FIGURE 7.

Competitive equilibrium binding titrations of NBD-ZPI-PZ complex with unlabeled mutant ZPIs. NBD-ZPI-PZ complex formed with 54 nm NBD-ZPI and 52 nm PZ was titrated with increasing concentrations of unlabeled ZPI variants with mutations in PZ binding site residues as indicated in pH 7. 1, I 0.15 Tris buffer at 25 °C, and the observed fluorescence was monitored at 545 nm as in Fig. 3. Fluorescence data corrected for buffer and dilution were fit by the cubic competitive binding equation as in Fig. 3 (solid lines). Additional details are provided under “Experimental Procedures.”