SPR for the measurement of the binding strength between PmrD and PmrA. The His-tagged wild-type and mutant PmrD were separately immobilized onto the NTA sensor chip in the running buffer (10 mM HEPES, 150 mM NaCl, 50 μM EDTA, 0.005% Tween 20, and pH 7.4). PmrA proteins with various concentrations (0.1, 1, 3, and 5 μM) were applied to a PmrD-modified channel under a continuous flow of 20 μL/min at 25°C using the Biacore T100 biosensor system. (A) Binding kinetics for wild-type PmrD with PmrA. (B) Plot based on the steady-state method for binding between wild-type PmrD and PmrA. (C) Binding kinetics for mutant PmrD with PmrA. (D) Plot based on the steady-state method for binding between mutant PmrD and PmrA.