Surface plasmon resonance (SPR) analysis of interactions between TRPV1-CT and PIP2-enriched liposomes.

Kinetic binding measurements of TRPV1-CT (A) and the TRPV1-CT-K770A/R778A/R785A triple mutant (B) to the sensor chip coated with PC/PIP2 (80∶20) liposomes. The proteins at indicated concentrations were injected in parallel over the lipid vesicles and the flow rate was maintained at 30 µl/min for both association and dissociation phases of the sensograms. (C) SPR equilibrium binding of the TRPV1-CT, TRPV1-CT-K770A/R778A/R785A, and TRPV1-CT-R778A proteins to the sensor chip coated with PC/PIP2 (80∶20) liposomes. The proteins were injected at 25 µl/min at different concentrations and washed over the lipid surface and Req values were deduced from steady state (equilibrium) SPR response. The solid lines represent binding isotherms determined by nonlinear least-squares analysis of the isotherm using an equation Req = Rmax/1+Kd/P0), where Req stands for SPR response value near -equilibrium, Rmax is the maximum response and P0 is the protein concentration. Values represent the mean ± S.D from four independent experiments.