Fig. 6.

Lipid binding kinetics of syb-2. Stopped-flow measurements with an Alexa Fluor 488-labeled N-terminal cysteine mutant of syb-2. (A) Time course of Texas Red fluorescence emission at increasing total lipid concentrations is shown for syb-2 (lines) and at highest total lipid concentration for the SNARE complex (red symbols). (B) Observed k_obs versus total lipid concentrations. The y-intercept provides the dissociation rate constant, k_off, and the slope yields the association rate constant, k_on. Bars represent the SEs of three to five technical repeats. The solid line shows a weighted linear fit (Eq. 2). (C) Fluorescence increase of Texas Red fluorescence emission is shown at different total lipid concentrations, in the absence (colored circles) and presence (black circles) of Ca²⁺, and fitted by a Hill equation (Eq. 3) with fixed n = 1. We obtained K_d = 602 ± 107 μM and K_d = 1,425 ± 119 μM, respectively. A.U., arbitrary unit.