Kolossov Vladimir L, Spring Bryan Q, Sokolowski Anna, Conour John E, Clegg Robert M, Kenis Paul J A, Gaskins H Rex
Institute for Genomic Biology, Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Exp Biol Med (Maywood). 2008 Feb;233(2):238-48. doi: 10.3181/0707-RM-192.
The ability to sense intracellular or intraorganellar reduction/oxidation conditions would provide a powerful tool for studying normal cell proliferation, differentiation, and apoptosis. Genetically encoded biosensors enable monitoring of the intracellular redox environment. We report the development of chimeric polypeptides useful as redox-sensitive linkers in conjunction with Förster resonance energy transfer (FRET). Alpha-helical linkers differing in length were combined with motifs that are sensitive to the redox state of the environment. The first category of linkers included a redox motif found in the thioredoxin family of oxidoreductases. This motif was flanked by two alpha-helices of equal length. The second and third categories of redox linkers were composed of alpha-helices with embedded adjacent and dispersed vicinal cysteine residues, respectively. The linkers containing redox switches were placed between a FRET pair of enhanced cyan and yellow fluorescent proteins and these constructs were tested subsequently for their efficacy. A robust method of FRET analysis, the (ratio)(A) method, was used. This method uses two fluorescence spectra performed directly on the FRET construct without physical separation of the fluorophores. The cyan/yellow construct carrying one of the designed redox linkers, RL5, exhibited a 92% increase in FRET efficiency from its reduced to oxidized states. Responsiveness of the cyan-RL5-yellow construct to changes in the intracellular redox environment was confirmed in mammalian cells by flow cytometry.
感知细胞内或细胞器内还原/氧化状态的能力将为研究正常细胞增殖、分化和凋亡提供一个强大的工具。基因编码的生物传感器能够监测细胞内的氧化还原环境。我们报道了与荧光共振能量转移（FRET）结合用作氧化还原敏感连接子的嵌合多肽的开发。将不同长度的α-螺旋连接子与对环境氧化还原状态敏感的基序相结合。第一类连接子包括在氧化还原酶硫氧还蛋白家族中发现的氧化还原基序。该基序两侧是两个等长的α-螺旋。第二类和第三类氧化还原连接子分别由嵌入相邻和分散的邻位半胱氨酸残基的α-螺旋组成。将含有氧化还原开关的连接子置于增强型青色和黄色荧光蛋白的FRET对之间，随后对这些构建体的功效进行测试。使用了一种强大的FRET分析方法，即（比率）（A）法。该方法直接对FRET构建体进行两个荧光光谱分析，而无需对荧光团进行物理分离。携带设计的氧化还原连接子之一RL5的青色/黄色构建体，其从还原态到氧化态的FRET效率提高了92%。通过流式细胞术在哺乳动物细胞中证实了青色-RL5-黄色构建体对细胞内氧化还原环境变化的响应性。
