Kd (dissociation constant) determination for 17-AAG and compound D binding to PfHSP90.

Tryptophan fluorescence analysis was performed using a PerkinElmer LS55 luminescence spectrometer. In order to determine the binding affinity of purified recombinant proteins, 25 μg/ml PfHSP90 or PfHSP90 N-terminal domain (PfHSP90-NTD) was incubated with concentrations of 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) or compound D ranging from 0 to 60 μM. The binding buffer used for the reaction contained 50 mM Tris-HCl, pH 7.4, and 1 mM EDTA. Samples were excited at 280 nm, and tryptophan fluorescence measurements were carried out by scanning the emission spectrum in the wavelength range 300 to 400 nm. A maximum wavelength (λ_max) of 346 nm was selected for all the calculations. The slit widths of excitation and emission were set at 2.5 and 5 nm, respectively. The difference in fluorescence intensity between protein alone and protein with various concentrations of 17-AAG or compound D was calculated and plotted against the different concentrations of the respective ligand used. The resultant hyperbolic curve was analyzed with GraphPad Prism software, using nonlinear regression analysis with single-site-specific binding as described previously (32).