Development, Characterization, and Structural Analysis of a Genetically Encoded Red Fluorescent Peroxynitrite Biosensor

ABSTRACT Boronic acid-containing fluorescent molecules have been widely used to sense hydrogen peroxide and peroxynitrite, which are important reactive oxygen and nitrogen species in biological systems. However, it has been challenging to gain specificity. Our previous studies developed genetically encoded, green fluorescent peroxynitrite biosensors by genetically incorporating a boronic acid-containing noncanonical amino acid (ncAA), p -boronophenylalanine ( p BoF), into the chromophore of circularly permuted green fluorescent proteins (cpGFPs). In this work, we introduced p BoF to amino acid residues spatially close to the chromophore of an enhanced circularly permuted red fluorescent protein (ecpApple). Our effort has resulted in two responsive ecpApple mutants: one bestows reactivity toward both peroxynitrite and hydrogen peroxide, while the other, namely pnRFP, is a selective red fluorescent peroxynitrite biosensor. We characterized pnRFP in vitro and in live mammalian cells. We further studied the structure and sensing mechanism of pnRFP using X-ray crystallography, 11 B-NMR, and computational methods. The boron atom in pnRFP adopts an sp 2 -hybridization geometry in a hydrophobic pocket, and the reaction of pnRFP with peroxynitrite generates a product with a twisted chromophore, corroborating the observed “turn-off” fluorescence response. Thus, this study extends the color palette of genetically encoded peroxynitrite biosensors, provides insight into the response mechanism of the new biosensor, and demonstrates the versatility of using protein scaffolds to modulate chemoreactivity.