ITC binding experiments were performed on an iTC200 (Malvern Instruments) and fitted using in-built software. Before performing the ITC experiments, all protein samples were dialyzed overnight in a buffer containing 20 mM sodium phosphate at pH 6.5 or 7.5. The protein samples were then filtered, and their concentrations were determined using absorbance at 205 nm. The ITC experiments were done at different temperatures (288, 293, 298, 303, and 308 K). Typically, 20 injections of 2 μl each of 300 μM CID domain solution were titrated into 280 μl of 30 μM NCBD domains. All samples were allowed to equilibrate for about 10 min at the experimental temperature before the actual measurements were initiated. For the determination of ΔCp, both ΔH and ΔS were plotted as a function of temperature following Eqs. 2 and 3.Embedded Image(2)Embedded Image(3)ΔCp is the specific heat capacity at constant pressure. ΔxH and ΔxS are the temperature-independent contributions of enthalpy and entropy, respectively. The ITC measurements for pH dependence of affinity were carried out essentially as above, with the following differences: The protein samples were dialyzed against 20 mM sodium phosphate buffer (pH 8.2, 7.7, 7.2, 6.7, and 6.2) or, alternatively, 20 mM sodium acetate buffer (pH 5.7 and 5.2), and the ionic strength was adjusted with NaCl to 58 mM for all buffers. All measurements were conducted at 298 K. The measurement for the extant human complex at pH 5.2 was repeated four times because of problems with noise in the isotherms, and these data points were excluded from the fitting of pKa values. The data were fitted to a two-state equation (Eq. 4) to extract apparent pKa values for the association (KA) and dissociation (KD) constants for the respective protein complex.Embedded Image(4)

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