ERαDBD-Ext multiphasic ligand dissociation is independent of competitor. Dissociation curves from FPCD versus FPJD experiments. FPJD experiments were performed for the ERαDBD-Ext–ΔERE interaction using 50 nM ligand and 50 nM protein (predilution), with protein–ligand reactions being incubated to equilibrium before dilution. The protein–polynucleotide reaction was then diluted ∼80-fold in buffer (at 4°C) and polarization was monitored over time postdilution (at 4°C) to quantify protein–ligand dissociation kinetics. FPCD experiments were performed as described in Figure 2. Anisotropy traces were normalized to the controls to give “Fraction Bound” over time, and then normalized data were fit with biexponential regression to determine rate constants. Dots are data points and solid lines are regression fits (Equation 5) from single experiments. Rate constants are the average values from all independent experiments (two for FPCD, three for FPJD), with percent contributions of fast and slow components to the biexponential curve in parentheses. Error analyses for FPCD values are in Table 1, and for FPJD kfast = 1.9 ± 0.91 × 10−2 sec−1, kslow = 4.2 ± 1.2 × 10−4 sec−1 (mean ± ½ range).
