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4 protocols using 1 palmitoyl 2 oleoyl sn glycero 3 phosphatidylethanolamine

1

Preparation of Lipid Vesicles for Biophysical Studies

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Lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid (POPA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), were obtained from Avanti Polar Lipids (Alabaster, AL). For circular dichroism, fluorescence and 1D NMR experiments, large unilamellar vesicles were prepared using extrusion as previously described [29 (link),30 (link)]. Vesicles with zwitterionic lipids, POPC (100%) and POPE/POPC (40/60 mol%), and with negatively charged lipids, POPG/POPC (40/60 mol%) and POPA/POPC (10/90, 20/80, 30/70, 40/60 and 50/50 mol%), were produced. Lipids were dissolved in chloroform and the chloroform was evaporated overnight under nitrogen gas. The lipid mixtures were resuspended in 1 ml of the appropriate buffer for the experiment for 30 min by vortexing the samples. Next, each sample went through five cycles of freezing in liquid nitrogen and thawing in a water bath (∼60 °C). Finally, samples were extruded through a 100 nm polycarbonate membrane (Avanti Polar Lipids, Alabaster, AL). The size of the extruded vesicles was analyzed by dynamic light scattering (ALV-laser, Langen, Germany) and vesicles were found to have a radius of around 65 nm.
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2

Lipid and Peptide Preparation for Biophysical Studies

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Lipids including 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE), 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol (Chol), and cardiolipin 1′,3′-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol (TOCL) are purchased from Avanti Polar Lipids (Alabaster, AL). 5-doxyl stearic acid (5-SASL), TmCl3·6H2O, and VC (vitamin C) are purchased from Sigma-Aldrich (St. Louis, MO). C-terminal amidated peptide PrP106-126 (K106TNMKHMAGAAAAGAVVGGLG126-NH2) is synthesized by the solid synthesis method. For simplicity, we will refer to the peptide as PrP106-126 without explicitly noting the amidation. Peptide purity is 98% as indicated by analytical HPLC elution. Fresh solutions containing monomeric PrP106-126 are prepared by dissolving lyophilized peptide in 10 mM HEPES pH 7.4. Centrifugation is used to remove preexisting insoluble aggregates.
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3

Lipid Composition for Membrane Studies

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1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE), soy l-α-phosphatidylinositol (PI), 1-oleoyl-2-(6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-2-propionyl)amino)-hexanoyl)-sn-glycero-3-phosphoinositol (TopFluor TMR PI), 1-oleoyl-2-(6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-2-propionyl)amino)-hexanoyl)-sn-glycero-3-phosphatidylethanolamine (TopFluor TMR PE), and 1-palmitoyl-2-{12-[(7-nitro-2–1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phosphatidylcholine (NBD PC) were purchased from Avanti Polar Lipids (Alabaster, AL). Buffer components tris(hydroxymethyl)-aminomethane (Tris), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium chloride, ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), and benzamidine were purchased from Fisher Scientific (Pittsburgh, PA). Dodecyl thiomaltopyranoside was purchased from Anatrace (Maumee, OH). The buffer solution used for all experiments contained 50 mM Tris, 600 mM HEPES, 300 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 4 mM benzamidine, and 24.7 μM Dodecyl thiomaltopyranoside (pH 8.0) (referred to as Amot ACCH domain elution buffer).
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4

Lipid Membrane Composition and Peptide Incorporation

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Lipids including 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and Chol were purchased from Avanti Polar Lipids (Alabaster, AL). 5-doxyl stearic acid (5-SASL) was purchased from Sigma-Aldrich (St. Louis, MO). The 16-residue peptide M2AH of sequence F47FKSIYRFFEHGLKRG62 was synthesized by Genscript. The vendor confirmed peptide purity >90% by HPLC and mass spectroscopy. Fresh peptide stock solution was prepared by dissolving lyophilized powder in 10 mM HEPES pH 7.4. Unless noted otherwise, all ratios and percentages are molar based in this paper.
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