The crystal structure of the Y66L variant of green fluorescent protein supports a cyclization–oxidation– dehydration mechanism for chromophore maturation, Biochemistry 43

ABSTRACT: The crystal structure of a colorless variant of green fluorescent protein (GFP) containing the Y66L substitution has been determined to 1.5 Å. Crystallographic evidence is presented for the formation of a trapped intermediate on the pathway of chromophore maturation, where the peptide backbone of residues 65-67 has condensed to form a five-membered heterocyclic ring. The hydroxyl leaving group remains attached to the ring as confirmed by high-resolution electrospray mass spectrometry. The R-carbon of residue 66 exhibits trigonal planar geometry, consistent with ring oxidation by molecular oxygen. Side chain positions of surrounding residues are not perturbed, in contrast to structural results obtained for th