Liposome preparation and protein reconstitution.

The lipids used in the study were 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phoshpo-l-serine (POPS), and Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Rh-DHPE) (Fig. S1). Proteoliposomes were prepared using detergent-mediated reconstitution as described elsewhere (32). POPC–POPS–POPE–Rh-DHPE were combined at a molar ratio of 80:10:9.5:0.5 to yield 1 μmol lipid. Lipids were dried under a stream of N₂ gas and spun in an Eppendorf VacFuge at 30°C for 1 h. The dried lipid film was hydrated in 500 μl of HEPES buffer (20 mM, pH 7.4) containing NaCl (0.5 M), CHAPS (40 mM), and glycerol (10% [vol/vol]) to a final lipid concentration of 2 mM. Detergent-solubilized CobS^WT was added at a lipid-to-protein molar ratio of 1,000:1 and incubated with rotation on a Benchmark RotoBot at 4°C for 1 h. Detergent removal and proteoliposome formation was achieved by four 20-h dialyses using Slide-A-Lyzer dialysis cassettes (20-kDa molecular mass cutoff) against HEPES buffer (20 mM, pH 7.4) containing NaCl (0.5 M) and glycerol (10% [vol/vol]). Dialysis was performed at a dialysate/sample volumetric ratio of 2,000:1. After dialysis, the proteoliposome suspension was applied to a HistoDenz (Sigma) gradient to perform a liposome flotation assay. The proteoliposome suspension was mixed with equal volumes HEPES buffer (20 mM, pH 7.4) containing NaCl (0.15 M), HistoDenz (80% [wt/vol]), and glycerol (10% [vol/vol]) and deposited in a Beckman Coulter ultracentrifuge tube (polyallomer, 13 by 51 mm). A 4-ml overlay of HEPES (20 mM, pH 7.4) containing NaCl (0.15 M), HistoDenz (30% [wt/vol]), and glycerol (10% [vol/vol]) was applied followed by a 200-μl overlay of HEPES buffer (20 mM, pH 7.4) containing NaCl (0.15 M) and glycerol (10% [vol/vol]). The gradient was subjected to centrifugation at 4°C for 3 h at 268,000 × g in a refrigerated Beckman Coulter Optima MAX-XP ultracentrifuge using an MLS-50 rotor. The size distribution of reconstituted liposomes was determined by dynamic light scattering in a 1.5-ml polystyrene cuvette using a Zetasizer Nano (Malvern Instruments). Lipid concentration of the reconstituted liposomes was determined by a standard curve generated from fluorescence of Rh-DHPE. Fluorescence measurements were read on a BioTek Gemini instrument set at an excitation wavelength of 540 nm and emission wavelength of 586 nm. Protein concentration in proteoliposomes was determined by generating a standard curve. Briefly, a range of known bovine γ-globulin (BGG) and lysozyme concentrations were separated by SDS-PAGE with unknown concentrations of CobS-containing proteoliposomes stained with Oriole fluorescent stain and analyzed using TotalLab TL100 software. The presence of CobS in liposomes was confirmed by Western blot and mass spectrometry analyses.