Fluorescence polarization (FP) assay

Purified AtERF96 proteins were desalted to FP buffer (30 mM HEPES pH 7.4, 250 mM NaCl, 10% glycerol) using a HiTrap™ Desalting column (GE Healthcare), and the concentration was determined by the Bradford protein assay. The AtERF96 protein samples were two-fold serially diluted in FP butter to 16 or 24 concentrations, and 50 µL of each diluted protein sample was added to a 96-well plate, along with 50 µL of 20 nM GCC12 probes in each sample well. A set of wells containing 100 µL FP buffer, and another set of wells containing 50 µL FP buffer mixed with 50 µL of 20 nM GCC12 probes were used as blanks and controls, respectively. FP enables the study of molecular interactions by monitoring changes in the apparent size of fluorescently-labelled or inherently fluorescent molecules, which are often referred to as the tracer or the ligand (Checovich et al. 1995; Heyduk et al. 1996; Moerke 2009). The samples of fluorescent molecules were excited by plane-polarized light, and the emission spectra were recorded and analyzed by PARADIGM™ (Beckman Coulter/Molecular Devices). Quantification of fluorescence polarization (FP) is defined as the difference between the emission intensities of horizontally (F‖) and perpendicularly polarized light (F⊥) to the excitation light plane normalized by the total fluorescence emission intensity (Moerke 2009). The formula of FP is described as follows:

where P is the polarization obtained by subtracting the blank value of both the horizontally and perpendicularly polarized light. The anisotropic levels of polarized fluorescence were plotted against the concentrations of protein samples using Prism 7 (GraphPad Software, Inc.) with the two-site binding equation. The dissociation constant (K_d) is determined by the correlation between polarizations and sample concentrations, and the formula of two-site binding is described as follows:

where x is the protein concentration, y is the polarized value. B_maxHi and B_maxLo are the maximum specific bindings to the two sites in the same units as y. K_dHi and K_dLo are the equilibrium binding constants, in the same units as x. All experiments were done from three independent replicates.