Supplementary Figure 1: Characterization of His₁₀-SNAP₅₀₅–liposomes interaction and the effect of PS on Lck phosphorylation of CD3ζ.

(a, b) FRET based kinetic analysis of the interaction between His₁₀-SNAP₅₀₅ and Ni²⁺-NTA-containing liposomes. Purified His₁₀-SNAP was fluorescently-labeled with SNAP-cell 505 (designated as His₁₀-SNAP₅₀₅), which served as a FRET donor for membrane-conjugated rhodamine. Panel a, shown in red is the representative time course of fluorescence (excitation: 504 nm; emission: 540 nm) change of 0.25 μM His₁₀-SNAP₅₀₅ upon mixing with 1.1 nM rhodamine-bearing Ni-NTA liposomes, as monitored by a plate reader. Single exponential fitting using Graphpad Prism 5.0 yielded an observed rate constant (k_obs) of 0.08 s^-1. The black trace corresponds to the fluorescence of His₁₀-SNAP₅₀₅ upon mixing buffer. Panel b, k_obs measured in a plotted as a function of liposome concentration. Assuming pseudo first order kinetics, the elementary rate constants of the binding reaction was determined by linear regression, as previously described¹. The on-rate (k_on) and off-rate (k_off) were 1.4 × 10⁶ M^-1s^-1 and 0.0009 s^-1, respectively, from which the dissociation constant (K_d) was computed (0.6 nM). Error bars represent s.e.m. from triplicate measurements. (c) Inclusion of PS in the membranes moderately accelerated the kinetics of Lck catalyzed phosphorylation of CD3ζ. The time course of CD3ζ phosphorylation was followed by FRET essentially as described in Fig. 1a,b. His₁₀-Lck (~280 μm^-2) and His₁₀-CD3ζ (~1300 μm^-2) were attached to liposomes that contained 10% DGS-NTA-Ni, 0.3% Rhod-PE and indicated molar fractions of PS (at the cost of PC). Shown are representative traces of SNAP₅₀₅-tSH2 fluorescence before and after the addition of ATP, under the three indicated PS content. The inset shows the first 1 min linear phase of the fluorescence changes, allowing the estimation of initial rates of phosphorylation under different conditions. Increasing PS content from 0% to 10% accelerated CD3ζ phosphorylation by 1.8-fold. No further rate enhancement was observed when PS content increased from 10% to 20%. A similar result was obtained in an independent experiment.