Quantum chemical modeling of structure and absorption spectra of the chromophore in green fluorescent proteins

As a first step towards modeling the green fluorescent protein (GFP), we have carried out absorption spectra calculations on chromophores of both native and mutant proteins using the semiempirical method INDO/S. A number of protonated and deprotonated states of the GFP fluorophore were considered. We find predicted and observed absorbance energies in very good agreement. Based on a comparison of calculated and experimental absorption spectra, we suggest structures for the ground and excited states of the chromophore of GFP. We assign the absorption maximum of GFP at 477 nm to an H-bonded complex of the zwitterion (O_Y, HN, O_X) involving the phenolic oxygen of Tyr66 and its environment; the nitrogen of the heterocyclic ring is protonated in this complex. Another peak at 397 nm is due to excitation of the corresponding protonated form (HO_Y, HN, O_X)⁺. Calculated transitions in the mutant chromophores (Tyr66→Phe), (Tyr66→Trp), and (Tyr66→His) at 355, 433, and 387 nm, respectively, are close to the corresponding experimental values of 360, 436, and 382 nm, suggesting cationic forms with the nitrogen protonated to be responsible for the absorbance. Proton transfer to or from the phenolic hydroxyl group of the chromophore is shown to be crucial for understanding the absorption and emission spectra of GFP.