Dipalmitoylglycerophosphocholine Research Articles

Double Electron-Electron Resonance of Spin-Labeled Cholestane in Model Membranes: Evidence for Substructures inside the Lipid Rafts.

Plasma membranes are assumed to be highly compartmentalized, which is believed to be important for the membrane protein functionality. The liquid ordered-disordered phase segregation in the membranes results in nanoscale liquid-ordered assemblies-lipid rafts. Double electron-electron resonance spectroscopy (DEER, also known as PELDOR) is sensitive to spin-spin dipolar interactions between spin labels at the nanoscale range of distances. Here, DEER is applied to spin-labeled cholestane, 3β-doxyl-5α-cholestane (DChl), diluted in bilayers composed of an equimolar mixture of dioleoyl-glycero-phosphocholine (DOPC) and dipalmitoyl-glycero-phosphocholine (DPPC) phospholipids, with cholesterol (Chol) added. The DEER data allowed us to detect clustering of the DChl molecules. Their lateral distribution in the clusters in the absence of Chol was found to be random, while in the presence of Chol it became quasi-regular. DEER time traces are fairly well simulated within a simple square superlattice model. For the 20 mol % Chol content, for which at physiological temperatures, the lipid rafts are formed, the found superlattice parameter was 3.7 nm. Assuming that lipid rafts are captioned upon shock freezing at the temperature of investigation (80 K), the found regularity of DChl lateral distribution was interpreted by raft substructuring, with the DChl molecules embedded between the substructures.

Lipid chain mobility and packing in DOPC bilayers at cryogenic temperatures

Low-temperature molecular mobility and packing in biological tissues are important for their survival upon cryopreservation. Electron paramagnetic resonance (EPR) in its pulsed version of electron spin echo (ESE) allows studying stochastic librations of spin-labeled molecules, the type of motion which dominates at low temperatures. These librations are characterized by the parameter <α2>τc where <α2> is the mean squared angular amplitude and τc is the correlation time for the motion. This parameter is known to be larger for higher temperature and for looser intermolecular structure. In this work, ESE data for the bilayers comprised of doubly-unsaturated DOPC (dioleoyl-glycero-phosphocholine) lipids and mono-unsaturated POPC (palmitoyl-oleoyl-glycero-phosphocholine) lipids with spin-labeled stearic acids added were obtained in the temperature range between 80 and 210 K; the results were compared also with the previously obtained data for fully-saturated DPPC (dipalmitoyl-glycero-phosphocholine) lipid bilayers [J. Phys. Chem. B2014, 118, 12,478–12,485; Appl. Magn. Reson. 2018, 49, 1369–1383]. It turned out that for DOPC bilayers the <α2>τc values are of intermediate magnitude between those for POPC and DPPC bilayers, which implies an intermediate density of lipid packing. A possible explanation of this result could be rearrangement at cryogenic temperatures of the DOPC lipid tails, with their terminal segments folding cooperatively. This interpretation is also in agreement with the known thermodynamic properties of gel-fluid transition for DOPC bilayer.

Clustering of spin-labeled cholesterol analog diluted in bilayers of saturated and unsaturated phospholipids

Clustering of spin-labeled cholesterol analog, 3β-doxyl-5α-cholestane (DChl), diluted in bilayers comprised of either saturated dipalmitoyl-glycero-phosphocholine (DPPC) or unsaturated dioleoyl-glycero-phosphocholine (DOPC) phospholipids was studied. DChl molar fraction X varied between 0.005 and 0.04. EPR spectroscopy applied at low temperatures (200 K) enabled exploring magnetic dipole-dipole (d-d) interaction between spin labels. For DOPC bilayers, EPR spectra were found to broaden remarkably with X increase. The broadening was simulated for the models of 2-dimentional (2-D) clusters with enhanced local concentration, Xloc, which was several times larger than X, and for 1-dimensional (1-D) DChl clusters. The distance of closest approach in these simulations attained the intermolecular lateral distance in the membrane (~0.7 nm). For DPPC bilayers, EPR spectra showed only small broadening, which in these simulations could not be reproduced even if Xloc was taken as small as X. However strong concentration dependence was found for electron spin echo (ESE) decays. Both the EPR and ESE data for DPPC bilayers were explained within the model assuming encapsulation of DChl molecules in lipid shells so preventing them to approach each other closer than a certain distance, Rmin. The Rmin value was found to vary between ~2.5 nm and 5 nm, for X varying between 0.04 and 0.005; Xloc in these simulations was several times larger than X. So the DChl clustering in DOPC bilayers is driven by attractive lipid-mediated forces, while in DPPC bilayers long-range nanoscale lipid-mediated repulsive/attractive forces take place for distances smaller and larger Rmin, correspondingly.

