| Aequorea victoria green fluorescent protein (GFP) and its fluorescent homologs, such as the Discosoma sp. red fluorescent protein (DsRed), exhibit bright, visible fluorescence. The fluorophore results from a spontaneous self-cyclization event involving three consecutive amino acid residues buried within the core of the folded protein. This unique post-translational modification has lead to the increased interest and popularity of these proteins for use as protein localization tags, markers of gene expression, and reporters of environmental conditions within cells, and has prompted the need to better understand chromophore formation, structure, and chemistry to produce fluorescent proteins with improved properties. The goals of this research are to investigate DsRed chromophore structure, chemistry and local neighborhood to gain further insight into the chromophore green-to-red maturation requirements, and to design and create novel protein metal ion binding sites within the green fluorescent protein scaffold to couple the chromophore and metal ion cofactor environments. Overall, we seek to examine chromophore structural chemistry and environment to gain an increased understanding and appreciation for the remarkable fluorescent chromophores of DsRed and GFP. Our discoveries provide a better understanding of chromophore structural chemistry and environment, and should prove useful in the ongoing effort to create novel or improved fluorescent proteins that can be used as molecular tools for cutting edge investigations into important biological processes. |
