Reconstitution and Characterization of Aquaporins

Purified AQPs were reconstituted into proteoliposomes by mixing them with Escherichia coli lipids (Avanti Polar Lipids, Alabaster, USA) solubilized in 5% OG. The lipid-to-protein ratio (LPR) was set at 30, and the reconstitution was performed in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2 mM dithiothreitol (DTT), and 0.03% NaN₃ with a lipid concentration of 2 mg mL⁻¹. The mixture was incubated with gentle mixing (10 min at RT). OG was removed with Bio-Beads (2 h of incubation). The reconstituted proteoliposomes were extruded 11 times through an extruder (Avanti Polar Lipids) using a 200-nm Whatman polycarbonate membrane. Control liposomes were made in the same manner without protein. The size and polydispersity index (PDI) were measured using dynamic light scattering (DLS) on a Malvern Zetasizer NanoZS instrument (Malvern, UK) at 25 °C (3 measurements of 13 runs). Immunoblotting against the 6xHis-tag was done to confirm the integrity of the proteins. To assess the functional characterization of both AQPs, the osmotic water permeability (Pf) was measured by stopped-flow spectrophotometry in a PiStar-180 Spectrometer at 20 °C (Applied Photophysics, Leatherhead, UK) [37 (link)]. Pf was computed according to the equation: Pf = k_exp V₀/A_v V_w C_out, where k_exp is the fitted exponential rate constant (Figure S2), V₀ is the initial mean vesicle volume, A_v is the mean vesicle surface area, V_w is the molar volume of water, and C_out is the external osmolarity. Measurements were performed at different points (0 h, 48 h, and 1 week) and at different storage temperatures (4 °C, RT, and 37 °C).