Quantitative Analysis of Peptide Uptake by Liposomes

TmrAB-containing liposomes (0.4 µM) were incubated with C4^ATTO655 peptides (1 µM), ATP or ADP (3 mM each), MgCl₂ (5 mM), and CP^Fs (1 µM) for 5 min at 45°C. Transport reactions were stopped by adding EDTA (10 mM). Samples were diluted to a final TmrAB concentration of 20 nM with transport buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 5% [v/v] glycerol) and analyzed by flow cytometry (FACSCelesta). For regression analysis, liposomes were loaded with increasing amounts of peptides to convert the fluorescence intensities into the number of peptides per liposome (Stefan et al., 2020 (link)). For this, liposomes were destabilized by adding Triton X-100 while increasing the C4^ATTO655 concentration. Equal amounts of carboxyfluorescein (Sigma-Aldrich) served as loading control. Detergent was removed by adding SM-2 Bio-beads (Bio-Rad) as detailed above. Liposomes were harvested for 30 min at 270,000 g and washed three times by centrifugation. Generally, 20,000–100,000 proteoliposomes were selected according to sideward and forward scatter areas. Single events were selected based on the height of forward scatter plotted against the area of forward scatter. Mean fluorescence intensities of fluorescein and ATTO655 were calculated using FlowJo 10.6.1 software. Data represent mean ± SD from three experiments.