Figure 2.

Biochemical characterization of Mtb DsbF. (a) Redox equilibrium of Mtb DsbF with glutathione. The y-axis represents the proportion of Mtb DsbF present in the reduced form at equilibrium with various mixtures of reduced (GSH) and oxidized (GSSG) glutathione (x-axis), which was measured by exploiting the difference in fluorescence (347 nm) of reduced Mtb DsbF compared to oxidized Mtb DsbF. Equilibrium concentrations of oxidized and reduced Mtb DsbF and GSH and GSSG were calculated as described ⁶⁶. The equilibrium constant was calculated to be 40 ± 5 μM, which is consistent with Mtb DsbF acting as an oxidant. (b) Analysis of the refolding activity of reduced and unfolded hirudin. 5 pmol reduced, unfolded hirudin was incubated alone or with stoichiometric quantities (5 pmol) of Mtb DsbF, E. coli DsbA and E. coli thioredoxin. Samples were removed as indicated at each time point and the reactions, quenched by the addition of iodoacetamide, were analyzed by MALDI-TOF mass spectrometry. The appearance of native hirudin (m/z 6765) is represented as a percentage of the total intensities of native and carbamidomethylated hirudin. (c) GdnHCl-dependent unfolding/folding of Mtb DsbF. Guanidine hydrochloride (GdnHCl)-dependent unfolding was monitored by circular dichroism spectroscopy at 222 nm for oxidized Mtb DsbF (shown as squares) and reduced Mtb DsbF (shown as triangles). Since the reduced form of Mtb DsbF unfolds at higher concentrations of GdnHCl than the oxidized form, the reduced form of Mtb DsbF is its more stable form. (d) Determination of the pK_a value of the active-site cysteine, Cys81, in Mtb DsbF. The absorption specific to reduced Mtb DsbF protein is shown as a function of pH (x-axis) compared to the fraction of Cys81 thiolate (y-axis). The pK_a of Cys81 was calculated by fitting the data points as described ⁶⁷. The pK_a of Cys81 in Mtb DsbF is 5.6 ± 0.3.