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Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors

Nature Chemical Biology volume 11, pages 332–338 (2015)Cite this article

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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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Figure 1: Design and spectra of MetSOx and MetROx sensors.
Figure 2: Response of recombinant MetSOx and MetROx to DTT and various oxidants.
Figure 3: Characterization of MetSOx and MetROx sensors in E. coli.
Figure 4: MetSOx and MetROx oxidation levels and the response to sodium hypochlorite in E. coli.
Figure 5: Characterization of MetROx in HEK293 cells.
Figure 6: MetROx response in subcellular compartments and physiological stimuli in HEK293 cells.

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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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Author notes

  1. Lionel Tarrago

    Present address: Present address: Aix Marseille Université, CNRS, iSm2 UMR 7313, Marseille, France.,

  2. Lionel Tarrago and Zalán Péterfi: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Lionel Tarrago, Zalán Péterfi, Byung Cheon Lee & Vadim N Gladyshev

  2. Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Thomas Michel

  3. College of Life Sciences and Biotechnology, Korea University, Seoul, South Korea

    Byung Cheon Lee

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Contributions

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.

Corresponding author

Correspondence to Vadim N Gladyshev.

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The authors declare no competing financial interests.

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Supplementary Results, Supplementary Figures 1–15 and Supplementary Tables 1–4. (PDF 1887 kb)

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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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