New red-shifted fluorescent biosensor for monitoring intracellular redox changes.

Maintenance of intracellular redox homeostasis is critical for cell survival, proliferation, differentiation, and signaling. In this regard, major changes in the intracellular redox milieu may lead to cell death whereas subtle increases in the level of certain oxidizing species may act as signals that regulate a plethora of cellular processes. Redox-sensitive variants of green fluorescent proteins (roGFP2 and rxYFP) were developed and proved useful to monitor intracellular redox changes in a non-invasive and online manner. With the aim to extend the spectral range of the fluorescent redox biosensors, we here describe the generation, biochemical characterization and biological validation of a new redox reporter based on the red-shifted mRuby2 protein (rxmRuby2). Spectrofluorimetric analysis performed with the recombinant biosensor shows a reversible redox response produced by two redox-active cysteine residues predicted by molecular modeling. rxmRuby2 is highly selective for the couple glutathione/glutathione disulfide in the presence of the oxidoreductase glutaredoxin. The estimated redox potential of rxmRuby2 (E° -265 ± 22 mV) makes it suitable for its use in reducing subcellular compartments. Titration assays demonstrated the capacity of rxmRuby2 to monitor redox changes within a physiological pH range. rxmRuby2 responded sensitively and reversibly to different redox stimuli applied to HeLa and HEK293 cells expressing transiently and/or stable the biosensor. Fusing rxmRuby2 to the Clover fluorescent protein allowed normalization of the redox signal to the expression level of the reporter protein and/or to other factors that may affect fluorescence. The new red-shifted redox biosensor show promises for deep-tissue and in vivo imaging applications.