Phosphocholine Lipids Research Articles

Role of Different Phospholipids in the Synthesis of Pearl-Necklace-Type Gold−Silver Bimetallic Nanoparticles as Bioconjugate Materials

A seed-growth (S-G) method has been used to synthesize gold (Au) and Au−silver (Ag) bimetallic pear-necklace-type nanoparticles (NP) as bioconjugate materials by using a series of phospho-glycerol (PG) and phospho-choline (PC) lipids as capping agents. All PG lipids produce a fine pear-necklace arrangement of Au NP with dimensions between 10 and 20 nm. Addition of Ag converts this arrangement into Au−Ag bimetallic NP with a diameter essentially close to that of Au NP. Use of PC lipids does not show this arrangement but promotes a significant anisotropic growth especially in the presence of Ag. This difference has been attributed to a difference in the capping ability of PG and PC lipids because of their anionic and zwitterionic nature, respectively. XPS results have demonstrated the presence of adsorbed PG and PC lipids on Au or Au−Ag bimetallic surfaces in their respective samples. The results also indicate a decrease in the capping amount of a lipid with an increase in the growth of Au−Ag bimetallic NP....

Nanoporous Microbead Supported Bilayers: Stability, Physical Characterization, and Incorporation of Functional Transmembrane Proteins

The introduction of functional transmembrane proteins into supported bilayer-based biomimetic systems presents a significant challenge for biophysics. Among the various methods for producing supported bilayers, liposomal fusion offers a versatile method for the introduction of membrane proteins into supported bilayers on a variety of substrates. In this study, the properties of protein containing unilamellar phosphocholine lipid bilayers on nanoporous silica microspheres are investigated. The effects of the silica substrate, pore structure, and the substrate curvature on the stability of the membrane and the functionality of the membrane protein are determined. Supported bilayers on porous silica microspheres show a significant increase in surface area on surfaces with structures in excess of 10 nm as well as an overall decrease in stability resulting from increasing pore size and curvature. Comparison of the liposomal and detergent-mediated introduction of purified bacteriorhodopsin (bR) and the human type 3 serotonin receptor (5HT3R) are investigated focusing on the resulting protein function, diffusion, orientation, and incorporation efficiency. In both cases, functional proteins are observed; however, the reconstitution efficiency and orientation selectivity are significantly enhanced through detergent-mediated protein reconstitution. The results of these experiments provide a basis for bulk ionic and fluorescent dye-based compartmentalization assays as well as single-molecule optical and single-channel electrochemical interrogation of transmembrane proteins in a biomimetic platform.

Defect formation of lytic peptides in lipid membranes and their influence on the thermodynamic properties of the pore environment

We present an experimental study of the pore formation processes of small amphipathic peptides in model phosphocholine lipid membranes. We used atomic force microscopy to characterize the spatial organization and structure of alamethicin- and melittin-induced defects in lipid bilayer membranes and the influence of the peptide on local membrane properties. Alamethicin induced holes in gel DPPC membranes were directly visualized at different peptide concentrations. We found that the thermodynamic state of lipids in gel membranes can be influenced by the presence of alamethicin such that nanoscopic domains of fluid lipids form close to the peptide pores, and that the elastic constants of the membrane are altered in their vicinity. Melittin-induced holes were visualized in DPPC and DLPC membranes at room temperature in order to study the influence of the membrane state on the peptide induced hole formation. Also differential scanning calorimetry was used to investigate the effect of alamethicin on the lipid membrane phase behaviour.

Trans and surface membrane bound zervamicin IIB: 13C-MAOSS-NMR at high spinning speed

