Figure 3
Griseoluteic acid binds EhpR. (A) Stopped-flow transient kinetic measurements demonstrate binding of griseoluteic acid to EhpR. Incubation of EhpR (5 μM) with an excess of griseoluteic acid (black circle: 250 μM; black triangle: 500 μM) leads to a time-dependent decrease of tryptophan fluorescence, whereas no change is observed in the absence of griseoluteic acid (cross) or EhpR (empty circle). (B) Microscale thermophoresis measurements of 25 nM fluorescein-labeled His6-EhpR incubated with the indicated amounts of griseoluteic acid. The relative fluorescence in the thermophoresis phase of the experiment has been plotted against the concentration of the ligand. (C) Stereo plot of |FO-FC| difference electron density at the ligand binding site of the high-resolution EhpR/griseoluteic acid (GA) complex before incorporation of the ligand, displayed at 3.5 σ.