Green Fluorescent Protein (GFP), from the jellyfish Aequorea Victoria, is a vital imaging tool in cellular and molecular biology.  In order to probe the mechanism of chromophore formation and facilitate the introduction of unnatural amino acids into the intrinsic chromophore, 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.  Spectroscopic studies demonstrate that this GFP variant closely resembles wild-type GFP.  Following purification from inclusion bodies, the GFP variant efficiently undergoes folding and de novo chromophore formation. N-terminal truncation of the chromophore comprising residues will result in a form of GFP to which short peptides can be ligated, thereby facilitating the incorporation of isotopically labeled and unnatural amino acids into the chromophore.  Additionally, novel chromophores may be studied inside the protein matrix using this method.