Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
Antioxid Redox Signal. 2012 Apr 1;16(7):698-704. doi: 10.1089/ars.2011.4251. Epub 2011 Oct 21.
Development of redox-sensing fluorescent proteins (redox probe proteins) have enabled live imaging of the physiological redox state within a cell, generating new strategies for detecting changes in the redox state during developmental, pathogenic, and aging processes. Several of the probe proteins utilize their characteristic redox-sensing segments as linkers in between two fluorophores, where structural alternations of the segments lead to changes in FRET efficiencies across the fluorophores. In this review we summarize two types of novel FRET-based redox probe proteins, namely redox linker (RL)-derived probes and Redoxfluor.
After these FRET-based redox probe proteins were generated, their responsiveness toward redox-related compounds as well as toward reactive oxygen species or reducing stimuli was investigated in vitro. Notably, both the RL-derived probe and Redoxfluor were found to directly respond to the redox state of glutathione, a main redox-formulating compound, showing a promising property for their use in subsequent in vivo analyses. Redoxfluor was not only used for redox sensing in the cytoplasm, but also utilized for assessing the redox state within peroxisomes.
In contrast to "one-fluorophore" redox probes such as roGFP and rxYFP proteins, whose usage has been established and widely expanded to various experimental systems, FRET-based redox probes were invented very recently and their applications to in vivo studies are still in their infancy.
FRET-based redox probes provide novel approaches for redox sensing that are complementary to other methodologies.
氧化还原感应荧光蛋白（氧化还原探针蛋白）的开发使人们能够对细胞内的生理氧化还原状态进行实时成像，为在发育、致病和衰老过程中检测氧化还原状态的变化提供了新策略。一些探针蛋白利用其特征氧化还原感应片段作为两个荧光团之间的连接子，其中片段的结构改变导致荧光团之间的 FRET 效率发生变化。在这篇综述中，我们总结了两种新型基于 FRET 的氧化还原探针蛋白，即氧化还原连接子（RL）衍生探针和 Redoxfluor。
这些基于 FRET 的氧化还原探针蛋白生成后，在体外研究了它们对氧化还原相关化合物以及活性氧或还原刺激的反应性。值得注意的是，RL 衍生探针和 Redoxfluor 都被发现可以直接响应谷胱甘肽的氧化还原状态，谷胱甘肽是一种主要的氧化还原形成化合物，这表明它们在随后的体内分析中具有很大的应用潜力。Redoxfluor 不仅用于细胞质中的氧化还原感应，还用于评估过氧化物酶体中的氧化还原状态。
与已经建立并广泛扩展到各种实验系统的“单荧光团”氧化还原探针（如 roGFP 和 rxYFP 蛋白）不同，基于 FRET 的氧化还原探针是最近才发明的，它们在体内研究中的应用仍处于起步阶段。
基于 FRET 的氧化还原探针为氧化还原感应提供了与其他方法互补的新方法。
