FIG. 4.

Disruption of lysine 175 prevents NemR^{C106 only} DNA binding, but does not affect its oligomerization state. (A) EMSAs of the binding of NemR^{C106 only} (0, 100, 200, 300, 400 nM) to nemR promoter DNA (P_nemR, 10 nM) before and after treatment with NCT. To test the reversibility of oxidative inactivation, NCT-oxidized NemR^{C106 only} was incubated with the thiol reductant, DTT (5 mM). Representative gels are shown along with quantification results (mean±SD, n=3). (B) EMSA of the binding of NemR^{C106 only, K175A} to 10 nM P_nemR DNA in the presence of 5 mM DTT (mean±SD, n=3). Concentration of NemR is plotted on a log₂ scale. (C) Size exclusion chromatography was used to compare the oligomeric state of reduced and oxidized NemR^{C106 only} and reduced NemR^{C106 only, K175A}. Dotted lines indicate elution volume of protein standards of known molecular weight. EMSA, electrophoretic mobility shift assay.