FIGURE 7.

Tpr^C229S is impervious to proteolysis by active forms of Tpr (A) and in the presence of Ca²⁺ also blocks degradation by Kgp (B) and Rgp (C) because the protein is stabilized (D). Recombinant Tpr^C229S was incubated with Tpr55/Tpr48 at 37 °C in the activity assay buffer at the molar ratio 100:1 for the indicated time periods (A) or with gingipains at different molar ratios in the presence or absence of Ca²⁺ for 1 h (B and C). Boiling in a denaturing sample buffer stopped the reaction, and samples were analyzed by SDS-PAGE followed by protein staining and analysis by N-terminal sequencing. The effect of increasing concentrations of calcium ions on the T_m of Tpr^C229S was measured by differential scanning fluorimetry (D).