Lipid Vesicle Preparation and Peptide Binding Assay

POPG, dimyristoylphosphatidylcholine, or dipalmitoylphosphatidylcholine (Avanti Polar Lipids), were prepared by extrusion as reported previously (66 (link)). The phospholipids solution was transferred to a glass vial, and the solvent was evaporated under a stream of nitrogen. The vial was placed in the lyophilizer under vacuum overnight. The lipid film was resuspended by adding PBS buffer to obtain a multilamellar vesicle suspension with a POPG concentration of 1 mg/ml. The lipid solution was subjected to a water bath sonication to disrupt the multilamellar vesicles. LUVs were then prepared by extrusion, applying 21 passes through a polycarbonate filter with a 100-nm average pore diameter (Avestin). Samples for membrane-binding studies were prepared by adding 5 molar eq of lipid to the Nt17 peptide solution (60 μm final concentration) and incubating the peptide/LUV samples at room temperature for 1 h before CD measurements.