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

Abstract

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.

Identification of Factors Complicating Bioluminescence Imaging

“…Dluciferin itself did not cause any PercevalHR fluorescence change(Figure S6A). Also, the bioluminescence reaction of FLuc-D-luciferin did not affect intracellular pH as monitored by pHRFP, a red fluorescent pH biosensor(Figure S6B) 27. It is worthwhile to note that DTZ does not induce fluorescence in live cells.…”

Monitoring intracellular redox metabolism with genetically encoded fluorescent sensors

“…针 对 第 三 类 的 巯 基 类 代 谢 物 , 研 究 者 也 发 展 了 多 种 多 样 的 荧 光 蛋 白 探 针 , 包 括 rxYFP [76] , roGFP 家族 [77~79] , rxRFP [80] 以及特异性检测GSSG/GSH氧化 还原状态的荧光蛋白探针Grx1-roGFP2 [81] , 它们可以原 位、实时地监测单细胞和亚细胞水平的巯基氧化还 原状态. [40] .…”

Live-cell imaging approaches for the investigation of xenobiotic-induced oxidant stress

“…The expansion of the color palette available to analyze oxidant events with genetically-encoded sensors is yet another contribution to the field, as in the case of rxRFP, wherein a cpRFP scaffold was utilized to develop a red fluorescent redox sensor. rxRFP is useful for monitoring the general redox health of the cell, since it has been used to detect GSSG, ${\mathrm{O}}_2^{•-}$, and ONOO− in live-cell studies [180]. Sugiura et al recently reported the development of a group of proteins similar to rxYFP, known as oxidation balance sensed quenching (Oba-Q) proteins [181].…”