14 research outputs found
Tuning the lipid bilayer: the influence of small molecules on domain formation and membrane fusion
Alle levende organismen bestaan uit cellen omgeven door een membraan. Dit membraan bestaat uit een lipide bilaag welke transport-, receptor- en kanaaleiwitten bevat. Verwacht wordt dat membranen domeinen bevatten, genaamd ‘rafts’. Eiwitten associeren met deze rafts of worden juist buitengesloten, waardoor bepaalde eiwitten dicht bij elkaar komen of juist bij elkaar uit de buurt worden gehouden. Omdat rafts zo klein zijn en dynamisch, zijn modelsystemen ontwikkeld om interacties tussen lipiden en eiwitlocalisatie te kunnen onderzoeken. Één van deze model membranen zijn gigantische unilamellaire vesikels (GUVs), welke tientallen micrometers groot kunnen zijn en daardoor bestudeerd kunnen worden met lichtmicroscopie. GUVs bestaande uit drie verzadigde en onverzadigde lipiden en cholesterol scheiden in twee fasen: een dichte geordende vloeibare Lo fase en een meer vloeibare Ld fase. In dit proefschrift heb ik laten zien dat kleine moleculen zoals suikers, koolwaterstoffen en bepaalde lipiden de fase scheiding beïnvloeden. De verdeling (partitie) van het model peptide WALP over de Lo en Ld domeinen van de membraan is bestudeerd. Het tweede deel van dit proefschrift is gericht op kleinere vesikels, zogenoemde grote-unilamellaire vesikels (LUVs). Vesikels gemaakt van niet-ionogene oppervlakte-actieve stoffen zijn gekarakteriseerd. Ik laat zien dat deze vetachtige stoffen gesloten vesikels kunnen vormen, niosomen genoemd, die qua eigenschappen te vergelijken zijn met vesikels gebaseerd op lipiden. Membraanfusie een delicate balans is tussen het verstoren van het membraan en handhaven van de permeabiliteitsbarriere. Ik ben er in geslaagd membraanfusie te realiseren zonder noemenswaardige lekkage van de vesikelinhoud door gebruik te maken van een enzym welke de kopgroepen van een deel van de lipiden afknipt
A trifunctional linker for palmitoylation and peptide and protein localization in biological membranes
Attachment of lipophilic groups is an important post-translational modification of proteins, which involves the coupling of one or more anchors such as fatty acids, isoprenoids, phospholipids, or glycosylphosphatidyl inositols. To study its impact on the membrane partitioning of hydrophobic peptides or proteins, we designed a tyrosine-based trifunctional linker. The linker allows the facile incorporation of two different functionalities at a cysteine residue in a single step. We determined the effect of the lipid modification on the membrane partitioning of the synthetic α-helical model peptide WALP with or without here and in all cases below; palmitoyl groups in giant unilamellar vesicles that contain a liquid-ordered (Lo) and liquid-disordered (Ld) phase. Introduction of two palmitoyl groups did not alter the localization of the membrane peptides, nor did the membrane thickness or lipid composition. In all cases, the peptide was retained in the Ld phase. These data demonstrate that the Lo domain in model membranes is highly unfavorable for a single membrane-spanning peptide
Lipid phase separation in the presence of hydrocarbons in giant unilamellar vesicles
Hydrophobic hydrocarbons are absorbed by cell membranes. The effects of hydrocarbons on biological membranes have been studied extensively, but less is known how these compounds affect lipid phase separation. Here, we show that pyrene and pyrene-like hydrocarbons can dissipate lipid domains in phase separating giant unilamellar vesicles at room temperature. In contrast, related aromatic compounds left the phase separation intact, even at high concentration. We hypothesize that this behavior is because pyrene and related compounds lack preference for either the liquid-ordered (Lo) or liquid-disordered (Ld) phase, while larger molecules prefer Lo, and smaller, less hydrophobic molecules prefer Ld. In addition, our data suggest that localization in the bilayer (depth) and the shape of the molecules might contribute to the effects of the aromatic compounds. Localization and shape of pyrene and related compounds are similar to cholesterol and therefore these molecules could behave as such
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A Trifunctional Linker for Palmitoylation and Peptide and Protein Localization in Biological Membranes
Attachment of lipophilic groups is an important post-translational modification of proteins, which involves the coupling of one or more anchors such as fatty acids, isoprenoids, phospholipids, or glycosylphosphatidyl inositols. To study its impact on the membrane partitioning of hydrophobic peptides or proteins, we designed a tyrosine-based trifunctional linker. The linker allows the facile incorporation of two different functionalities at a cysteine residue in a single step. We determined the effect of the lipid modification on the membrane partitioning of the synthetic α-helical model peptide WALP with or without here and in all cases below; palmitoyl groups in giant unilamellar vesicles that contain a liquid-ordered (Lo) and liquid-disordered (Ld) phase. Introduction of two palmitoyl groups did not alter the localization of the membrane peptides, nor did the membrane thickness or lipid composition. In all cases, the peptide was retained in the Ld phase. These data demonstrate that the Lo domain in model membranes is highly unfavorable for a single membrane-spanning peptide. © 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA
Membrane permeability of niosomes and liposomes.
