In vitro analysis of the ClpX R230A mutation. (A) Native-PAGE assessment of oligomerization state of the indicated ClpX isoforms. The expected size of a hexamer is ~282.6 kDa. MW, molecular weight. (B) Kinase Glo assay to assess ATPase activity of ClpX wild type (WT), Walker B mutant (E187A), RKH mutant (R230A), and double mutant (R230A/E187A). Values are reported as % of starting ATP depleted. (C) GFP(SsrA) degradation assay for the indicated ClpX isoforms. Measured as change in fluorescence from starting value. For both B and C, ***, P < 0.001; ****, P < 0.0001; and ns, not significant by ordinary one-way analysis of variance (ANOVA) with Tukey’s post hoc comparisons test. Data in B and C are the averages of three biological replicates. RFU, Relative Fluorescence Units.