Figure 7.

Binding of PA-824 to Ddn and Its Mutants Studied by Tryptophan Fluorescence Spectroscopy

(A) Emission spectra in relative fluorescence units (RFUs) of Ddn in the presence of varying concentrations of PA-824 (0–500 μM) were recorded upon excitation at 280 nm. The intrinsic spectrum of the NΔ30 Ddn construct is also shown. For this experiment, untagged WT Ddn protein was used.

(B) Titration of PA-824 in saturation binding analysis with the MBP fusion of Ddn WT and mutants monitored at 330 nm. The magnitude of fluorescence differences (1 − F/F₀) was used for K_d calculation, as described in Experimental Procedures.

(C) Values determined for MBP-Ddn mutants. Asterisks indicate the triple mutant which, even at saturating concentration of PA-824, showed less than 40% quenching, unlike WT MBP-Ddn, for which 80% quenching was observed. See Figure S8 and Table S5 for CD data on MBP-Ddn mutant proteins.