Illuminating Biology With Engineered Fluorescent Proteins

Fluorescent proteins (FPs) emerged in the mid-90s as a powerful tool for life science research. Among its numerous applications, FPs is best known as reporter genes to study the localization and dynamics of proteins. In this dissertation, we explore new uses of FPs as genetically encoded fluorescent probes to image cellular chemical species. In chapter 2, we incorporated of p-azidophenylalanine (pAzF) into circularly permuted GFP (cpGFP), which gave rise to the first genetically encoded fluorescent probe for H2S. Further engineering of the cpGFP scaffold, followed by directed evolution, has resulted in an improved fluorescent probe for H2S–hsGFP. In chapter 3, we incorporated p-boronophenylalanine (pBoF) into different cpGFP templates, which serendipitously resulted in pnGFP, the first genetically encoded fluorescent probe for the selective detection of peroxynitrite (ONOO–). In chapter 4, we examined the molecular mechanism underlying the unusual chemoselectivity of pnGFP towards ONOO– over H2O2 by using site-directed mutagenesis, X-ray crystallography, 11B NMR, and computational simulation studies. Surprisingly, the data collectively support an sp3-hybridized boron in pnGFP through a water bridge mediated N-B interaction between His 9 and the boronic acid moiety on the chromophore. In chapter 5, we described the engineering and biological application of “G-CaMP”-type indicators for zinc ion (Zn2+). Insertion of a naturally evolved zinc-binding domain into the β-barrel of single green or red FP, followed by directed protein evolution and functional screening, we identified single-FP based green and red Zn2+ indicators, ZnGreen and ZnRed. These optically compatible indicators enabled dual color imaging of Zn2+ at single cell resolution. We further demonstrated the use of ZnGreen1 in imaging of glucose-stimulated Zn2+ secretion in pancreatic β cells. Finally, in chapter 6, we laid out challenges in the field and provide future outlooks on possible new directions