFP chromophores. For the deprotonated RFP chromophore there is evidence that dissociation occurs through a non-trivial dissociation with substantial rearrangement.