GTPase activity assay

FtsZ GTPase activity was monitored using the PiColorLock Gold kit (Expedeon), a malachite-green-based assay. SepH and FtsZ were diluted to the desired concentration in buffer P (50 mM HEPES pH 7.2, 50 mM KCl, 5 mM MgCl₂). The protein solution was incubated for 10 min at 30°C and the reaction was started by adding 50 µM GTP. Samples were taken at 0, 2.5, 5, 7.5, and 10 min. Reactions were stopped by adding an equal volume of 0.6 M perchloric acid. Absorbance at 620 nm was measured and plotted using Microsoft Excel. GTPase activity was determined from the linear range of the curves (Wasserstrom et al., 2013). GTPase activity assays for M. smegmatis FtsZ_Ms and SepH_Ms were performed as described above but using a modified buffer P (50 mM HEPES pH 6.8, 100 mM KCl, 5 mM MgCl₂) and incubating the protein solutions at 37°C. Samples were taken at 0, 5, 10, 15, and 20 min and data was analyzed as described above.

To determine the critical concentration, the GTPase activity was determined for several FtsZ concentrations (4.5, 3.5, 2.5, and 2 µM) in the absence or presence of 0.6 µM SepH. To accommodate for the number of samples and higher FtsZ concentrations, reactions volumes were reduced to accommodate measurements using 96-well plates and samples were taken every 1.5 min instead of 2.5 min as described above. Each reaction was performed in duplicate. A linear regression was calculated for the GTPase rate with and without SepH to determine the value of the X-intercept (the critical concentration).