Fluorescence Polarization (FP) Assays

Two types of fluorescence polarization measurements were used in this study. Solution phase, direct binding assays (henceforth FP saturation binding assays) were performed to measure affinity (K_D) between the PDZ domains of NHERF1 and FITC-labeled PTHR peptides following the protocol described by Madden and co-workers.¹¹ Assays were performed in FP buffer (storage buffer, supplemented to a final concentration of 0.1 mg/ml with bovine IgG (Sigma) and 1 mM DTT) at pH 7.4, 7.0, or 6.4 by applying increasing amounts of the recombinant proteins to a fixed concentration of the FITC-labeled peptide (0.5 μM). Indicated FP experiments were performed in FP buffer supplemented with 300 mM NaCl.

In FP competition assays the binding affinity (Ki) of unlabeled peptide (competitor) corresponding to the concentration that inhibits 50% binding of the FITC-labeled peptide was measured. Competition assays were performed in FP buffer (DTT was avoided for Cys-PTHRct-9) containing fixed concentrations of both fluorescent labeled peptide and protein following the protocol described by Madden and co-workers¹¹. This mixture was equilibrated for 20 min in the dark at room temperature. Unlabeled competitor peptide was dissolved and serially diluted in storage buffer supplemented with 5% DMSO (Sigma). Each serial dilution was aliquoted at 1/10 final volume, to which was added 9/10 volume of the protein:peptide mixture. All FP assays were performed in a 96-well format. Polarized fluorescence intensities were measured at 23 °C with a Perkin Elmer Wallac Victor3 multilabel counter using excitation and emission wavelengths of 485 nm and 535 nm, respectively, for the FITC-labeled peptide. Experimental data were analyzed using Prism (GraphPad). All measurements are reported as fluorescent anisotropy (FA) rather than polarization. Anisotropy was computed using equation 1 from the measured fluorescence emission intensities that are polarized parallel (I_║) and perpendicular (I_┴) to the plane of the incident light¹²:

The equilibrium dissociation constant (K_D) for interaction between the indicated PDZ domain and labeled peptide was determined by fitting the FA data to a quadratic equation¹² as we described previously.¹³ The K_D obtained from direct binding experiments was used to calculate the dissociation constant of the interaction between unlabeled peptide (competitor) and PDZ domain (K_i) using equation 17 described previously.¹² FP saturation and competition binding assays were performed in triplicate and repeated independently at least three times unless otherwise noted. The mean values were plotted. Error bars represent the S.E.M.