Green Fluorescent Proteins (GFP) and GFP-like proteins all undergo an autocatalytic post-translational modification to form a centrally located chromophore. Structural analyses of all the GFP and GFP-like proteins in the protein databank were undertaken to determine the role of the tight-turn, broken hydrogen bonding, Gly67, Glu222 and Arg96 in the biosynthesis of the imidazolone group from 65SYG67. The analysis was supplemented by computational generation of the conformation adopted by uncyclized wild-type GFP. The data analysis suggests that Arg96 interacts with the Tyr66 carbonyl, stabilizing the reduced enolate intermediate that is required for cyclization; the carboxylate of Glu 222 acts as a base facilitating, through a network of two waters, the abstraction of a hydrogen from the alpha-carbon of Tyr66; a tight-turn conformation is required for autocatalytic cyclization. This conformation is responsible for a partial reduction in the hydrogen bonding network around the chromophore-forming region of the immature protein.