Transcription factors ERα and Sox2 have differing multiphasic DNA- and RNA-binding mechanisms

Surface plasmon resonance confirms multiphasic ERα_DBD-Ext-dsDNA binding kinetics. (A) Normalized SPR traces are shown for the association and dissociation phases. The change in RU signal after ERα_DBD-Ext injection (ΔRU), which excludes changes observed in the empty flow cell, is shown as a function of time postinjection. Legends indicate the concentration of ERα_DBD-Ext used during protein injection. Lines are data, not regression fits. (B) ERα_DBD-Ext ligand association curves. SPR association phase curves, taken from A with the same color scheme. (C) Association rate analysis. Association curves from B had their initial slopes normalized to their signal dynamic range to calculate their apparent association rates (see Materials and Methods). Plots of apparent initial association rates versus protein concentrations were fit with zero-intercept linear regression to calculate apparent initial association rate constants (). Dots are data and solid lines are linear regression fits. Bracketed data point is an outlier from the initial slope quantification of the 16 nM association curve in B, which has negligible signal dynamic range. (D) Dissociation rate analysis. SPR dissociation phase curves, taken from A, with the same color scheme. Two key protein concentrations (1 µM and 30 nM) are shown. Data were fit with biexponential regression (Equation 5) to determine rate constants. Gray dots are data and solid black/purple lines are regression fits; data points are mostly obscured by regression lines. Percent contributions of fast versus slow components to the biexponential curve are in parentheses. All SPR data in this figure are from a single experiment per ligand.