FIGURE 1.

Equilibrium binding experiments. a–c, ITC data for PDZ₃ (a), PDZ₁₂ (b), and PDZ₁₂₃ (c) with E6_L151V at 10 °C. Top panels, raw data. Bottom panels, integrated titration curves. See Table 1 for fitted parameters. d and e, SEC-MALS analysis of PDZ₁₂₃ in the presence and absence of a 3-fold molar excess of HPV E6. d, the elution volumes from an analytical Superdex 75 10/30 column were very similar for the apo- and saturated PZD₁₂₃-E6 complex (red and black traces, respectively). For clarity, the UV signals of PDZ₁₂₃ peaks are normalized to have similar intensities. The additional peak at ∼19.4 ml corresponds to unbound HPV E6 (and reductants in the E6 samples used to prevent thiol-mediated oligomerization). e, SEC-MALS was used to determine the molar mass across the peaks for the apo- and saturated PZD₁₂₃-E6 complex (trace colors are as in d). PDZ₁₂₃ exists as a discrete monomer, even at high protein concentrations (>8 mg/ml). Despite having a similar elution volume to apo-PDZ₁₂₃, the saturated PDZ₁₂₃-E6 complex has a significantly increased molar mass. This mass difference was consistent with each PDZ₁₂₃ binding three E6 molecules, which agrees with the number of binding sites available and independent ITC and kinetic measurements (Table 1).