The interaction between TtClpP and bortezomib was assessed by ITC using an Auto-iTC200 microcalorimeter (MicroCal-Malvern Panalytical, Malvern, UK). Calorimetric titrations were performed with a 1.4 mM bortezomib solution in the injecting syringe and a 10 μM ClpP solution in the calorimetric cell in 50 mM Hepes (pH 7.6) and 50 mM NaCl. All solutions were properly degassed to avoid bubble formation during stirring. In each titration, a sequence of 19 2-μl injections was programmed, with reference power of 10 μcal/s, initial delay of 60 s, spacing between injections of 150 s, and stirring speed of 750 rpm.

The heat evolved after each ligand injection was obtained from the integral of the calorimetric signal. Experiments were performed in replicates, and data were analyzed using in-house developed software implemented in Origin 7.0 (OriginLab, Northampton, MA). The binding isotherms (ligand-normalized heat as a function of the molar ratio) were analyzed considering the MWC model for ClpP, an oligomeric macromolecule consisting of 14 identical subunits containing a single ligand-binding site each (52). Nonlinear least squares regression allows the determination of the following binding parameters: equilibrium constants (KR, KT, γ), enthalpies (ΔHR, ΔHT, ΔHγ), and fraction of active protein (N). A fitting routine was implemented in Origin 7.0 (OriginLab).

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