Abstract
Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological and pathophysiological conditions, but its use as a redox marker suffers from the lack of tools to detect and quantify MetO within cells. In this work, we created a pair of complementary stereospecific genetically encoded mechanism-based ratiometric fluorescent sensors of MetO by inserting a circularly permuted yellow fluorescent protein between yeast methionine sulfoxide reductases and thioredoxins. The two sensors, respectively named MetSOx and MetROx for their ability to detect S and R forms of MetO, were used for targeted analysis of protein oxidation, regulation and repair as well as for monitoring MetO in bacterial and mammalian cells, analyzing compartment-specific changes in MetO and examining responses to physiological stimuli.
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Acknowledgements
We thank P. Rey (Laboratoire d'Ecophysiologie Moléculaire des Plantes, UMR7265 CEA-CNRS-Aix-Marseille Université) for the kind gift of dabsyl-MetO and V. Belousov and V. Verkhusha for discussion. This study was supported by US National Institutes of Health grants AG021518 and GM065204 to V.N.G. and HL48743 to T.M.
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L.T. designed, created and characterized the sensors; performed experiments with E. coli cells; analyzed the data and wrote the paper. Z.P. performed experiments with HEK293 cells, analyzed the data and wrote the paper. B.C.L. performed experiments with MICAL1-oxidized actin and analyzed the data. T.M. contributed reagents and tools and analyzed the data. V.N.G. designed the sensors, supervised the research and wrote the paper.
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Tarrago, L., Péterfi, Z., Lee, B. et al. Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors. Nat Chem Biol 11, 332–338 (2015). https://doi.org/10.1038/nchembio.1787
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DOI: https://doi.org/10.1038/nchembio.1787
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