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 fluorophor e were considered. We find predicted and observed absorbance energies in very good agreement. Based on a comparison of calculated and experi mental absorption spectra, we suggest structures for the ground and ex cited states of the chromophore of GFP. We assign the absorption maxim um 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 ni trogen of the heterocyclic ring is protonated in this complex. Another peak at 397 nm is due to excitation of the corresponding protonated f orm (HOY,HN,O-X)(+). Calculated transitions in the mutant chromophores (Tyr66 --> Phe), (Tyr66 --> Trp), and (Tyr66 --> His) at 355, 433, an d 387 nm, respectively, are close to the corresponding experimental va lues of 360, 436, and 382 nm, suggesting cationic forms with the nitro gen protonated to be responsible for the absorbance. Proton transfer t o or from the phenolic hydroxyl group of the chromophore is shown to b e crucial for understanding the absorption and emission spectra of GFP . (C) 1998 Elsevier Science B.V. All rights reserved.