Monitoring Redox Dynamics in Living Cells with a Redox-Sensitive Red Fluorescent Protein

Redox signaling and homeostasis are important for all forms of life on Earth. There has been great interest in monitoring redox dynamics in living cells and organisms as a mean to better understand redox biology in physiological and pathological conditions. Herein we report our recent results on the development of a genetically encoded redox-sensitive red fluorescent protein (rxRFP). We first identified a circularly permuted RFP (cpRFP) scaffold, which maintained its autocatalytic fluorescence, from a red fluorescent Ca²⁺ sensor, R-GECO1. We then introduced cysteine residue pairs to the N- and C- termini of the cpRFP scaffold, and subsequently optimized the length and composition of the sequences adjacent to the cysteine residues. From these libraries, we identified rxRFP, showing up to a 4-fold fluorescence increase in the oxidized state compared to the reduced state at pH 7.4. We thoroughly characterized rxRFP in vitro, and expressed it in living mammalian cells to monitor redox dynamics. With its excitation peak at 576 nm and emission peak at 600 nm, rxRFP is one of the first genetically encoded red fluorescent probes that can sense general redox states