Understanding GFP Chromophore formation: Refolding Circularly Permutated Green Fluorescent Protein From Inclusion Bodies

Abstract

The Green Fluorescent Protein(GFP) which is derived from the jellyfish Aequorea Victoria has become an invaluable tool in molecular and cellular biology research due to its ability to form a chromophore in vivo and in vitro. Its amino acid components, Ser65, Tyr66, and Gly67 undergo post-translational, autocatalytic reaction to create a five membered ring chromophore. This mature P-hydroxybenzylidene imidazolinone chromophore is formed in exposure to molecular oxygen via an intermolecular reaction. In accelerating the chromophore formation, the mechanisms of chromophore biosynthesis must be understood. Even though several possible mechanisms have been suggested, none have been proven definitive. The chemical ligation of unnatural amino acids onto the chromophore residues will halt the chromophore formation without affecting protein refolding process. For example, adding a methyl group or a proton to the pre-chromophore residues, then halting the chromophore formation at each stage will allow us to examine its structure and mechanism. Because the chromophore is located in the center of the beta-barrel protein, making any changes to the chromophore is difficult to accomplish. Therefore, a permutated variant of GFP has been constructed where N and C termini of the protein are linked and a new N terminus closer to S65, T66, G67 is created. The truncation of N terminus results in GFP where unnatural amino acids can be ligated in place of the residues that comprise the chromophore. Subsequent refolding of this protein will allow us to better understand the mechanisms of chromophore formation.