Polycarbonate filter

POPE and POPG (3:1) pure lipids (Avanti) were mixed with MMK buffer [10
mM MOPS (pH-6.5), 100 mM KCl, 5 mM MgCl₂]. The final concentration
was kept at 10 mg/ml. Lipids were vortexed until completely homogenized with no
visible clumps. Lipids were flash frozen in liquid nitrogen and thawed in warm
water for eight cycles. Liposomes were then extruded using 200 nm polycarbonate
filters (Whatman) for 21 cycles. For reconstitution 250 μl liposomes were
destabilized by 0.65% (final concentration) of sodium cholate. QacA and ATPase
(2:1 molar ratio) were incubated with the liposomes for 30 min at RT; equal
amount of ATPase was incubated with liposomes as a ATPase control. Detergent was
removed using sephadex G25 beads (Sigma) and the sample was collected in a final
volume of 1.5 ml. For assay, liposomes (100 μl) were added in 2 ml of MMK
buffer (pH 6.5) containing 130 nM valinomycin and 200 nM ACMA
(λ_Ex=410 nm, λ_Em=480 nm), ATP (130
μM) was added at 50^th second, substrate (TPP 0.5 mM, Pm 250
μM, Dq 1 mM) was added at 250^th second, NH₄Cl (4
mM) at 300^th second was added as a uncoupler to abolish the pH
gradient.

To prepare samples for solid-state NMR experiments, 8 mg of DMPC dissolved in CHCl₃ was used for each sample preparation. The samples were dried under a stream of nitrogen gas, followed by overnight drying under a vacuum (30 °C) to completely remove any residual solvent. Tris buffer (10 mM Tris, pH 7.4) was added to hydrate the lipid film, vortexed for 2 min above the lipid’s phase transition temperature, and freeze-thawed between liquid nitrogen temperature and 35 °C for at least four times. To obtain a uniform size of large unilamellar vesicles (LUVs) of 1 μm in diameter, the LUVs were extruded through polycarbonate filters (pore size of 1 μm, Nuclepore, Whatman, NJ, USA) mounted on a mini extruder (Avanti Polar Lipids, AL, USA) fitted with two 1.0 mL Hamilton gastight syringes (Hamilton, NV, USA). The samples were typically subjected to 23 passes through the filter. An odd number of passages were used to avoid contamination of the sample by vesicles that have not passed through the first filter. The SMA-QA sample, dissolved in Tris buffer, was added to LUVs to prepare the desired polymer concentration by making a final sample volume of 150 μL. The mixture was freeze-thawed between liquid nitrogen and 35 °C for three to five times to get a transparent solution.