Nature Methods: doi: /nmeth Supplementary Figure 1. Salipro lipid particles.
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1 Supplementary Figure 1 Salipro lipid particles. (a) Gel filtration analysis of Saposin A after incubation with the indicated detergent solubilised lipid solutions. The generation of Saposin A-lipid complexes was unfavorable using PE and an E. coli total lipid extract. The void volume is marked with an asterisk, the peak for monomeric Saposin with (^). (b) Negative-stain electron microscopy images of lipid-only Salipro particles. The scale bar=50 nm. (c) Gel filtration analysis of Saposin A after incubation with varying amounts of lipids ("Lipids 5": 5 µg, "Lipids 12.5": 12.5 µg, "Lipids 25": 25 µg, "Lipids 50": 50 µg, "Lipids 100": 100 µg) from a brain-lipid solution (5 mg/ml brain lipids) in a final reaction volume of 65 µl. Peaks for monomeric Saposin are marked with (^) and for Saposin-lipid complexes with (#). (d) Gel filtration analysis of lipid Salipro particles after a 10 min incubation step at the indicated temperatures. The void volume is marked with an asterisk, the peaks for monomeric Saposin with (^) and Saposin-lipid complexes with (#).
2 Supplementary Figure 2 Incorporation of the bacterial POT transporter. (a) Gel filtration analysis of purified Salipro-T2 that has been concentrated, frozen, thawed, diluted and subjected to SEC. The void volume is marked with an asterisk and the peak for Salipro-T2 with (<>). (b) Gel filtration analysis of the incorporation of the tetrameric PepT channel into Salipro nanoparticles. The void volume is marked with an asterisk, the peaks for monomeric Saposin with (^), Saposin-lipid complexes with (#) and Salipro-POT with (<>). Note that the gel filtration buffer is 1xPBS ph 7.4. In the absence of Saposin and lipids, the membrane protein POT completely aggregates under these conditions (green line). (c) Gel filtration analysis of the incorporation of the tetrameric PepT channel into Salipro nanoparticles at various ph conditions, as indicated. The void volume is marked with an asterisk, the peaks for monomeric Saposin with (^), Saposin-lipid complexes with (#) and Salipro-POT with (<>). (d) Thermal unfolding analysis with dye-free differential scanning fluorimetry (using two samples each for POT in detergent (1x PBS, ph 7.4, 0.4% NM) and Salipro-POT (1x PBS ph 7.4). Deviation of the mean is indicated. The bacterial peptide transporter is significantly more stable when embedded in Salipro nanoparticles (Tm 72 C) as compared to detergent micelles (POT in Nonyl-β-D- Maltopyranoside, Tm 43 C). (c) Stoichiometry of Salipro-POT. SDS-PAGE of purified Salipro-POT1 and BSA-standards in concentrations as indicated, suggesting a 1:1 ratio of Saposin / POT. Given a tetrameric POT, this means each Salipro-POT contains four Saposin proteins, as confirmed by the cryo-em structure.
3 Supplementary Figure 3 Salipro-POT density displayed in two different isosurface levels. Left: Top and side view of the 3D reconstruction displayed at two different isosurface levels (high in orange, low in grey mesh). At low isosurface level, the density of the Saposin-lipid scaffold is visible. Right: Side and top view, cut within the plane of the membrane as indicated (dashed line).
4 Supplementary Figure 4 Solubilization of radioactively labeled HIV-1 VLPs with high critical micelle concentration (CMC) detergents. HIV-1 VLPs were solubilised in 1x HNC buffer containing 25 mm Anameg-7 (CMC 19.5 mm) (lane 2), 9 mm HEGA-10 (CMC 7 mm) (lane 3), 14 mm C-HEGA-11 (CMC 11.5 mm) (lane 4), 9 mm MEGA-10 (CMC 6-7 mm) (lane 5), 12 mm n18 Octyl-beta-D- Thiomaltopyranoside (OT) (CMC 9 mm) (lane 6), or 10 mm Tetraethylene Glycol Monooctyl Ether (C8E4) (CMC 10 mm) (lane 7), for 10 min on ice (a) or for 30 min at 37 C (b) and analysed by BN-PAGE. VLPs solubilised in TX100 (TX) on ice were analyzed as control (lane 1). Migration of spikes and gp monomers are indicated. Note the dissociation of spikes into monomers by the 37 C incubation.
