FIGURE 1.

Thermodynamic double mutant cycle for the PDZ-peptide interaction. As described in the text, we measured the association and dissociation rate constants (k_on and k_off) for the binding of peptide ligands to PDZ domains. Each thermodynamic cycle involves four different species: PA (wild type protein with residue A in green), P (single mutant protein at position A), LB (wild type peptide ligand with residue B in blue), and L (single mutant peptide ligand at position B) and their four complexes: PA-LB, PA-L, P-LB, and PL. The free energy of interaction between the two probed positions A on the protein and B on the peptide ligand is calculated from the determined rate constants. In the scheme, positions A and B are exemplified as Val⁴⁴ of the PDZ2 domain (highlighted in green) and Val⁰ of the peptide ligand (highlighted in blue).