Concentrations of ClpP and ClpX were calculated using the molecular weight of a tetradecamer and a hexamer, respectively. PKMamc degradation by TtClpP and EcClpP was measured as described (45). Briefly, TtClpP in Hepes buffer [50 mM Hepes (pH 7.6) and 50 mM NaCl] was mixed with indicated concentrations of PKMamc and bortezomib/Bz-LL [or respective dimethyl sulfoxide (DMSO) volume] at 60°C (or indicated temperature), and fluorescence (340-nm excitation, 462-nm emission) was measured at defined time intervals. FITC-casein degradation by TtClpP was measured as described (45); in a typical assay, FITC-casein (0.15 μM) was mixed with TtClpP14 (0.1 μM) and fluorescence increase (440-nm excitation, 509-nm emission, 495-nm cutoff) was monitored. GFPssrA (0.5 μM) degradation by TtClpX6 (0.2 μM) and TtClpP14 (0.1 μM) in the presence of ATP (10 mM) was monitored, taking advantage of the intrinsic fluorescence of GFPssra (440-nm excitation, 509-nm emission, 495-nm cutoff) at the indicated temperatures. FtsZ degradation by TtClpP was measured at 37°C. Briefly, TtClpP14 (1 μM) was mixed with purified FtsZ (20 μM) in the presence of the indicated concentrations of bortezomib. Aliquots were removed at the indicated time points, and the samples were loaded in a 15% SDS agarose gel. It is important to note the substantial differences in TtClpP concentration used for the different techniques, with 10-fold more protease being used for the FtsZ protein degradation study compared with FITC-casein degradation, as a consequence of the lower sensitivity of SDS–polyacrylamide gel electrophoresis staining versus fluorescein fluorescence methods. TtClpP reaction with ActivX TAMRA-FP was executed according to the manufacturer’s instructions (Thermo Fisher Scientific). TtClpP14 (1 mg/ml) was incubated in the indicated conditions with 1 μl of TAMRA-FR reagent (final concentration of 5 μM). At defined time intervals, aliquots were removed and loaded in a 4 to 20% SDS gel. Gels were visualized in a Bio-Rad ChemiDoc XRS system. TtClpP (20 μM) unfolding was measured in a Varian Cary Eclipse spectrofluorimeter monitoring tryptophan intrinsic fluorescence (280-nm excitation, 350-nm emission). TtClpP14 analytical SEC was performed using a Superdex 200 10/300 GL column.

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