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![[Figure 3]](gm5029fig3mag.jpg)
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Figure 3
A disulfide bond in the −35 promoter-binding domain of σK acts as a redox sensor. (a) The experimental electron-density map at 2.4 Å resolution (1.0σ level) reveals a disulfide between Cys133 of helix H5 and Cys183 of helix H8 in the σK4 domain. (b) Intrinsic tryptophan fluorescence was monitored from 300 to 400 nm for freshly purified σK–RskAcyto as well as the oxidized, reduced and denatured protein samples. These spectra reveal a 10 nm shift between the oxidized and reduced samples. The oxidized and reduced forms of the cysteine residues in σK were confirmed by mass spectrometry (Supplementary Fig. S1). (c) The redox potential was measured with a glutathione-coupled assay using the intrinsic tryptophan fluorescence of this protein (Wunderlich & Glockshuber, 1993  ). The plot shows the amount of σK–RskAcyto complex equilibrated with varying concentrations of GSH. (d) The Keq calculated for the σK–RskAcyto complex at different ratios of [GSH]2/GSSG was used in the Nernst equation to calculate the redox potential. The mean redox potential of −265 mV for the σK–RskAcyto complex suggests the cysteines are likely to be oxidized in the cytosolic environment. |
![[Figure 3]](gm5029fig3.jpg)
 | STRUCTURAL BIOLOGY |
ISSN: 2059-7983
