dioleoyl-phosphatidylethanolamine and 1-palmitoyl-2-oleoylphosphatidylcholine

Understanding how small molecules cross cell membranes is crucial to pharmaceutics. Several methods have been developed to evaluate such a process, but they need improvement since many false-positive candidates are often selected. Robust tools enabling rapid and reproducible screening can increase confidence on hits, and artificial membranes based on droplet interface bilayers (DIBs) offer this possibility. DIBs consist in the adhesion of two phospholipid-covered water-in-oil droplets which reproduce a bilayer. By having donor and acceptor droplets, the permeability of an analyte can be studied. However, the relevance of this system relies on the comprehension of how well the physical chemistry of the produced bilayer recapitulates the behavior of cell membranes. This information is missing, and we address it here. Taking small fluorophores as model analytes, we studied their permeation through DIBs made of a wide range of phospholipids. We found that both the phospholipid acyl chain and polar head affect permeability. Overall, these parameters impact the phospholipid shape and thereupon the membrane lateral pressure, which is a major factor modulating with permeability in our system. These results depend on the nature of the chosen oil. We thereupon identified relevant physical chemistry conditions that best mimic the compactness and subsequent permeability of biological membranes.

Topics: Caco-2 Cells; Cell Membrane; Fluorescent Dyes; Humans; Lipid Bilayers; Oils; Permeability; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Water

The structural characteristics of liposomes have been widely investigated and there is certainly a strong understanding of their morphological characteristics. Imaging of these systems, using techniques such as freeze-fracturing methods, transmission electron microscopy, and cryo-electron imaging, has allowed us to appreciate their bilayer structures and factors which can influence this. However, there are few methods which all us to study these systems in their natural hydrated state; commonly the liposomes are visualized after drying, staining, and/or fixation of the vesicles. Environmental Scanning Electron Microscopy (ESEM) offers the ability to image a liposome in its hydrated state without the need for prior sample preparation. Within our studies we were the first to use ESEM to study liposomes and niosomes and we have been able to dynamically follow the hydration of lipid films and changes in liposome suspensions as water condenses on to, or evaporates from, the sample in real time. This provides insight into the resistance of liposomes to coalescence during dehydration, thereby providing an alternative assay of liposome formulation and stability.

Topics: Antigens; Cations; DNA; Imaging, Three-Dimensional; Lipid Bilayers; Liposomes; Microscopy, Electron, Scanning; Pharmaceutical Preparations; Phosphatidylcholines; Phosphatidylethanolamines

Nonlamellar lipid membranes are frequently induced by proteins that fuse, bend, and cut membranes. Understanding the mechanism of action of these proteins requires the elucidation of the membrane morphologies that they induce. While hexagonal phases and lamellar phases are readily identified by their characteristic solid-state NMR line shapes, bicontinuous lipid cubic phases are more difficult to discern, since the static NMR spectra of cubic-phase lipids consist of an isotropic (31)P or (2)H peak, indistinguishable from the spectra of isotropic membrane morphologies such as micelles and small vesicles. To date, small-angle X-ray scattering is the only method to identify bicontinuous lipid cubic phases. To explore unique NMR signatures of lipid cubic phases, we first describe the orientation distribution of lipid molecules in cubic phases and simulate the static (31)P chemical shift line shapes of oriented cubic-phase membranes in the limit of slow lateral diffusion. We then show that (31)P T2 relaxation times differ significantly between isotropic micelles and cubic-phase membranes: the latter exhibit 2 orders of magnitude shorter T2 relaxation times. These differences are explained by the different time scales of lipid lateral diffusion on the cubic-phase surface versus the time scales of micelle tumbling. Using this relaxation NMR approach, we investigated a DOPE membrane containing the transmembrane domain (TMD) of a viral fusion protein. The static (31)P spectrum of DOPE shows an isotropic peak, whose T2 relaxation times correspond to that of a cubic phase. Thus, the viral fusion protein TMD induces negative Gaussian curvature, which is an intrinsic characteristic of cubic phases, to the DOPE membrane. This curvature induction has important implications to the mechanism of virus-cell fusion. This study establishes a simple NMR diagnostic probe of lipid cubic phases, which is expected to be useful for studying many protein-induced membrane remodeling phenomena in biology.

Topics: Deuterium; Lipids; Magnetic Resonance Spectroscopy; Membranes, Artificial; Micelles; Parainfluenza Virus 5; Phase Transition; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Phosphorus Radioisotopes; Scattering, Small Angle; Temperature; Viral Fusion Proteins; X-Ray Diffraction

Fluorescence quenching of lipid-bound pyrene was used to assess the binding of cytochrome c (cyt c) to liposomes that mimic the inner mitochondrial membrane (IMM) POPC/DOPE/TOCL, with the conditions that it did or did not contain oxidized phosphatidylcholine molecules, i.e., 1-O-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), or a mixture of two hydroperoxide isomers derived from POPC (POPCOX). The binding isotherms reveal two dissociation constants, K(D)(1) and K(D)(2), representing, respectively, the low- and high-affinity states of the membrane. These dissociation constants probably are due to the lipid reorganization promoted by cyt c, as observed in giant unilamellar vesicles that contain fluorescent cardiolipin (CL). The presence of PazePC, which has a nonreactive carboxylic group, increased the K(D)(1) and K(D)(2) values 1.2- and 4.5-fold, respectively. The presence of POPCOX which has a reactive peroxide group, decreased the K(D)(1) value 1.5-fold, increased the K(D)(2) value 10-fold, and significantly reduced the salt-induced detachment of cyt c. MALDI-TOF spectrometry analysis of cyt c incubated with liposomes containing POPCox demonstrated a mass increase corresponding to the formation of nonenal adducts as hydrophobic anchors. Electronic absorption spectroscopy, circular dichroism, and magnetic circular dichroism demonstrated that all of the lipids studied promoted changes in the cyt c coordination sphere. Therefore, in the presence of CL, the oxidation of zwitterionic lipids also promotes changes in the cyt c structure and in the affinity for lipid bilayers.

