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. 2014 Dec 10;290(5):2919–2937. doi: 10.1074/jbc.M114.611251

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FIGURE 2. — Binding of ExoU to vesicles containing PI(4,5)P₂ and PI(4)P measured by SPR analysis. A, determination of K_d for ExoU binding to PC/PS/PI(4,5)P₂ (77:20:3) vesicles by equilibrium SPR analysis. The binding isotherm was generated from the R_eq (average of triplicate measurements) versus the concentration (P₀) of ExoU plot. A solid line represents a theoretical curve constructed from R_max (= 1300 ± 120) and K_d (= 110 ± 30 nm) values determined by nonlinear least squares analysis of the isotherm using the following equation: R_eq = R_max/(1 + K_d/P₀). B, determination of K_d values for ExoU binding to PC/PS/PI(4)P (77:20:3) vesicles by equilibrium SPR analysis. R_max (= 1100 ± 60) and K_d (= 290 ± 30 nm) values were calculated as described for B. C, kinetic SPR sensorgrams for ExoU_{P. aer} binding to PC/PS/PI(4,5)P₂ (77:20:3) and PC/PI(4,5)P₂ (97:3) vesicles. D, kinetic SPR sensorgrams for ExoU_{P. aer}, ExoU_{P. flu}, and ExoU_{P. asy} binding to PC/PS/PI(4,5)P₂ (77:20:3) vesicles. ExoU_{P. flu} showed the highest degree of vesicle binding. Equilibrium SPR analysis confirmed that it has the highest affinity (K_d = 30 ± 6 nm). RU, resonance units. Error bars represent S.D.