In recent years, green fluorescent protein (GFP) technology has revolutionized molecular and cellular biology. GFP has proven extremely useful as a fusion tag for detection of gene products, and as a biosensor for monitoring specific cellular events. Yet, the level of understanding of the underlying mechanism of fluorophore generation is lagging behind the development of novel applications. This project is designed to learn more about the structural and catalytic requirements for chromophore formation in GFP. When the protein folds into its native three-dimensional structure, a spontaneous, autocatalytic peptide backbone cyclization occurs in the protein's interior, initiating a series of chemical events that lead to the generation of the fluorophore. This highly unusual backbone cross-link has been identified only in GFP and in histidine ammonia lyase, though it might be present in a number of other, less well-characterized proteins. In this project, structural and catalytic requirements for GFP chromophore formation will be explored, using techniques such as site-directed mutagenesis, kinetics, and x-ray crystallography. Crystal structures of GFPs without chromophore will be analyzed with respect to preorganization towards chromophore formation. A detailed kinetic investigation of the individual steps leading to fluorescence will be carried out, with the aim of clarifying how backbone cyclization is coupled to protein folding and oxidation. Improved understanding of the factors leading to chromophore formation in GFP will aid in predicting similar unusual post-translational modifications and chemistries in unrelated proteins.