Comparative Experimental and Computational Study of Monoalkyl Chain Phosphatidylcholine-Containing Thermoresponsive Liposomes.

Liposomes containing lysophospholipids are intensively studied as drug delivery systems that are stable at normal body temperature but exhibit fast release of their drug load at slightly elevated temperatures. In this study, the stability and release properties of dipalmitoylglycerophosphocholine (DPPC)-based liposomes incorporating the commonly used lysophosphatidylocholine (lyso-PC), and a series of monoalkyl chain ether-linked phosphatidylcholine, i.e., the biologically relevant monoalkyl chain platelet activating factor (PAF) and its derivatives lyso-PAF and methyl-PAF, were investigated. To this end a series of PEGylated small unilamellar liposomes with DPPC:monoalkyl lipid compositions of 5% and 10% molar ratio were prepared and compared with regard to stability (37 °C) and release properties at elevated temperatures (38-43 °C). All systems were characterized with respect to size distribution, ζ-potential, and phase transition characteristics. The presence of ether-lipids endows liposomes with superior (∼10% increase) release properties at 5% incorporation compared to lyso-PC, while at 10% molar ratio the formulations do not differ significantly, the release being close to 90%. The findings are supported by atomistic molecular dynamics simulations that suggest a correlation between the enhanced permeability and increased penetration of water molecules within the bilayers with density fluctuations resulting from the increased area-per-lipid and the disorder of the lysolipids alkyl chains.

Effects of the Size, Shape, and Structural Transition of Thermosensitive Polypeptides on the Stability of Lipid Bilayers and Liposomes

We performed all-atom and coarse-grained (CG) molecular dynamics (MD) simulations of lipid bilayers grafted with elastin-like polypeptides (ELPs; [VPGVG]n). All-atom simulations of a single ELP in water show that ELPs become more collapsed and folded as the temperature increases from 293 up to 353 K, in agreement with experiments. All-atom simulations of lipid bilayers composed of dipalmitoylglycerophosphocholine (DPPC), cholesterol, and fatty acids grafted with ELPs show that ELPs insert into the bilayer and significantly disorder lipids, to an extent that depends on the ELP length over the temperature range 293–323 K. In the bilayer, ELPs are mainly, but not entirely, random coil in character at temperatures between 293 and 315 K and, in contrast to the behavior in water, become increasing random coil and extended in length over the range 315–323 K, over which the bilayer is in the disordered liquid phase. The insertion of ELPs into the lipid-tail region is mediated by the interaction of hydrophobic Pro...

Coarse-grained model for PEGylated lipids: effect of PEGylation on the size and shape of self-assembled structures.

Self-assembly of polyethylene glycol (PEG)-grafted lipids at different sizes and concentrations was simulated using the MARTINI coarse-grained (CG) force field. The interactions between CG PEG and CG dipalmitoylglycerophosphocholine (DPPC)-lipids were parametrized by matching densities of 19-mers of PEG and polyethylene oxide (PEO) grafted to the bilayer from all-atom simulations. Mixtures of lipids and PEG-grafted (M(w) = 550, 1250, and 2000) lipids in water self-assembled to liposomes, bicelles, and micelles at different ratios of lipids and PEGylated lipids. Average aggregate sizes decrease with increasing PEGylated-lipid concentration, in qualitative agreement with experiment. PEGylated lipids concentrate at the rims of bicelles, rather than at the planar surfaces; this also agrees with experiment, though the degree of segregation is less than that assumed in previous modeling of the experimental data. Charged lipids without PEG evenly distribute at the rim and planar surfaces of the bicelle. The average end-to-end distances of the PEG on the PEGylated lipids are comparable in liposomes, bicelles (edge or planar surface), and micelles and only slightly larger than for an isolated PEG in solution. The ability of PEGylated lipids to induce the membrane curvature by the bulky headgroup with larger PEG, and thereby modulate the phase behavior and size of lipid assemblies, arises from their relative concentration.

