0.1 μm polycarbonate membrane

A total 5 μg of the lipid with a ratio of 50:20:3:4:8:15 PC/PE/SM/PS/PI/LBPA (Sigma) in 1 ml of the calcein buffer (50 mM calcein, 100 mM NaCl, 10 mM Na₂PO₄ and 2 mM KH₂PO₄) was prepared using a mini extruder with a 0.1 μM polycarbonate membrane (Avanti Polar Lipids). Unencapsulated calcein was removed by size exclusion chromatography. Wild-type or mutant VP5 protein were purified the same way as describe before^{14 (link)} and validated by gel-filtration chromatography in a Superdex 200 Increase 10/300 GL column in the buffer containing 10 mM Tris-HCl at pH 8.0, 150 mM NaCl and 5% (w/v) glycerol. The wild-type protein was further verified by negative stain with 2% tungsten phosphate 7.5 for the high-pH condition and sodium citrate 5.5 and 2% uranyl acetate for the low-pH condition. Proteins were mixed with calcein-loaded liposomes at a final concentration of 0.1 mg ml⁻¹ and incubated at room temperature for 10 min. The mixture was then acidified to pH 5.5 with 1 N HCl and incubated for 20 min at 37 °C. Fluorescence was measured and then the percentage of calcein release was calculated as previously described^{14 (link)}.