Pyrene actin was purchased from Hypermol (Bielefeld, Germany) and diluted to a 1 mg/mL (24 μM) stock solution. Before use, spontaneously formed actin aggregates were removed by ultracentrifugation for 1 h at 40,000 rpm and 4 °C. 50 μL samples for the pyrene assay consisted of: 30 μL H2O, 10 μL 10 mM MgCl2 or 250 mM KCl, 5 μL F-actin Buffer (100 mM Imidazole-Cl pH 7.4, 10 mM ATP, Hypermol, Germany) as well as 5 μL DMSO (containing the indicated concentrations of MiuA). 10 μL pyrene actin (end concentration 1 µM) were rapidly added to start polymerization. Pyrene fluorescence was monitored at 400 nm every 20 s over 1 h in a 96-well fluorescence plate reader (Tecan) with 360 nm excitation.
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