Synthetic Chromophore Maturation by Split Green Fluorescent Protein (GFP)

Abstract

Green fluorescent protein (GFP) undergoes a self-catalyzed cyclization, dehydration, oxidation reaction sequence to form a fluorescent chromophore in the protein's interior. We have developed a system for studying chromophore maturation and the photophysical properties of novel chromophores in vitro. Intact GFPs, circularly permutated to locate the interior α-helix at the N-terminus, were expressed with a flexible proteolytic loop inserted on the C-terminal side of the chromophore-containing helix. With trypsin, the helix was severed and subsequently removed by size exclusion chromatography after denaturation. The split GFP was then renatured in the presence of a synthetic peptide which underwent chromophore maturation to acquire fluorescence. Since the complementary helix peptide is fully synthetic we have unprecedented control through specific incorporation of multiple unnatural amino acids. We are studying these effects with a range of spectroscopic techniques including steady state fluorescence, time-correlated and upconversion time-resolved fluorescence in order to better understand the process of chromophore maturation, the photochemistry of the protein, and the kinetics and efficiency of fluorescence reconstitution to inform the strategies for producing more robust in vivo probes.View Large Image | View Hi-Res Image | Download PowerPoint Slide