The effects of L‐ to D‐isomerization and C‐terminus deamidation on the secondary structure of antimicrobial peptide Anoplin in aqueous and membrane mimicking environment

AbstractUV resonance Raman spectra of the antimicrobial peptide (AMP) Anoplin (L‐Anoplin‐NH2) and two of its derivatives (enantiomer D‐Anoplin‐NH2 and C‐terminus deamidated L‐Anoplin‐OH) were measured in aqueous buffer solution and in membrane‐mimetic environments including 2,2,2‐trifluoro ethanol (TFE), zwitterionic lipid dipalmitoylglycerophosphocholine (DPPC) and anionic lipid dipalmitoylglycerophosphoglycerol (DPPG) vesicle solutions. All three peptides were found to adopt random‐coil/β turn‐like conformation in aqueous solution over the temperature range of 1–60 °C. The conformation was found to become more α‐helical in membrane‐mimetic solutions such as TFE and DPPG but not in DPPC for all Anoplin derivatives. The data demonstrate that Anoplin preferentially binds to the anionic over the zwitterionic model cell membranes. Results also showed that deamidation does not change the conformation of L‐Ano‐NH2 very significantly, but does alter membrane rupturing and antimicrobial activities thus confirming that it is the physicochemical properties rather than the peptide conformation that define the mechanism of AMP action. Copyright © 2010 John Wiley &amp; Sons, Ltd.

UV resonance Raman spectroscopy probes the localization of tryptophan‐containing antimicrobial peptides in lipid vesicles

AbstractIn this work we employed UV resonance Raman spectroscopy with 229 nm excitation to study two tryptophan‐containing antimicrobial peptides with a broad‐spectrum activity against Gram‐positive and Gram‐negative bacteria: lactoferricin B (LfB, RRWQWRMKKLG) and pEM‐2 (KKWRWWLKALAKK). UV resonance Raman spectra of both peptides are dominated by tryptophan bands. Raman spectra of LfB and pEM‐2 in D2O and 2,2,2‐trifluoro ethanol (TFE) have been measured and used to identify the hydrogen‐bond strength marker bands W6 and W17. The tryptophan doublet, W7, at 1340 and 1360 cm−1 was used to detect an increase in the hydrophobicity of Trp environment in TFE. The spectra of LfB in complex with model cell membranes composed of zwitterionic dipalmitoylglycero‐phosphocholine (DPPC) or anionic dipalmitoyglycero‐phosphoglycerol (DPPG) lipid vesicles revealed a more hydrophobic Trp environment in DPPG, suggesting stronger interactions between the cationic peptide and anionic model cell membrane. Copyright © 2008 John Wiley &amp; Sons, Ltd.

Interactions of lactoferricin B derivatives with model cell membrane studied by Raman spectroscopy

AbstractIn this work, we employed Raman spectroscopy to study the effect of the antimicrobial peptide lactoferricin B (LfB) on model cell membranes. We used two derivatives of LfB (RRWQWRMKKLG and RRWQWR) with broad‐spectrum activity against gram‐positive and gram‐negative bacteria, fungus, viruses and tumors. Raman spectra of the peptides showed no conformational change in the temperature range 4–60 °C. The positions of the amide I and amide III bands suggest that in an aqueous solution these peptides preferentially adopt a random coil‐like conformation. We also investigated the effect the peptides had on the melting behavior of model cell membranes composed of zwitterionic lipid dipalmitoylglycero‐phosphocholine (DPPC) and anionic lipid dipalmitoylglycero‐phosphoglycerol (DPPG). Raman CH stretching bands were used to follow the melting of the lipid vesicles. We found that the melting of DPPC lipid vesicles is not affected by the presence of the peptides, while the presence of the peptides reduced cooperativity of the phase transition for anionic DPPG vesicle, suggesting that both peptides interact strongly and specifically with this model cell membrane composed of anionic lipid. Copyright © 2006 John Wiley &amp; Sons, Ltd.

