VIBRATIONAL ACTIVITY AND PHOTOISOMERIZATION PATHS OF THE GREEN FLUORESCENT PROTEIN CHROMOPHORE: VACUO AND SOLVENT EFFECTS BY QM-COMPUTATIONS

The Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria has become a useful tool in molecular biology and biochemistry. Most of these applications take advantage of the highly fluorescent chromophore generated auto-catalytically via post-translational internal cyclization, dehydration and oxidation of the Ser65-Tyr66-Gly67 tri-peptide unit. Vibrational spectroscopy (infrared, Raman and resonance Raman spectra) of GFP and model chromophores in several protonation states has been extensively studied to achieve information about its molecular structure and activity. However, these spectroscopic data have not yet been satisfactorily reproduced and interpreted on the basis of quantum-chemical calculations, and a convincing well supported model for the lack of fluorescence in solution vs. the fluorescence observed in the protein is still lacking. For this reason, the objective of this work is to simulate the vibrational structure and the photochemical reaction paths of a realistic GFP chromophore model, the 4’-hydroxybenzylidene-2,3-dimethyl-imidazolinone (HBDI), which includes the complete conjugate system of the real chromophore and has been investigated in several studies in solution. In this work vibrational activities in the infrared, Raman and resonance Raman spectra of the cationic, neutral and anionic forms of HBDI have been obtained from quantum-chemical calculations in vacuo and with the inclusion of solvent effects through the polarizable continuum model (PCM) at different levels of theory (HF, CIS and CASSCF, in conjunction with the 6-31G* and 6-31+G* basis sets). It is found that inclusion of solvent effects improves the agreement with experimental data for all the species considered, but it is crucial to reproduce correctly the vibrational activities of the anionic form and its photochemical reaction paths. Photoisomerization routes for the anionic form in solution have been computed, which account for the ultrafast radiationless decay and lack of fluorescence observed for the chromophore in solution. Finally, a model for the photochemical behaviour of the chromophore in the protein is presented which explains its fluorescence and biological activity.