Figure 5. Conformational stability of WhiD.

(A) Chemical denaturation of apo- (gray circles) and native-WhiD (black circles) (14.1 μM protein) in 50 mM Tris-HCl, 25 mM NaCl, pH 8.0 with GdnHCl between 0 to 6 M. Fraction of unfolded protein (as normalised intensity at 222 nm) is plotted as a function of GdnHCl concentration. Apo-WhiD displayed a non sigmoidal transition suggesting it is partly unfolded in the absence of denaturant. Assuming apo-WhiD is ~25% unfolded at zero denaturant, both apo- and native-WhiD fitted to a two-state unfolding model (solid lines, see main text). (B) Thermal denaturation of apo- (open circles) and native- WhiD (filled circles) followed by tryptophan fluorescence changes. The thermal stability of the iron-sulfur cluster was followed by plotting the ratio of A_{420 nm} to A_{350 nm} (gray squares, see Table 1). Lines represent fits of the data obtained using the Cary Eclipse Bio Software or Origin (Microcal) to obtain T_m and ΔG_unfold values.