Unnatural Amino Acid Mutagenesis of Fluorescent Proteins

Tyrosine 66 of a green fluorescent protein (GFP) was substituted with unnatural amino acids carrying boronate, azido, nitro, and keto substituents. In general, the values of these GFP mutants is blue-shifted relative to that of GFP, and the fluorescence intensity of the boronate variant increases upon oxidation (see scheme). The X-ray crystal structures of the keto and boronate GFP mutants provide explanations of their altered fluorescence properties.