Interactions between (15)N-labelled peptides or proteins and lipids can be investigated using membranes aligned on a thin polymer film, which is rolled into a cylinder and inserted into the MAS-NMR rotor. This can be spun at high speed, which is often useful at high field strengths. Unfortuantely, substrate films like commercially available polycarbonate or PEEK produce severe overlap with peptide and protein signals in (13)C-MAOSS NMR spectra. We show that a simple house hold foil support allows clear observation of the carbonyl, aromatic and C(alpha) signals of peptides and proteins as well as the ester carbonyl and choline signals of phosphocholine lipids. The utility of the new substrate is validated in applications to the membrane active peptide zervamicin IIB. The stability and macroscopic ordering of thin PC10 bilayers was compared with that of thicker POPC bilayers, both supported on the household foil. Sidebands in the (31)P-spectra showed a high degree of alignment of both the supported POPC and PC10 lipid molecules. Compared with POPC, the PC10 lipids are slightly more disordered, most likely due to the increased mobilities of the shorter lipid molecules. This mobility prevents PC10 from forming stable vesicles for MAS studies. The (13)C-peptide peaks were selectively detected in a (13)C-detected (1)H-spin diffusion experiment. Qualitative analysis of build-up curves obtained for different mixing times allowed the transmembrane peptide in PC10 to be distinguished from the surface bound topology in POPC. The (13)C-MAOSS results thus independently confirms previous findings from (15)N spectroscopy [Bechinger, B., Skladnev, D.A., Ogrel, A., Li, X., Rogozhkina, E.V., Ovchinnikova, T.V., O'Neil, J.D.J. and Raap, J. (2001) Biochemistry, 40, 9428-9437]. In summary, application of house hold foil opens the possibility of measuring high resolution (13)C-NMR spectra of peptides and proteins in well ordered membranes, which are required to determine the secondary and supramolecular structures of membrane active peptides, proteins and aggregates.

The effect of adsorbed lipid on pyrite oxidation under biotic conditions.

The chemolithoautotrophic bacterium, Acidithiobacillus ferrooxidans, commonly occurs in acid mine drainage (AMD) environments where it is responsible for catalyzing the oxidation of pyrite and concomitant development of acidic conditions. This investigation reports on the growth of this bacterial species on the pyrite surface and in the aqueous phase at a pH close to 2 as well as the role of adsorbed lipid in preventing pyrite dissolution. Both acid washed pyrite and acid-washed pyrite coated with lipids were used as substrates in the studies. The choice of lipid, 1,2-bis(10,12-tricosadiynoyl)-sn-Glycero-3-Phosphocholine lipid (23:2 Diyne PC), a phosphocholine lipid, was based on earlier work that showed that this lipid inhibits the abiotic oxidation rate of pyrite. Atomic force microscopy showed that under the experimental conditions used in this study, the lipid formed ~4–20 nm layers on the mineral surface. Surface-bound lipid greatly suppresses the oxidation process catalyzed by A. ferrooxidans. This suppression continued for the duration of the experiments (25 days maximum). Analysis of the bacterial population on the pyrite surface and in solution over the course of the experiments suggested that the pyrite oxidation was dependent in large part on the fraction of bacteria bound to the pyrite surface.

High resolution crystallographic studies of alpha-hemolysin-phospholipid complexes define heptamer-lipid head group interactions: implication for understanding protein-lipid interactions.

The alpha-hemolysin is an archetypal pore-forming protein that is secreted from Staphylococcus aureus as a water-soluble monomer. When the monomer binds to the membrane of a susceptible cell, the membrane-bound molecules assemble into the lytic heptamer. Although a bilayer or a bilayer-like environment are essential to toxin assembly, there is no high resolution information on toxin-phospholipid complexes. We have determined the structures of detergent-solubilized alpha-hemolysin heptamer bound to glycerophosphocholine or dipropanoyl glycerophosphocholine at 1.75-1.80 A resolution and 110 K. The phosphocholine head group binds to each subunit in a crevice between the rim and the stem domains. The quaternary ammonium group interacts primarily with aromatic residues, whereas the phosphodiester moiety interacts with a conserved arginine residue. These structures provide a molecular basis for understanding why alpha-hemolysin preferentially assembles on membranes comprised of phosphocholine lipids.

Structure, Location, and Lipid Perturbations of Melittin at the Membrane Interface

Melittin is arguably the most widely studied amphipathic, membrane-lytic α-helical peptide. Although several lines of evidence suggest an interfacial membrane location at low concentrations, melittin’s exact position and depth of penetration into the hydrocarbon core are unknown. Furthermore, the structural basis for its lytic action remains largely a matter of conjecture. Using a novel x-ray absolute-scale refinement method, we have now determined the location, orientation, and likely conformation of monomeric melittin in oriented phosphocholine lipid multilayers. Its helical axis is aligned parallel to the bilayer plane at the depth of the glycerol groups, but its average conformation differs from the crystallographic structure. As observed earlier for another amphipathic α-helical peptide, the lipid perturbations induced by melittin are remarkably modest. Small bilayer perturbations thus appear to be a general feature of amphipathic helices at low concentrations. In contrast, a dimeric form of melittin causes larger structural perturbations under otherwise identical conditions. These results provide direct structural evidence that self-association of amphipathic helices may be the crucial initial step toward membrane lysis.

