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

Abstract

The Green Fluorescent Protein (GFP), from the jellyfish Aequorea Victoria, is a vital imaging tool in cellular and molecular biology. The p-hydroxybenzylidine imidazole chromophore is formed post-translationally within the beta-barrel protein from three centrally located amino acids; S65, Y66, and G67. In order to probe the mechanism of chromophore formation and facilitate the introduction of unnatural amino acids into the chromophore, a permutated variant of GFP was constructed in which the existing N- and C- termini of the protein are linked and a new N-terminus is created close 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 permuted GFP protein resulted 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. A novel and efficient method for the generation of such a truncation variant under the partially denaturing conditions was developed. The production of thioester peptides for ligation to the truncated GFP variant using Fmoc solid phase synthesis was explored and successfully implemented. Ligation reactions of the bacterially expressed truncation GFP variant to the synthetically generated thioester peptides under denaturing conditions were refined using existing methodology and de novo folding of the resultant ligation product was carried out. Modified unnatural amino acids and dipeptides were synthetically generated and incorporated into the thioester peptide to explore formation of the chromophore. In order to probe the excited state proton transfer mechanism essential for the fluorescence of GFP, a fluorinated tyrosine substituted into the Y66 position that lowers the pKa of the resulting chromophore phenol was biosynthetically generated and synthetically protected for introduction into a thioester peptide by solid phase synthesis. Further studies using the techniques developed will allow novel chromophores to be studied inside the protein matrix. Lastly, this general methodology may be extended to other protein systems, thereby expanding the chemical space within a protein system beyond the genetically encoded amino acid set.