Heat shock protein 90 localizes to the surface and augments virulence factors of Cryptococcus neoformans
Fig 3
Binding of Hsp90 inhibitor 17-AAG and its effect on CnHsp90 ATPase activity.
(a) Binding affinity of competitive inhibitor 17-AAG to CnHsp90 using tryptophan fluorescence was determined. Change in intrinsic fluorescence intensity upon ligand binding was plotted against ligand concentration. Dissociation constant, Kd, for 17-AAG binding was calculated to be 12.92 μM. (b) IC50 for inhibition of ATPase activity of CnHsp90 was determined by incubating the pure protein with fixed, saturating concentration of ATP and varying concentrations of 17-AAG. The reaction was carried out at 25°C and 37°C. The percent activity remaining was plotted against concentration of 17-AAG in logarithmic scale to obtain the inhibition curve. 17-AAG mediates inhibition of CnHsp90 activity at both temperatures tested. (c) IC50 values obtained at 25°C and 37°C for CnHsp90 inhibition by 17-AAG was found to be 26.89 μM and 117.15 μM respectively. Therefore, 17-AAG inhibits CnHsp90 more potently at 37°C.