Monitoring Redox Dynamics in Living Cells with a Redox-Sensitive Red Fluorescent Protein
Monitoring Redox Dynamics in Living Cells with a Redox-Sensitive Red Fluorescent Protein
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DOI：
10.1021/ac5041988
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发表时间：
2015-03-03
影响因子：
7.4
通讯作者：
Ai, Hui-wang
中科院分区：
文献类型：
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作者：
Fan, Yichong;Chen, Zhijie;Ai, Hui-wang
氧化还原信号和稳态对地球上所有形式的生命都很重要。作为更好地理解生理和病理条件下的氧化还原生物学的手段，在活细胞和生物体中监测氧化还原动力学已经引起了极大的兴趣。在此，我们报告了我们最近的研究结果的发展，基因编码的氧化还原敏感的红色荧光蛋白（rxRFP）。我们首先从红色荧光Ca 2+传感器R-GECO 1中鉴定出一种环状排列的RFP（cpRFP）支架，该支架保持其自催化荧光。然后，我们将半胱氨酸残基对引入cpRFP支架的N-和C-末端，随后优化了与半胱氨酸残基相邻的序列的长度和组成。从这些文库中，我们鉴定了rxRFP，在pH 7.4下，与还原状态相比，氧化状态下的荧光增加高达4倍。我们在体外彻底表征了rxRFP，并在活的哺乳动物细胞中表达它以监测氧化还原动力学。rxRFP的激发峰位于576 nm，发射峰位于600 nm，是首批可以检测一般氧化还原状态的基因编码红色荧光探针之一。
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.