Measuring the ADP production by P_II.

<p><b>A:</b> P_II-V was incubated with ATP for 1 h at 37°C, then NAGK-C and PipX were added and FRET was measured after 15 min of additional incubation. <b>B:</b> P_II-V was incubated without ATP for 1 h at 37°C, then ATP, NAGK-C and PipX were added and FRET was measured after 15 min of additional incubation. <b>C:</b> P_II-V was incubated without ATP for 1 h at 37°C, then a mixture of ATP, ADP, NAGK-C and PipX was added and FRET was measured after 15 min of additional incubation. Used concentrations: P_II-V, NAGK-C: 0.1 μM, PipX: 1 μM; ATP (A, B): 10 μM (dark blue), 100 μM (blue), 1000 μM (light blue); ATP+ADP mixture (C): 5 μM each (dark blue), 50 μM each (blue) 500 μM each (light blue). Mean values of 3 experiments with standard deviation are shown.