Surface plasmon resonance (SPR) analysis

All SPR measurements were performed at 23 °C in 20 mM Tris-HCl, pH 7.4, containing 0.16 M KCl using a lipid-coated L1 chip in the BIACORE X system as described previously^44,45. PM-mimetic vesicles containing cholesterol (or 7-dehydrocholesterol) and POPC vesicles were used as the active surface and the control surface, respectively. Vesicles were injected at 5 μl/min onto the corresponding sensor chip surfaces to yield the identical resonance units (RU), ensuring the equal concentration of the coated lipids. Equilibrium measurements were performed at a flow rate of 5 μL/min, which allowed enough time for the R values of the association phase to reach near equilibrium levels (R_eq)⁴⁶. Each sensorgram was background-corrected by subtracting the control surface response from the active surface response. A minimum of 5 different protein concentrations was injected to collect a set of R_eq values plotted against the protein concentrations (P_o). An apparent dissociation constant (K_d) was then determined by nonlinear least squares analysis of the binding isotherm using the equation, R_eq = R_max / (1 + K_d/P_o) where R_max indicates the maximal R_eq value⁴⁷. Since the concentration of lipids coated on the sensor chip cannot be accurately determined, K_d is defined as P_o yielding half-maximal binding with a fixed lipid concentration. Each measurement was repeated ≥ 3 times to determine average and standard deviation values. For kinetic measurements, the flow rate was maintained at 20–30 μL/min.