Acyl-Homoserine Lactone Recognition and Response Hindering the Quorum-Sensing Regulator EsaR
Figure 4
In vitro relative binding affinities of HMGE and EsaR* variants.
Various protein concentrations (0–100 nM) of A) HMGE, B) A32V, C) D83E, D) F94Y were incubated with 3 nM T-esabox (dsDNA concentration) for 20 min at 25°C in the presence (triangles) and absence (circles) of 1 µM AHL. Fluorescence anisotropy was measured with a Tecan F200 Pro fluorometer with a G factor of 1 and excitation and emission wavelengths of 540 and 590 nm, respectively. Background anisotropy was subtracted and the resulting data were used to generate a fit curve and calculate the apparent Kd. Duplicate samples were analyzed from experiments performed in triplicate. Average of and standard deviation across three experiments for each protein is shown.