<p>A: Graphical representation of the ion permeability of the vesicles. B: Proton permeability measured by fluorescence of the pH-sensitive dye pyranine in liposomes composed of unsaturated lipids plus cholesterol in the presence and absence of the sodium ionophore ETH-157. ETH-157 (5 μM, final concentration) or ethanol (0.1% v/v) were present from the start of the experiment. At time point 0 (indicated by an arrow), the medium pH was decreased from 7.5 to 6.3 by the addition of 10 mM HCl (large pulse) or from 7.5 to 7.0 by the addition of 4 mM HCl (small pulse). Black line: ethanol, small pulse; red line: ETH-157, small pulse; green line: ethanol, large pulse; blue line: ETH-157, large pulse. For comparison, liposomes composed of saturated lipids plus cholesterol subjected to a large HCl pulse (in the absence of ETH-157) are shown in grey. Average values of two experiments are shown. C: Proton permeability of niosomes composed of unsaturated surfactants plus cholesterol in the absence (0.1% v/v ethanol) or presence of the sodium ionophore ETH-157 (5 μM, final concentration). At time point 0 (indicated by an arrow), the medium pH was decreased from 7.5 to 6.3 by the addition of 10 mM HCl (large pulse) or from 7.5 to 7.0 by the addition of 4 mM HCl (small pulse). Black line: ethanol, small pulse; red line: ETH-157, small pulse; green line: ethanol, large pulse; blue line: ETH-157, large pulse. Niosomes composed of saturated surfactants plus cholesterol subjected to a large HCl pulse (in the absence of ETH-157) are shown in grey. Average values of two independent experiments are shown. D: KCl permeability of liposomes and niosomes filled with the fluorescent dye calcein (5 mM) after osmotic upshift by KCl. The arrow at 50s indicates the moment 0.4 M KCl (final concentration) was added. Green lines: niosomes composed of unsaturated surfactants plus cholesterol; red lines: niosomes composed of saturated surfactants plus cholesterol; black lines: liposomes composed of unsaturated lipids plus cholesterol; blue lines: liposomes composed of saturated lipids plus cholesterol. Representative traces of one out of three independent experiments are shown. E: Stability of liposomes and niosomes filled with the fluorescent dye calcein (5 mM) after osmotic upshift by glycerol. At 50s (indicated by a black arrow), 0.667 M glycerol was added (osmolarity comparable to that of 0.4M KCl); line color as indicated under B. Representative traces of one out of two independent experiments are shown. F: Stopped-flow measurements of the effects of osmotic upshift elicited by glycerol (red line) or KCl (green line) in niosomes composed of unsaturated surfactants plus cholesterol. Buffer (black line) is shown as a control. Representative traces of one out of two independent experiments are shown.</p
The L-arginine/L-ornithine antiporter ArcD2 is active in liposomes with anionic lipids but not in vesicles that do not contain lipids or surfactants with anionic headgroups.
<p>ArcD2 was reconstituted in liposomes and niosomes at 1 to 400 protein to lipid ratio (w/w). A: Schematic representation of the transport reaction. B: ArcD activity was measured using radiolabeled arginine. Green lines: niosomes composed of unsaturated surfactants plus cholesterol; blue lines: liposomes composed of unsaturated lipids plus cholesterol; black lines: liposomes composed of unsaturated lipids of which 38% is anionic (phosphatidylglycerol). Representative traces of one out of three independent experiments are shown. C: Incorporation of ArcD2 into vesicles was confirmed by Western blot analysis. 1: solubilized ArcD2, purified protein before reconstitution; 2: Unsaturated surfactants + cholesterol; 3: Saturated surfactants + cholesterol; 4: Unsaturated lipids + cholesterol.</p
Filter-extruded niosomes decrease in size upon freezing and thawing.
<p>A: Size of vesicles composed of unsaturated surfactants plus cholesterol (green: without freezing and thawing; red: with freezing and thawing), and vesicles composed of unsaturated lipids plus cholesterol (black: without freezing and thawing; blue: with freezing and thawing), measured by dynamic light scattering. Prior to the analysis the vesicles were extruded 15 times through a 200 nm polycarbonate filter. B-C: Cryo-EM pictures of niosomes composed of unsaturated surfactants plus cholesterol without (B) and with five freeze and thaw cycles (C). Niosomes appear smaller due to the freezing and thawing steps. As guidance, all niosomes are indicated with a red arrow in the right picture. In contrast, cryo-EM pictures of liposomes composed of unsaturated lipids plus cholesterol without (D) and with five freeze and thaw cycles (E) appear similar in size but the degree of multilamellarity decreases by the freezing-thawing and subsequent extrusion step.</p
Lipid composition of niosomes and liposomes used in this study.
<p>Lipid composition of niosomes and liposomes used in this study.</p
Examples of non-ionic surfactants used in this study.
<p>Examples of non-ionic surfactants used in this study.</p