Analysis of oxidized glycerophosphocholine lipids using electrospray ionization mass spectrometry and microderivatization techniques

Oxidized low-density lipoprotein (LDL) is thought to play an important role in atherogenesis and cardiovascular disease in humans. Oxidized LDL is a complex mixture of many oxidized species, including numerous oxidized glycerophospholipids. Electrospray ionization and tandem mass spectrometry as well as microchemical derivatization of high-performance liquid chromatographically purified fractions derived from oxidized LDL were investigated as means to determine the structure of individual components present in oxidized LDL. One major oxidized phosphocholine lipid had an [M + H](+) ion at m/z 650. Derivatization to the trimethylsilyl ether and methoxime caused shifts in mass which, along with negative ion collision-induced dissociation mass spectra, were consistent with the presence of three species, 1-palmitoyl-2-(9-oxononanoyl)glycerophosphocholine and two isomeric 1-octadecanoyl-2-(hydroxyheptenoyl)glycerophosphocholines. These species were chemically synthesized. Trimethylsilylation of free hydroxyl groups increased the mass of the phospholipid acyl chains containing hydroxyl groups by 72 u. Conversion of carbonyl groups to the methoxylamine derivative increased the mass by 29 u. Ozonolysis of those products which contained double bonds proved to be a facile technique to determine the position and number of double bonds present. The use of these techniques was illustrated in the structural characterization of one major component (m/z 650, positive ions) in oxidized LDL as 1-octadecanoyl-2-(7-hydroxyhepta-5-enoyl)glycerophosphocholi ne. A possible mechanism for the formation of this unique chain-shortened glycerophospholipid is proposed.

DNA-induced endocytosis upon local microinjection to giant unilamellar cationic vesicles.

We suggest a novel approach for direct optical microscopy observation of DNA interaction with the bilayers of giant cationic liposomes. Giant unilamellar vesicles, about 100 microns in diameter, made of phosphatidyl-cholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine, were obtained by electroformation. "Short" DNAs (oligonucleotide 21 b and calf thymus 250 bp) were locally injected by micropipette to a part of the giant unilamellar vesicle (GUV) membrane. DNAs were injected native, as well as marked with a fluorescent dye. The resulting membrane topology transformations were monitored in phase contrast, while DNA distribution was followed in fluorescence. We observed DNA-induced endocytosis due to the DNA/lipid membrane local interactions and complex formation. A characteristic minimum concentration (Cendo) of D-erythrosphingosine (Sph+) in the GUV membrane was necessary for the endocytic phenomenon to occur. Below Cendo, only lateral adhesions between neighboring vesicles were observed upon DNA local addition. Cendo depends on the type of zwitterionic (phosphocholine) lipid used, being about 10 mol% for DPhPC/Sph+ GUVs and about 20 mol% for SOPC/Sph+ or eggPC/Sph+ GUVs. The characteristic sizes and shapes of the resulting endosomes depend on the kind of DNA, and initial GUV membrane tension. When the fluorescent DNA marker dye was injected after the DNA/lipid local interaction and complex formation, no fluorescence was detected. This observation could be explained if one assumes that the DNA is protected by lipids in the DNA/lipid complex, thereby inaccessible for the dye molecules. We suggest a possible mechanism for DNA/lipid membrane interaction involving DNA encapsulation within an inverted micelle included in the lipid membrane. Our model observations could help in understanding events associated with the interaction of DNA with biological membranes, as well as cationic liposomes/DNA complex formation in gene transfer processes.

Conformational constraints on the headgroup and sn-2 chain of bilayer DMPC from NMR dipolar couplings.

This paper presents new NMR constraints on the conformation of the headgroup, glycerol backbone, and sn-2 chain of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) in the liquid-crystalline bilayer. Using two-dimensional 13C-1H chemical shift correlation spectroscopy, we find significant dipolar couplings between the carboxyl carbon CO2 and the headgroup protons. This indicates that a conformation in which the DMPC headgroup and the beginning of the sn-2 chain bend toward each other is significantly populated in the fluid bilayer. The predominance of this headgroup orientation can be further confirmed by 31P-13C dipolar couplings from the literature, which constrain the glycerol G2-G3 torsion angle to be close to trans, excluding a significant presence of one of the two conformations found in the DMPC crystal. Combining and reexamining 20 known NMR couplings for the glycerol backbone and its adjacent segments of L alpha-DMPC, we find that several torsion angles and bond orientations in the core of the DMPC molecule are constrained severely and must differ from those in the crystal structure. We propose a consistent molecular model for phosphocholine lipids in the liquid-crystalline phase, with a rigid backbone in the core of the molecule, a bent-back headgroup, and increasing mobility toward the ends of the acyl chains and the headgroup.

