An interesting aspect of the green fluorescent protein (GFP) is its autocatalytic chromophore maturation. Numerous experimental studies have indicated that dehydration is the last step in the chromophore maturation process of wild-type GFP. Based on the crystal structure of wild-type GFP, the mechanism of the reverse reaction of dehydration was investigated by using density functional theory (DFT) in this study. Our results proposed that the dehydration is exothermic. Moreover, the rate-limiting step of the mechanism is the proton on guanidinium of Arg96 transferring to the beta-carbon anion of Tyr66, which is consistent with the experimental observation. (C) 2015 Elsevier B.V. All rights reserved.