Electronic excitations of green fluorescent proteins: protonation states of chromophore model compound in solutions

Green fluorescent proteins (GFPs) are widely used as tools in biochemistry, cell biology, and molecular genetics due to their unusual optical spectroscopic characteristics. The spectrophotometric and fluorescence properties of GFPs are controlled by the protonation states and possibly cis-trans isomerization of the chromophore (p-hydroxybenzylideneimidazolinone). In this work, we have investigated electronic structures, liquid structures, and solvent shifts of the three possible protonated states (neutral, anionic, and zwitterionic) and their cis-trans isomerization of a model compound 4'-hydroxybenzylidene-2-methyl-imidazolin-5-one-3-acetate (HBMIA) in aqueous solutions. Our calculated results suggest that HBMIA could adopt both cis and trans conformations in a solution, and it exists in three different protonation states depending on the pH conditions. The absorption spectrum observed in neutral solution is thus assigned to the electronic excitations within the neutral form and the cis isomer of the zwitterionic form, while the absorption band at 425 nm in the basic solution is due to the excitations within the anionic form and the trans isomer of the zwitterionic form. Some technical problems related to the computation of electronic excitations within the HBMIA at the anionic state are also discussed.