TABLE 2.
Binding constants for unlabeled wild-type and variant ZPI interactions with PZ
Dissociation constants (KD) for ZPI-PZ interactions were determined in pH 7.1 Tris buffer containing 0.1 m NaCl (I 0.15) or 0.25 m NaCl (I 0.30) at 25 °C from competitive binding titrations of NBD-ZPI-PZ with unlabeled ZPIs as shown in Fig. 7 and described under “Experimental Procedures.” All recombinant ZPIs contained the two wild-type cysteine residues.
| ZPI variant | KD I 0.15 | KD I 0.30 | ΔG0 I 0.15 | ΔΔG0 I 0.15 | Unitary binding free energya I 0.15 |
|---|---|---|---|---|---|
| nm | nm | kcal/mol | kcal/mol | % | |
| WT ZPI | 2.1 ± 1.0 | 16 ± 4 | −11.8 | ||
| WT cZPI | 5.2 ± 0.2 | 23 ± 2 | −11.3 | ||
| M71A | 17 ± 4 | −10.6 | 1.2 | 8.7 | |
| D74A | 84 ± 41 | −9.6 | 2.2 | 15 | |
| D238A | 4.2 ± 0.8 | 52 ± 11 | −11.4 | 0.4 | 2.9 |
| K239A | 0.1 ± 0.1 | 0.3 ± 0.2 | −13.9 | −2.0 | |
| K239C | 0.1 ± 0.1 | 1.1 ± 0.3 | −13.6 | −1.8 | |
| Y240A | 330 ± 30 | −8.8 | 3.0 | 21 | |
| D293A | 1200 ± 500 | −8.1 | 3.8 | 26 |
a The unitary binding energy represents the apparent standard free energy of binding of ZPI and PZ (ΔG0) corrected for the entropic energy required to bring two molecules together in solution (2.38 kcal/mol at 25 °C) (23).