New Insights on the Mechanism of Cyclization in Chromophore Maturation of Wild-Type Green Fluorescence Protein: A Computational Study

Abstract

Cyclization is the first step in the chromophore maturation process of the green fluorescent protein (GFP). In our previous paper [J. Phys. Chem. B 2012, 116, 1426-1436], the results of molecular dynamics simulation suggested the possibility that the amide nitrogen atom of Gly67 attacks the carbonyl carbon of Ser65 directly to complete the cyclization process (one-step mechanism). In this paper, density functional theory (DFT) and quantum mechanical/molecular mechanical (QM/MM) calculations were undertaken to study this step reaction in detail. Three cluster model systems (model A, model B, and model C) and large protein system were set up to investigate the cyclization process. Our results indicate that the one-step mechanism only exists in the two minimum models. However, in model C and the large protein system, the cyclization mechanism involves two steps: the first step is proton of Gly67 amide nitrogen transferring to carbonyl oxygen of Ser65, generating protonated amide, which is stabilized by a hydrogen bond interaction with a crystallographic water molecule, and the second step is Gly67 amide nitrogen attacking the carbonyl carbon of Ser65. Arg96 plays an important role in promoting the cyclization. The energy of cyclized product relative to reactant is about 10.0 kcal/mol endothermic, which is in line with the experimental results.

Molecular Modeling Clarifies the Mechanism of Chromophore Maturation in the Green Fluorescent Protein

“…The highest computed barrier at the INT 3 → INT 4 (dehydration) step is 17 kcal/mol, and the highest barrier for cyclization ( REAG → INT 1) is 13 kcal/mol. The latter value is more than twice lower than the estimates of previous computational studies − assuming a direct attack of N 67 on C 65 and a simultaneous (or multistep) transfer of proton (H 67 ) from N 67 to O 65 . In this work, we found another reaction route for cyclization with a reasonable energy barrier.…”

“…Prompted by the calculation results they put forward an alternative suggestion, that dehydration of Tyr66 to dehydrotyrosine (removal of a proton from C α66 ) occurs prior to cyclization, but this hypothesis was not supported by the experiments. Ma et al explored ,, several options to model the cyclization step for wt-GFP: they used the approach of a quantum-chemical cluster and the QM/MM ONIOM method . Molecular models were constructed on the basis of crystal structure PDB ID 2AWJ; the original wt-GFP residues were restored, and the structure was refined by using molecular dynamics and quantum-chemical calculations.…”

“…In particular, the authors considered the mechanisms studied by Siegbahn et al as well as the original suggestions, most of which were rejected by the authors due to the computed high-energy barriers. The most recent paper proposed a two-step mechanism according to which, first, a proton on the amide nitrogen of Gly67 (H 67 in our designation, see Figure ) transfers to O 65 ; second, the amide nitrogen (N 67 ) attacks the carbonyl carbon of Ser65 (C 65 ). The computed energy profile (calculated with the ONIOM­(B3LYP/6-31G**:AMBER) electronic-embedding scheme) shows barriers as high as 33 kcal/mol, which is significantly higher than the experimentally derived activation energy of 21 kcal/mol. , …”

“…This is the first calculation of the entire reaction pathway, including the rate-determining oxidation step. Previous theoretical studies have only attempted to characterize the cyclization − and some aspects of the dehydration steps; however, the corresponding quantum chemical calculations yielded reaction barriers that were too high and inconsistent with the experimental kinetics. As shown below, our simulations lead to results consistent with the available experimental data.…”

Computational Challenges in Modeling of Representative Bioimaging Proteins: GFP-Like Proteins, Flavoproteins, and Phytochromes

“…From the theoretical side, the first complete computational description of all elementary reaction steps resulting in the chromophore’s maturation in GFP was reported in ref . Other computational studies attempted to characterize the cyclization − and some aspects of dehydration and oxidation steps; however, the reported quantum chemical calculations yielded the reaction barriers that were too high and inconsistent with the experimental kinetics data.…”