Figure 9: Titration calorimetry of Pop5-RPP30 binding reveals net 1 : 1 stoichiometry but higher-order binding. Top panels: Representative ITC thermograms obtained upon titration of Pfu Pop5 into Pfu RPP30 at 55°C. Bottom panels: Time-integration for each peak in the thermogram after normalizing per mol of injectant (squares). Titrations, (a, no salt) and (b, 130 mM KCl), were performed with 9.89 and 4.95 μM RPP30, respectively, in the cell and can be fit by a single site binding model, with apparent dissociation constants of 124 ± 13 and 3.5 ± 0.6 nM, respectively. Experiments performed with 14.8 μM RPP30 in the cell (c: no salt, d: 150 mM NaCl) yielded more complex thermograms that could only be fit by invoking two-site, or more complex binding models, consistent with higher-order assembly. Comparison of data at lower and higher ionic strength indicates binding is strongly favored by inclusion of moderate salt (e.g., 130 mM KCl) in the solutions.