Theoretical study of the mechanism of peptide ring formation in green fluorescent protein

Density functional calculations using hybrid functionals (B3LYP) have been performed to study the mechanism of peptide ring formation in green fluorescent protein (GFP). Several different chemical models were used ranging from a minimal model of the ring formation to a model including the full side chains of the groups involved in forming the peptide ring. The surrounding protein was described using a dielectric cavity model. The previously most accepted mechanism was found to lead to an endothermic cyclization of about 10 kcal/mol, independent of chemical model used. The formation of the required dihydro‐imidazolone intermediate was found to be even more endothermic with 16–18 kcal/mol. In contrast, another mechanism where the dehydration of residue 66 precedes cyclization was found to be exothermic by 1.9 kcal/mol and to go over an endothermic intermediate of only 6.7 kcal/mol. Correcting these results using the more accurate G2‐M scheme leads to an intermediate with an energy of only +3.7 kcal/mol and an overall exothermicity of 4.7 kcal/mol. Possible transition states involving proton transfer steps were also investigated. Comparisons are made to the similar and more well‐known deamination reaction of Asn‐Gly sequences in peptides, for which good agreement is obtained with experiments. The results are discussed with respect to available experiments. © 2001 John Wiley & Sons, Inc. Int J Quant Chem 81: 169–186, 2001