N/C Terminal Relocation, Truncation, and Native Chemical Ligation; Accessing the Chromophore of Green Fluorescent Protein

Abstract

The Green Fluorescent Protein (GFP), derived from the marine organism Aequorea Victoria, is a vital imaging tool in cellular and molecular biology. The four hour, autocatalytic, post-translational reaction of residues Ser65, Tyr66, and Gly67 into the mature p-hydroxybenzylidene imidazolinone chromophore is not completely understood. To better understand the multi-step chromophore formation process, a permutated variant of GFP has been constructed in which the existing N and C termini of the protein are linked and a new N terminus is created closer to the tripeptide chromophore region. Initial spectroscopic data on this variant of GFP show that it is highly similar to wild-type GFP. Importantly, N-terminal truncation of the residues that comprise the chromophore result in a form of GFP to which short peptides can be ligated allowing the incorporation of isotopically labeled and unnatural amino acids into the chromophore. Refolding of the ligated protein and subsequent analysis will then elucidate the steps in chromophore formation. An understanding of this mechanism may then provide insight into improving the rate of chromophore formation.