Isothermal Titration Calorimetry

The procedure previously described (Raaf et al., 2013) was a adapted to perform ITC measurements. In this work, C-terminally truncated variants of the CK2 subunits were used which previously had been demonstrated to be fully competent to perform the CK2α/CK2β interaction (Raaf et al., 2008). The standard background buffer (500 mM NaCl, 25 mM TRIS/HCl, pH 8.5) was used to dilute the stock solutions of either CK2α^1–335 or the mutant CK2α^{1–335,Lys198Arg} to a concentration of 7 µM and that of CK2β^1–193 to 149 µM. The latter was filled into the injection syringe. The measurements were carried out using a VP-ITC (Microcal) at 35°C. In each run, 25 injections of CK2β^1–193 solution with a volume of 2 µL for the first, and 10 µL for all subsequent injections were applied; the off-time between two single injections was 300 s. Three independent measurements were performed for the CK2α^1–335/CK2β^1–193 interaction and two for the CK2α^{1–335,Lys198Arg}/CK2β^1–193 interaction.

The raw ITC data were processed with ORIGIN (version 7), Origin Lab (OriginLab Corporation, Northampton, MA, United States), assuming a binding model of a single set of sites, and with the “ligand is present in the cell” option. For curve fitting, the stoichiometry between CK2α and CK2β was fixed to 1 according to the structure of the CK2α₂β₂ holoenzyme (Niefind et al., 2001).