FIGURE 11.
Determination of the binding of recombinant PDZ3, PDZ4, PDZ123, and PDZ1234 to plasma membrane mimetic vesicles by equilibrium SPR analysis. A, left panel, sensorgrams of the time courses of binding of PDZ3 (2 μm, broken line) and PDZ4 (0.25, 0.5, 1, 2, 3, and 5 μm, lowest to highest sensorgram responses, respectively). Equilibrium SPR measurements were performed in 20 mm Tris-HCl, pH 7.4, containing 0.16 m NaCl. Proteins were injected over an L1 chip coated with PM mimetic vesicles at 5 μl/min. For PDZ4, the membrane association was too slow for the sensorgrams to reach saturation within the injection period, and thus the final resonance units (RU) values were arbitrarily taken as Req. Right panel, concentration dependence of binding of PDZ4 generated from Req values (averages of triplicate measurements). The solid line represents the curve determined by nonlinear least squares fit of the data to the equation Req = Rmax/(1 + Kd/P0), where P0 is the concentration of PDZ4, Rmax = 34 ± 2, and Kd = 1.2 ± 0.1 μm. B, left panel, sensorgrams of the time courses of binding of PDZ123 (2 μm, broken line) and PDZ1234 (0.25, 0.5, 1, 2, 3, and 5 μm, lowest to highest sensorgram responses, respectively) as described above. Right panel, concentration dependence of binding of PDZ1234 with the solid line representing the nonlinear least squares fit of the data (Rmax = 38 ± 1 and Kd = 1.1 ± 0.1 μm).