Development of a high-dynamic range, GFP-based FRET probe sensitive to oxidative microenvironments.
Development of a high-dynamic range, GFP-based FRET probe sensitive to oxidative microenvironments.
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DOI：
10.1258/ebm.2011.011009
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发表时间：
2011-06-01
期刊：
影响因子：
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通讯作者：
Gaskins HR
中科院分区：
文献类型：
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作者：
Kolossov VL;Spring BQ;Clegg RM;Henry JJ;Sokolowski A;Kenis PJ;Gaskins HR
我们报告了一种新型氧化还原敏感探针的优化，该探针具有增强的动态范围和位置极佳的氧化中点氧化还原电位。目前的工作描述了有助于改善这种基于绿色荧光蛋白 (GFP) 的氧化还原传感器的福斯特共振能量转移 (FRET) 性能的因素。 α-螺旋连接体将 FRET 供体和受体分开，在新探针中得到了扩展，并导致连接体减少的“FRET 关闭”状态下的 FRET 效率降低。出乎意料的是，尽管接头序列较长，但与母体探针相比，新接头氧化“FRET-on”状态下的 FRET 效率有所提高。降低基线“FRET-off”和增加“FRET-on”信号的组合显着提高了探针的动态范围，从而更强有力地区分其还原和氧化的接头状态。 α-螺旋接头内半胱氨酸残基的诱变揭示了第四个（C 端半胱氨酸）的重要性，以及第二个半胱氨酸在形成二硫键以将接头夹紧到高 FRET 氧化状态方面的相对重要性。为了进一步优化氧化还原探针的性能，对放置在改进的氧化还原接头（RL7）相对两端的各种青色荧光蛋白（CFP）/黄色荧光蛋白（YFP）FRET对进行定量比较和交换。我们发现，CyPet/YPet 和 ECFP/YPet FRET 对连接至 RL7 时，不能很好地发挥敏感氧化还原探针的作用，因为它们很容易形成异二聚体，从而破坏 α 螺旋。然而，CyPet 和 YPet（mCyPet 和 mYPet）的单体版本消除了二聚化并恢复了探针的氧化还原敏感性。性能最佳的探针 ECFP-RL7-EYFP 在体外从氧化态转变为还原态时，FRET 效率提高了约六倍。我们确定探针的中点氧化还原电位为-143 ± 6 mV，这是测量哺乳动物细胞氧化室（例如内质网）中谷胱甘肽（GSH/GSSG）氧化还原电位的理想选择。
We report the optimization of a novel redox-sensitive probe with enhanced dynamic range and an exceptionally well-positioned oxidative midpoint redox potential. The present work characterizes factors that contribute to the improved Förster resonance energy transfer (FRET) performance of this green fluorescent protein (GFP)-based redox sensor. The α-helical linker, which separates the FRET donor and acceptor, has been extended in the new probe and leads to a decreased FRET efficiency in the linker’s reduced, ‘FRET-off’ state. Unexpectedly, the FRET efficiency is increased in the new linker’s oxidized, ‘FRET-on’ state compared with the parent probe, in spite of the longer linker sequence. The combination of a lowered baseline ‘FRET-off’ and an increased ‘FRET-on’ signal significantly improves the dynamic range of the probe for a more robust discrimination of its reduced and oxidized linker states. Mutagenesis of the cysteine residues within the α-helix linker reveals the importance of the fourth, C-terminal cysteine and the relative insignificance of the second cysteine in forming the disulfide bridge to clamp the linker into the high-FRET, oxidized state. To further optimize the performance of the redox probe, various cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) FRET pairs, placed at opposite ends of the improved redox linker (RL7), were quantitatively compared and exchanged. We found that the CyPet/YPet and ECFP/YPet FRET pairs when attached to RL7 do not function well as sensitive redox probes due to a strong tendency to form heterodimers, which disrupt the α-helix. However, monomeric versions of CyPet and YPet (mCyPet and mYPet) eliminate dimerization and restore redox sensitivity of the probe. The best performing probe, ECFP-RL7-EYFP, exhibits an approximately six-fold increase in FRET efficiency in vitro when passing from the oxidized to the reduced state. We determined the midpoint redox potential of the probe to be −143 ± 6 mV, which is ideal for measuring glutathione (GSH/GSSG) redox potentials in oxidative compartments of mammalian cells (e.g. the endoplasmic reticulum).