Preparation of proteoliposomes

Liposomes were created from E. coli polar lipid extract (Avanti Polar Lipids) by slow detergent removal using BioBeads SM-2 (BioRad). A 25 mg/mL stock solution of E. coli polar lipids was created by suspending the lipids in reconstitution buffer (50 mM NaCl, 50 mM Tris-HCl pH 8.0). Lipids were diluted to 4 mg/mL in reconstitution buffer and β-OG was added to a final concentration of 2% (w/v). Protein was added to a theoretical lipid-to-protein ratio (LPR) of 50 and the solutions were briefly incubated on ice. Biobeads were then added to a bead-to-detergent weight ratio of 30:1 and the pre-liposome mixtures were incubated at 10°C with slight agitation overnight. The mixtures were centrifuged for 30 minutes at 138,000 x g. The supernatant was discarded and the pellet slowly resuspended to 2 mg lipid/mL using either reconstitution buffer or reconstitution buffer supplemented with glycerol (ca 580 mOsm, for the analysis of glycerol transport), and then filtered through a 0.2 μm filter. The liposome samples were allowed to rest for at least an hour at room temperature before measurements. A western blot was performed to ensure incorporation of the aquaporins into the liposomes (S10 Fig).