FIGURE 3.

Factors affecting the extent of glutathionylation of DnaK. Alexa Fluor 350 dye staining was used to detect the presence or absence of a free thiol in DnaK or its mutants in non-glutathionylated and glutathionylated states. Unmodified cysteine residues for untreated control, glutathionylated, and deglutathionylated samples were labeled with fluorescent dye. Equal loading of samples is shown by Coomassie Brilliant Blue staining of the same SDS-PAGE gel. A, WT DnaK, Δ553–638DnaK, and Δ606–638DnaK before and after treatment with diamide and GSH. The C-terminal truncation mutant Δ553–638DnaK mimics substrate binding. B, DnaK in the absence or presence of ATP or ADP before and after treatment with diamide and GSH. The DnaK mutant T199A binds but does not hydrolyze ATP. C, catalytic deglutathionylation of DnaK by Grx3 in the presence of GSH. Glutathionylated DnaK (DnaK-G) was treated with a range of concentrations of reducing agents and Grx3 enzyme alone or in combination as indicated.