Probing Phospholipid Main Phase Transition by Fluorescence Spectroscopy and a Surface Redox Reaction

Headgroup and acyl chain NBD-labeled phospholipid derivatives, viz. 1,2-dipalmitoyl-sn-glycero-3-phospho-[N-(4-nitrobenz-2-oxa-1,3-diazole)-ethanolamine] (DPPN) and 1-acyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (NBD-PC), were employed to study lipid conformations and organization in the course of the main phase transition of dipalmitoylglycerophosphocholine (DPPC) liposomes. The differential sensitivity of the 335 and 470 nm absorption bands to environment polarity was used to explore the ground-state NBD environment. Anisotropy measurements with excitation at those bands revealed the presence of two NBD-PC populations below Tm. The fluorescence maximum emission intensity, the ratio of emission intensities with excitation at 470 and 335 nm, and the rate coefficient k1 for the reduction of NBD by dithionite to nonfluorescent derivatives undergo complex changes in the course of the main phase transition of DPPC. For DPPN, the alterations in k1 and intensity ratio are likely to originate from a redistribution of this fluorophore at the transition and suggest a maximum in the bilayer transverse compressibility. For NBD-PC, the changes in these properties appear to be related to a shift in the fluorophore distribution at the percolation threshold for the two-phase region and self-quenching of the NBD moiety.

Control of a Redox Reaction on Lipid Bilayer Surfaces by Membrane Dipole Potential

Nitro-2,1,3-benzoxadiazol-4-yl (NBD) group is a widely used, environment-sensitive fluorescent probe. The negatively charged dithionite rapidly reduces the accessible NBD-labeled lipids in liposomes to their corresponding nonfluorescent derivatives. In this study both the phospholipid headgroup and acyl chain NBD-labeled l- α-1,2-dipalmitoyl- sn-glycero-3-phospho-[ N-(4-nitrobenz-2-oxa-1,3-diazole)-ethanolamine] (DPPN) and 1-acyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecanoyl]- sn-glycero-3-phosphocholine (NBD-PC), respectively, were employed. The correlation of both the rate coefficient k 1 of the redox reaction and the fluorescence properties of the two probes with the membrane dipole potential Ψ in fluid dipalmitoylglycerophosphocholine (DPPC) liposomes is demonstrated. When Ψ of the bilayer was varied (decreased by phloretin or increased by 6-ketocholestanol), the value for k 1 decreased for both DPPN and NBD-PC with increasing Ψ. For both fluorophores a positive correlation to Ψ was evident for the relative fluorescence emission intensity (RFI, normalized to the emission of the fluorophore in a DPPC matrix). The relative changes in emission intensity as a function of Ψ were approximately equal for both NBD derivatives. Changes similar to those caused by phloretin were seen when dihexadecylglycerophosphocholine (DHPC) was added to DPPC liposomes, in keeping with the lower dipole potential for the former lipid compound compared with DPPC. These effects of Ψ on NBD fluorescence should be taken into account when interpreting data acquired using NBD-labeled lipids as fluorescent probes.