Topics: Animals; Cardiolipins; Circular Dichroism; Cytochromes c; Fish Proteins; Fluorescence; Horses; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Liposomes; Mitochondrial Membranes; Models, Biological; Molecular Structure; Myocardium; Oxidation-Reduction; Phosphatidylcholines; Phosphatidylethanolamines; Phosphorylcholine; Pyrenes; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tuna

The magnetic field dependence of the 31P spin-lattice relaxation rate, R1, of phospholipids can be used to differentiate motions for these molecules in a variety of unilamellar vesicles. In particular, internal motion with a 5- to 10-ns correlation time has been attributed to diffusion-in-a-cone of the phosphodiester region, analogous to motion of a cylinder in a liquid hydrocarbon. We use the temperature dependence of 31P R1 at low field (0.03-0.08 T), which reflects this correlation time, to explore the energy barriers associated with this motion. Most phospholipids exhibit a similar energy barrier of 13.2 +/- 1.9 kJ/mol at temperatures above that associated with their gel-to-liquid-crystalline transition (Tm); at temperatures below Tm, this barrier increases dramatically to 68.5 +/- 7.3 kJ/mol. This temperature dependence is broadly interpreted as arising from diffusive motion of the lipid axis in a spatially rough potential energy landscape. The inclusion of cholesterol in these vesicles has only moderate effects for phospholipids at temperatures above their Tm, but significantly reduces the energy barrier (to 17 +/- 4 kJ/mol) at temperatures below the Tm of the pure lipid. Very-low-field R1 data indicate that cholesterol inclusion alters the averaged disposition of the phosphorus-to-glycerol-proton vector (both its average length and its average angle with respect to the membrane normal) that determines the 31P relaxation.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; Cattle; Cholesterol; Dimyristoylphosphatidylcholine; Motion; Nuclear Magnetic Resonance, Biomolecular; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Phosphorus Isotopes; Sphingomyelins; Temperature; Unilamellar Liposomes; Water

The release of cytochrome c from mitochondria to the cytosol is a crucial step of apoptosis that involves interactions of Bax and tBid proteins with the mitochondrial membrane. We investigated Bax and tBid interactions with (i) phosphatidylcholine (PC) monolayer as the main component of the outer leaflet of the outer membrane, (ii) with phosphatidylethanolamine (PE) and phosphatidylserine (PS) that are present in the inner leaflet and (iii) with a mixed PC/PE/Cardiolipin (CL) monolayer of the contact sites between the outer and inner membranes. These interactions were studied by measuring the increase of the lipidic monolayer surface pressure induced by the proteins. Our measurements suggest that tBid interacts strongly with the POPC/DOPE/CL, whereas Bax interaction with this monolayer is about 12 times weaker. Both tBid and Bax interact moderately half as strongly with negatively charged DOPS and non-lamellar DOPE monolayers. TBid also slightly interacts with DOPC. Our results suggest that tBid but not Bax interacts with the PC-containing outer membrane. Subsequent insertion of these proteins may occur at the PC/PE/CL sites of contact between the outer and inner membranes. It was also shown that Bax and tBid being mixed in solution inhibit their insertion into POPC/DOPE/CL monolayer. The known 3-D structures of Bax and Bid allowed us to propose a structural interpretation of these experimental results.

Topics: Animals; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Calcium; Cardiolipins; Cattle; Humans; Lipid Metabolism; Phosphatidylcholines; Phosphatidylethanolamines; Protein Structure, Tertiary

We studied the effects of the addition of a series of 1, 2-dioctadecenoyl-sn-glycerol-3-phosphoethanolamines to vesicles composed of 1-palmitoyl-2-oleoylphosphatidylserine and 1-palmitoyl-2-oleoylphosphatidylcholine on the activity and membrane binding of protein kinase C (PKC). The three phosphatidylethanolamines (PEs) were dipetroselinoyl-PE, dioleoyl-PE, and divaccenoyl-PE, which have double bonds in positions 6, 9, and 11, respectively. These lipids represent a group of structurally homologous compounds whose physical properties have been compared. We also used a fluorescent probe, 4-[(n-dodecylthio)methyl]-7-(N, N-dimethylamino)coumarin to measure the relative interfacial polarities of LUVs containing each of the three PEs. We find dipetroselinoyl-PE allows the least access of the fluorescent probe to the membrane. This is also the lipid that shows the lowest activation of PKC. The activity of PKC was found to correlate best with the interfacial properties of the three PEs rather than with the curvature energy of the membrane. The results show the sensitivity of the activity of PKC to small changes in lipid structure.

Topics: Animals; Binding Sites; Coumarins; Enzyme Activation; Fatty Acids, Unsaturated; Fluorescence Polarization; Fluorescent Dyes; Membrane Lipids; Membrane Proteins; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipids; Protein Kinase C; Rats; Spectrometry, Fluorescence; Surface Properties