Preparation of CL/DPPC liposome

We adopted 20 mol% of (18:1)₄CL/DPPC as the experimental system. Considering the critical micelle concentration (CMC) difference between DOPC and DPPC^56–58, the DPPC liposome can be formed readily. Plus, lipids containing lower saturation of the fatty acyl chains were considered to exist in inner membrane contact sites⁵⁹. Furthermore, the literature also showed that 20 mol% of DPPC/CL formed the most thermodynamically stable binary monolayer⁶⁰. CL-containing liposome was prepared by dissolving DPPC (7.56 mg) and 18:1 CL (3.75 mg) at a molar ratio of 4:1 in chloroform in a glass vial. The chloroform was then evaporated using a rotary evaporator at 45 °C, leaving a liposomal gel film. The dried constituents were reconstituted with 1 mL of 100 mM KCl (for HDXMS) or 50 mM Tris–HCl (pH 7.0, for QCM-D), and sonicated at 60 °C for 30 min. The resulting liposome was sequentially extruded through a polycarbonate filter (100 nm, Whatman) to form liposomal capsules. Before HDXMS experiments, the CL/DPPC liposome was equilibrated in a 30 °C water bath for 30 min.