Green fluorescent protein (GFP), the most widely used fluorescent protein for in vivo monitoring of biological processes, is known to undergo photooxidation reactions. However, the most fundamental property underpinning photooxidation, the electron detachment energy, has only been determined for the deprotonated GFP chromophore in the gas phase. Here, we develop a methodology for calculating the electron detachment energy (VDE) of the GFP chromophore in aqueous solution and, for the first time, determine the first four VDEs, which are consistent with the experimental data.
The first VDE are calculated using two developed approaches - the hybrid DFT/EFP/MD and XMCQDPT2/SA(10)-CASSCF(14,14)/EFP//DFT/EFP/MD methods. The vertical excitation energies (VEEs) of the first seven excited states of the GFP chromophore in aqueous solution are obtained using XMCQDPT2/SA(10)-CASSCF(16,14)/(aug)-cc-pVDZ/EFP calculations.
We show that the first VDE strongly depends on the system size as it relies on the accuracy of the absolute energy estimation of long-range interactions between the negatively charged chromophore and polar water molecules. It converges for a system with 12500 water molecules (R = 40 Å). The calculated VDE is 6.9 eV. Notably, the first VDE is more than double that of the deprotonated chromophore in vacuo (2.7 eV) as a result of solvent stabilization of the anion. At the same time, the VDE of the GFP protein is similar to that in aqueous solution and equals 7.1 eV. Although the VEE of the first electronically excited singlet state of the protein in its anionic form is very similar to that of the deprotonated chromophore in vacuo, the VDE and the pattern of higher lying electronically excited states in the protein are very similar to those of the deprotonated chromophore in aqueous solution. We also show that higher-lying excited states of the solvated GFP chromophore may act as a gateway for electron transfer processes in the condensed phase, in particular those that are excited shape resonances with respect to the quasi-continuum of the solvated electron.
This work is supported by the Russian Foundation for Basic Research (grant no. 20-33-90183). The research is carried out using the equipment of the shared research facilities of HPC computing resources at Lomonosov Moscow State University as well as the local resources provided through the Lomonosov Moscow State University Program of Development.