5 Supplementary Figure 5 Schematic illustration of the Salipro HIV spike purification. For detailed description see material and methods. Saposin A (blue) is mixed together with HEGA-10 and VLPs containing HIV-1 proteins such as, gp120 subunit (brown), gp41 subunit (grey), CA protein (white circle) and MA protein (green). HEGA-10 is then removed using two subsequent spin SEC columns (Molecular weight cut-off 7 kda) and the Salipro-HIV-spike particles are purified using a lectin affinity column. The lectin binds to high mannose sugar moieties on the spike protein. The column was washed with 50 column volumes HN buffer followed by elution using 0.3 M methyl-αd-mannopyranoside.
6 Supplementary Figure 6 Optimization of the amount of saposin A for efficient Salipro HIV spike nanoparticle formation. Radioactively labeled VLPs were mixed with Saposin A ( µg/ml) followed by 10 min solubilisation on ice using 9 mm HEGA-10 in 1x HNC buffer. HEGA-10 was then removed using a SEC spin column and the amount of reconstituted Salipro-HIV-spike particles was monitored by BN-PAGE. About 100 µg/ml Saposin A was found to be optimal.
7 Supplementary Table 1: Selection of scaffolding systems for membrane proteins Name Scaffold Type Adjusts to size of Lipid Reference membrane protein environment Peptitergents Peptide, 24-residue amphipathic α-helix No Schafmeister, C. E., Miercke, L. J. & Stroud, R. M. Structure at 2.5 A of a designed peptide that maintains solubility of membrane proteins. Science 262, (1993). Lipopeptide detergents β-sheet peptides Peptide, 25-residue amphipathic α-helix with fatty acyl chains linked to side chains acetyl-(octyl)gly- Ser-Leu-Ser-Leu- Asp-(octyl)Gly-Asp- NH2 No (fatty acyl chains to mimic lipid environment) McGregor, C. L. et al. Lipopeptide detergents designed for the structural study of membrane proteins. Nature biotechnology 21, , doi: /nbt776 (2003). No Tao, H. et al. Engineered nanostructured beta-sheet peptides protect membrane proteins. Nature methods 10, , doi: / nmeth.2533 (2013). Amphipols Amphiphilic polymer No Tribet, C., Audebert, R. & Popot, J. L. Amphipols: polymers that keep membrane proteins soluble in aqueous solutions. Proceedings of the National Academy of Sciences of the United States of America 93, (1996). SMALPs Styrene Maleic Acid Copolymer No Knowles, T. J. et al. Membrane proteins solubilized intact in lipid containing nanoparticles bounded by styrene maleic acid copolymer. Journal of the American Chemical Society 131, , doi: /j a810046q (2009). Nanodiscs Apolipoprotein A-1 No Bayburt, T. H., Carlson, J. W. & Sligar, S. G. Reconstitution and imaging of a membrane protein in a nanometer-size phospholipid bilayer. Journal of structural biology 123, 37-44, doi: / jsbi (1998). Macrodiscs ΔMSP ΔH-MSP variants 14-residue peptide derived from Apolipoprotein A-1 Truncated version of Apolipoprotein A-1 Truncated versions of Apolipoprotein A- 1 No (Diameter can be varied by at least 3- fold by changing the lipid:peptide molar ratio) No (Diameter can be varied by changing the molar ratio of ΔMSP to lipid) No (Diameter can be varied by changing the molar ratio of scaffold protein to lipid) Park, S. H. et al. Nanodiscs versus macrodiscs for NMR of membrane proteins. Biochemistry 50, , doi: /bi201289c (2011). Wang, X., Mu, Z., Li, Y., Bi, Y. & Wang, Y. Smaller Nanodiscs are Suitable for Studying Protein Lipid Interactions by Solution NMR. The protein journal 34, , doi: /s (2015). Hagn, F., Etzkorn, M., Raschle, T. & Wagner, G. Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins. Journal of the American Chemical Society 135, , doi: /ja310901f (2013). Salipro Saposin lipoproteins Frauenfeld, J. et al. A saposinlipoprotein nanoparticle system for membrane proteins. Nature Methods (2016) Nature Methods doi: /nmeth.3801
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