Molecular modeling clarifies the mechanism of chromophore maturation in the green fluorescent protein

B.L. Grigorenko, A.I. Krylov, and A.V. Nemukhin
J. Am. Chem. Soc. 139, 10239 – 10249 (2017)

We report first complete theoretical description of the chain of elementary reactions resulting in chromophore maturation in the green fluorescent protein (GFP). All reaction steps including cyclization, dehydration and oxidation are characterized at the uniform quantum mechanics/molecular mechanics (QM/MM) computational level using density functional theory in quantum subsystems. Starting from a structure of the wild-type protein with the non-cyclized Ser65-Tyr66-Gly67 tripeptide, cyclization and dehydration reactions were modeled. Next, molecular oxygen was added to the system and oxidation reaction was modeled resulting in the mature protein-bound chromophore. Computationally derived structures of the reaction product and several reaction intermediates were compared to the relevant crystal structures showing good agreement between theory and experiment. The highest computed energy barriers at the cyclization-dehydration (17 kcal/mol) and oxidation (21 kcal/mol) steps correlate well with the values derived from the kinetics measurements (20.7 and 22.7 kcal/mol, respectively). Cyclization-dehydration reactions were also modeled for the GFP mutant Ser65Gly and Tyr66Gly (GGG mutant) for which experimental data were also well reproduced. The results of simulations provide a solid basis for predictions of maturation mechanisms in other fluorescent proteins.

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