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. 2017 Jun 29;53(53):7385-7388.
doi: 10.1039/c7cc02285a.

Chemoselective Ratiometric Imaging of Protein S-sulfenylation

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Free PMC article

Chemoselective Ratiometric Imaging of Protein S-sulfenylation

Christopher T M B Tom et al. Chem Commun (Camb). .
Free PMC article

Abstract

Here we report a ratiometric fluorescent probe for chemoselective conjugation to sulfenic acids in living cells. Our approach couples an α-fluoro-substituted dimedone to an aminonaphthalene fluorophore (F-DiNap), which upon sulfenic acid conjugation is locked as the 1,3-diketone, changing the fluorophore excitation. F-DiNap reacts with S-sulfenylated proteins at equivalent rates to current probes, but the α-fluorine substitution blocks side-reactions with biological aldehydes.

Figures

Fig. 1
Fig. 1
F-DiNap is an excitation ratiometic probe for protein S-sulfenylation. (a) Comparison between 3a-c conjugation to AhpC (C166S) by in-gel fluorescence (Ex. 488 nm, Em: 555/20 nm). (b) Methyl methane thiosulfonate (MeO2S-SMe) reacts with 3a and 3c to yield conjugated standards in aqueous buffer. (c) F-DiNap fluorescent excitation and emission spectra before and after conjugation to MeO2S-SMe (S-methyl methanethiosulfonate, MMTS).
Fig. 2
Fig. 2
F-DiNap is does not react with protein-linked aldehydes. (a) α-Fluoro substituted probes do not form condensation products with aldehydes. (b) Reactive oxygen species promote the formation of cysteine S-sulfenylation, but also generate protein-linked aldehydes and lipid peroxidation ene-al products. (b) GAPDH oxidized under Fenton conditions reacts with H-DiNap, but not F-DiNap. (c) Addition of acrolein (1 mM) or 4-hydroxynonenal (HNE) (1 mM) to GAPDH increases labelling by H-DiNap, but not F-DiNap. In-gel fluorescence ex. 488 nm, em. 555/20 nm.
Fig. 3
Fig. 3
Live cell excitation ratiometric imaging of sulfenic acids in CH27 B-cells with DiNap probes. (a) Images were acquired by TIRF microscopy 15 minutes after probe addition, showing spatially resolved ratio changes with F-DiNap. The ratios were calculated after background subtraction of regions outside of cells, and dividing the emission intensities from each separate excitation. Scale bars are displayed to highlight scale and range of ratios displayed. (b) Hydrogen peroxide (1 mM) induces rapid F-DiNap ratio changes within a few seconds. Data is normalized to excitation ratios measured after 9 minutes of probe labeling. Images are representative of 3 biological replicates. Scale bar = 20 μm.
Fig. 4
Fig. 4
Snail expression attenuates F-DiNap labeling in MCF10A cells. (a) Confocal ratiometric imaging of F-DiNap in Snail-expressing cells. Images are representative of 3 technical replicates. Scale bar = 50 μm. (b) In-gel F-DiNap fluorescence of attenuated S-sulfenylation in Snail-expressing MCF10A cells. (c) Snail over-expression protects MCF10A cells from peroxide-dependent reduction in cell viability. Cell viability was measured after 24 hours by resorufin-AM fluorescence. Data is representative of 3 biological replicates each with 6 technical replicates.
Scheme 1
Scheme 1
DiNap probes react with S-sulfenylated proteins. After sulfenic acid conjugation, unsubstituted probes undergo a second deprotonation to return to the enolate form. However, α-substituted Me- and F-DiNap probes are locked in the diketone form after conjugation.

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