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

Abstract

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