Conformational changes of the lecithin headgroup in monolayers at the air/water interface

The headgroup conformation of the phospholipid dipalmitoyl-glycero-phosphocholine (DPPC) in monolayers at the air/water interface has been studied by neutron reflection in the fluid like liquid-expanded (LE) and in the crystal like solid (S) phase. Information on the headgroup conformation in the two phases has been obtained by scattering contrast variation of the lipid monolayer using four differently deuterated species of DPPC: perdeuterated, chain perdeuterated, choline group perdeuterated and selectively headgroup deuterated. Since the measurements were done mainly on a subphase of null reflecting water (i.e. water scattering contrast matched to the air) there is no subphase contribution to reflectivity and the simplest one layer model can be employed for the data analysis, thus minimising the number of free parameters. A remarkable change of the headgroup orientation was observed between the LE and the S phase. We found that the phosphate-nitrogen dipole of the DPPC headgroup exhibits an in-plane orientation with respect to the monolayer in the LE phase but it assumes a more parallel orientation to the surface normal at lateral pressures above 30 mN/m (S phase). Moreover, this conformational change is accompanied by a significant alteration of the headgroup hydration.

Micelle formation of diacylglycerophosphocholine in organic solvents. II. Measurements of microenvironmental characteristics by fluorescence quenching

Reversed micelles formed by dipalmitoylglycerophosphocholine (DPPC) in cyclohexane were investigated by using fluorescence quenching technique. Anthroyloxy derivatives (ACA, 2AP, 12AS, 16AP) were used as fluorescent probes in this study. It was found that they were largely distributed in the micelles, and strongly quenched by dimethylaniline (DMA) and dihexadecylviologen (HDV) in DPPC-reversed micelles. In DMA system, their quenching rates follow the positions of the fluorophore in micelles in the order of 16AP, 12AS, 2AP, ACA. In the HDV system, on the contrary, the reversed order of quenching rates was obtained, which means that these probes are oriented with their carboxyl groups to the micelle core. ACA and 2AP are also quenched by DPPC monomer at the concentrations below the CMC, but the fluorescence is largely recovered above the CMC. This fact was utilized to estimate the CMC (1.2 × 10 5 M).

Sterol orientations in phosphatidylcholine liposomes as determined by deuterium NMR.

Deuterium magnetic resonance spectra (55.26 MHz) of cholesterol-3 alpha-d1 and epicholesterol-3 beta-d1 in dipalmitoylglycerophosphocholine (DPPC) liposomes were measured as a function of sterol-to-phospholipid ratio below (24 degrees C) and above (60 degrees C) the phase transition temperature of DPPC. From the quadrupolar splittings delta vq, the molecular order parameters S describing the motions of the sterols in the bilayer were calculated, and the most probable angle of tilt alpha 0 of the molecular axis of the sterols relative to the bilayer normal was determined. We observed that the molecular axis of cholesterol in DPPC liposomes at both 24 and 60 degrees C is tilted at an angle of 16-19 degrees with the 3 beta-hydroxyl group projecting parallel to the bilayer normal into the aqueous interface. In contrast, at 24 degrees C, epicholesterol is aligned parallel (0 degrees) to the bilayer normal, placing the 3 alpha-hydroxyl group essentially perpendicular to the bilayer normal along the aqueous interface. At 60 degrees C, the average angle of epicholesterol (16-18 degrees) is similar to that of cholesterol, which can project the 3 alpha-hydroxyl group into the hydrophobic bilayer region. On the basis of the observed tilt angles of the two isomeric sterols in DPPC liposomes, a model is proposed that can rationalize the differential effects of cholesterol and epicholesterol on membrane properties.(ABSTRACT TRUNCATED AT 250 WORDS)

Mixed monolayers of dipalmitoylglycerophosphocholine, distearoylglycerophosphocholine and 1-palmitoylglycerol

Mixed monolayer systems of dipalmitoylglycerophosphocholine (DPPC)-distearoylglycerophosphocholine (DSPC), palmitoylglycerol-DPPC, and palmitoylglycerol-DSPC have been investigated by a previously described thermodynamic treatment. The surface pressures of these systems were measured at various compositions and temperatures. The two-dimensional phase diagrams and the apparent molar entropy and energy changes were evaluated. It was found that the phase diagrams of the DPPC-DSPC, palmitoylglycerol-DSPC and palmitoylglycerol-DPPC systems are all of different types. According to our phase diagram classification, DPPC-DSPC, palmitoylglycerol-DSPC, and palmitoylglycerol-DPPC systems exhibited a modified cigar type, a eutectic type, and a negative azeotropic type, respectively.

