Maturation efficiency, trypsin sensitivity, and optical properties of Arg96, Glu222, and Gly67 variants of green fluorescent protein.

Spontaneous chromophore biosynthesis in green fluorescent protein (GFP) is initiated by a main-chain cyclization reaction catalyzed by the protein fold. To investigate the structural prerequisites for chromophore formation, we have substituted the conserved residues Arg96, Glu222, and Gly67. Upon purification, the variants can be ordered based on their decreasing extent of chromophore maturation according to the series EGFP, E222Q, R96K, G67A, and R96M.

The crystal structure of the Y66L variant of green fluorescent protein supports a cyclization-oxidation-dehydration mechanism for chromophore maturation.

The crystal structure of a colorless variant of green fluorescent protein (GFP) containing the Y66L substitution has been determined to 1.5 A. 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.