FIGURE 4.

The asymmetry of giant unilamellar LPS R45/PL vesicles is confirmed by leaflet-specific fluorescence quenching with KI. (A,a) Asymmetric vesicles with LPS R45 in the outer leaflet and PL (PE:PG:CL; 81:17:2; w/w/w) in the inner leaflet; 5% (w/w) of the LPS R45 molecules in the outer leaflet were fluorescent FITC-LPS R45; (B,b) Quenched fluorescence of the vesicles after the addition of KI. The inlet shows the vesicle in phase contrast; (C,c) Asymmetric vesicles with LPS R45 in the outer leaflet and PL in the inner leaflet; 2% (w/w) of the PL molecules in the inner leaflet were fluorescent FITC-PE; (D,d) Vesicles after the addition of KI. The fluorophores in the inner leaflet were protected from quenching. Experiments were carried out at 25°C in 100 mM KCl, 5 mM MgCl₂, 5 mM HEPES buffer at pH 7.0 using a Leica TCS SP3 confocal microscope. Final KI concentration was 0.83 M; Scale bar applies to all images.