The role of the phospholipid phase transition in the regulation of glucagon binding to lecithin

Glucagon can interact rapidly with multilamellar vesicles of dimyristoyl glycerophosphocholine over a narrow temperature range around or above the phase transition temperature of the pure phospholipid. The temperature dependence of the rates arises, in large part, from glucagon-induced alterations in the phase transition properties of the phospholipid. Similar effects are observed with dilaury glycerophosphocholine but the rate of reaction of glucagon with multilamellar dipalmitoyl glycerophosphocholine is too slow to measure. The rate of reaction of glucagon with equimolar mixtures of two phospholipid molecules has also been studied. Mixtures of dilauryl glycerophosphocholine and distearoyl glycerophosphocholine are known to exhibit lateral phase separation in the gel state. The presence of distearoyl glycerophosphocholine has no effect on the rate of reaction with glucagon, despite the increased number of phase boundaries present. In the case of mixtures of dilauryl glycerophosphocholine and dimyristoyl glycerophosphocholine, glucagon appears to induce some lateral phase separation. This is demonstrated by the ability of glucagon to react rapidly with this lipid mixture, even at temperatures well below the phase transition temperature of the mixture and by differential scanning calorimetry. The thermodynamics of the binding of glucagon to dimyristoyl glycerophosphocholine and dilauryl glycerophosphocholine were analyzed with Scatchard plots calculated from measurements of the fluorescence enhancement caused by lipids. Equilibrium binding constants of glucagon to dimyristoyl glycerophosphocholine and dilauryl glycerophosphocholine are 1·10 5 and 5·10 4 M −1, respectively. These values are relatively insensitive to temperature, indicating that the equilibrium being measured is between lipid-bound glucagon and free lipid which has had its phase transition properties altered. The number of moles of lipid bound per mole of glucagon decreases markedly above the phase transition temperature. In the water-soluble complex formed between glucagon and dimyristoyl glycerophosphocholine, the peptide binds directly to only 40% of the lipid molecules but, nevertheless, is able to modify the phase transition properties of all of the lipid in the particle.

Preferential interaction of pentagastrin with the gel state of dimyristoyl glycerophosphocholine.

Fluorescence and circular dichroism spectra indicate that pentagastrin interacts with dimyristoyl glycerophosphocholine more strongly below the phase transition temperature of the lipid than above it. Studies on the interaction of several peptides with dimyristoyl glycerophosphocholine suggest that this property may be related to the ability of these peptides to form amphipathic structures containing two hydrophobic amino acids separated by two other amino acids. Pentagastrin has a marked effect on the proton magnetic resonance spectra of dipalmitoyl glycerophosphocholine below the phase transition temperature indicating the the peptide decreases the motional freedom of the lipid.

Some physical properties of free and esterified cholesterol mixtures: Studies on relative stability of cholesterol ester ratio in blood plasma

Some physical properties of the mixtures composed of esterified and free cholesterol at various ratios were measured. Addition of a small proportion of free cholesterol, lowered slightly the melting point of the esterified cholesterol fraction from human plasma and it was kept nearly constant at this level by the addition of 30–40% of free cholesterol. Further addition of free cholesterol resulted in an abrupt rise in the melting point. The ranges of the stable plateau were clearly wider in esterified cholesterol with more polyunsaturated acids. The extent of inhibition of monolayer formation of free cholesterol with cholesterol palmitate was found to be greatest at a mixing ratio of 60–70%. In the case where cholesterol palmitate was added to an equimolar mixture of dipalmitoyl glycerophosphocholine and free cholesterol, a trimolecular mixed monolayer was formed above the mixing ratio, 7.5 : 2.5, of esterified and free cholesterol. The present studies clearly showed that there were distinct changes in some physical properties examined at the molar ratio of esterified cholesterol : free cholesterol of about 7 : 3.