Protonation states of the chromophore of denatured green fluorescent proteins predicted by ab initio calculations

Green fluorescent proteins (GFPs) are being intensively investigated due to both their unusual optical spectroscopic characteristics and the extraordi nary utility of GFPs as tools in biochemistry, cell biology, and molecular genetics. Recent studies have suggested that the spectrophotometric and flu orescence characteristics of GFPs are controlled through protonation states of the GFP chromophore (p-hydroxybenzylideneimidazolinone). However, of th ree protonation sites in the chromophore, only two have been studied. To un derstand the structural origin of the observed spectrophotometric and fluor escence characteristics of GFPs, employing ab initio methods, we have inves tigated all the possible protonation sites of the chromophore of denatured GFPs under different pH conditions. Our results suggest that the denatured GFP chromophore exists in not just two protonation states, as widely assume d in the literature, bur in five different protonation states that depend o n pH over the range -3.2. to 9.4 as assessed from the predicted pK(a) value s and the self-consistent reaction field continuum calculations of solvatio n employing Schrodinger's Jaguar 3.5 program. The unexpected complexity of the protonation states of the denatured GFP chromophore postulated here may provide a useful starting point Tor a further investigation of the protona tion states of the intact GFP chromophore responsible for the experimentall y observed UV absorption and fluorescence emission properties of structural ly intact GFPs.