Figure 6
Zn++ and Cu++ ions interfere with the stability of HpHtrA via an allosteric loop. To investigate the effect of ZnCl2 and CuCl2 on the stability and activity of HpHtrA oligomers, HpHtrA wt was incubated with increasing concentrations of ZnCl2 (A) and CuCl2 (B) and then analyzed by a Coomassie-stained SDS-PAGE under non-reducing conditions (left panels). 4 µM recombinant HpHtrA wt (black circles), HpHtrA S164A (red squares), HpHtrA D165A (green inverted triangles), HpHtrA S166A (yellow triangles), and HpHtrA D168A (blue rhombus) were incubated with SYPRO Orange and increasing concentrations of ZnCl2 and CuCl2 at a temperature ramp from 25–95 °C (increase of 0.5 °C per minute). Changes in melting temperature ΔTm are presented normalized to the intrinsic Tm of the individual HtrA mutant (right panels).
