Figure 1
Characterization of proteopliposomes. (A) Diagrams illustrating the compositions of T1, V1, T2 and V2 liposomes. The light blue color in the membranes of the V1 and T2 liposomes represents the mixture of fluorescent lipids (NBD-PE and MB-PE). The PhycoE-Biotin trapped in the lumen of the T1 and V2 liposomes is represented by a black square with a yellow circle, whereas the Cy5-SA trapped in the lumen of the V1 and T2 liposomes is represented by a pie shape with a red circle. Note that syntaxin-1 and SNAP-25 are shown forming a heterodimer with a 2:1 stoichiometry (reviewed in52). (B) Equal amounts of T1, V1, T2 and V2 liposomes were analyzed by SDS-PAGE and coomassie blue staining. The positions of molecular weight markers are indicated on the left, and those of synaptobrevin (Syb), SNAP-25 (SN25), syntaxin-1 (Syx), SA and PhycoE (PhE) are indicated on the right. The uncropped gel is shown in Supporting Fig. S1. (C) DLS analysis of a sample of T2 liposomes illustrating the particle size distribution typical of the liposome preparations. (D) Average radii of representative samples of T1, V1, T2 and V2 liposomes obtained from three independent determinations by DLS. Error bars indicate standard deviations. (E) Fluorescence emission spectra of V1 and T2 liposomes before and after addition of β-OG. The MB fluorescence was excited at 370 nm. (F) Fluorescence emission spectra of the same liposomes as in panel (E), exciting the NBD fluorescence at 465 nm. (G) Fluorescence emission spectra of T1 and V2 lipososomes before and after addition of β-OG. The PhycoE fluorescence was excited at 488 nm. (H) Fluorescence emission spectra of V1 and T2 liposomes before and after addition of β-OG. The Cy5 fluorescence was excited at 645 nm.