An investigation of miscibility in monolayer films of phosphocholine-phosphoglycerol binary mixtures

A surface thermodynamic analysis was applied to the experimentally measured pressure-area isotherms of mixed monolayer films of phosphocholine (PC) and phosphoglycerol (PG) lipids in order to quantitatively describe the phase miscibility of these physiologically important binary mixtures. We have calculated the excess free energies of mixing ( ΔG xs π ) of binary mixtures of acyl chain saturated PC (1,2-dipalmitoyl- sn-glycero-3-phosphocholine, DPPC) with saturated PG (1,2-dipalmitoyl- sn-glycero-3-phosphoglycerol, DPPG), as well as saturated PC (DPPC) with acyl chain unsaturated PG (1,2-dioleoyl- sn-glycero-3-phosphoglycerol, DOPG) at various mole fractions and under different subphase Ca 2+ ion conditions. ΔG xs π for the binary mixture of DPPC + DPPG on a NaCl subphase indicates that this binary system approximates an ideal mixture at almost all mole fractions studied. The presence of 5 mM CaCl 2 in the subphase results in negative values of ΔG xs π for the DPPC/DPPG mixture, indicating a miscible, non-ideal monolayer film, where increased intermolecular interactions are stabilized in the presence of Ca 2+. The mixture of DPPC and DOPG on NaCl was found to have positive deviations from ideality at all mole fractions studied, indicating a phase separated binary mixture. The magnitude of the phase separation increased as the surface pressure increased. The presence of Ca 2+ ions in the subphase was found to have little effect on the mixing of the DPPC and DOPG binary monolayer. This system remained phase separated at all mole fractions in the presence of Ca 2+, with ΔG xs π values increasing as the isotherm surface pressure increased.

The determination of binding constants of micellar-packaged gramicidin A by 13C-and 23Na-NMR

Based on the malonyl gramicidin A structure of a single-stranded head-to-head hydrogen bonded right-handed, β 6.3-helix in dodecyl phosphocholine (DPC) lipid micelles (Jing et al. (1994) Biophys. J. 66, A353), the determination of cation binding sites for gramicidin A (GA) in DPC micelles becomes a significant step in the study of ion transport through the model channel. First, the investigation of cation binding sites in DPC micellar packaged gramicidin A was achieved by 13C-NMR experiments at 30°C using four C-13 labeled GA samples. Then, the analyses based on two different equations, one for single and one for double occupancy, were employed to evaluate the correct occupancy model for GA in DPC micelles. The results clearly indicate double occupancy to be correct for Na + ion as well as for K +, Rb +, Cs + and Tl + ions. Finally, the binding constants for Na + ion were also estimated by the measurement of the longitudinal relaxation time ( T 1) using 23Na-NMR of the same sample at the same temperature as used for the 13C-NMR study. The binding constants obtained from 23Na-NMR are essentially equivalent to those determined from the 13C-chemical shifts.

Membrane hydration and structure on a subnanometer scale as seen by high resolution solid state nuclear magnetic resonance: POPC and POPC/C12EO4 model membranes

The position on a subnanometer scale and the dynamics of structurally important water in model membranes was determined using a combination of proton magic-angle spinning NMR (MAS) with two-dimensional NOESY NMR techniques. Here, we report studies on phosphocholine lipid bilayers that were then modified by the addition of a nonionic surfactant that is shown to dehydrate the lipid. These studies are supplemented by 13C magic-angle spinning NMR investigations to get information on the dynamics of segmental motions of the membrane molecules. It can be shown that the hydrophilic chain of the surfactant is positioned at least partially within the hydrophobic core of the lipid bilayer. With the above NMR approach, we are able to establish molecular contacts between water and the lipid headgroup as well as with certain groups of the hydrocarbon chains and the glycerol backbone. This is possible because high resolution proton and 13C-NMR spectra of multilamellar bilayer membranes are obtained using MAS. A phase-sensitive NOESY must also be applied to distinguish positive and negative cross-peaks in the two-dimensional plot. These studies have high potential to investigate membrane proteins hydration and structural organization in a natural lipid bilayer surrounding.

Hydration force parameters of phosphatidylcholine lipid bilayers as determined from 2H-NMR studies of deuterated water

The continuous decrease of the quadrupolar splitting of deuterated water interacting with phosphocholine lipid bilayers with growing water concentration is analyzed as a function of the water activity. From the apparent linear dependence on water activity a measure for hydration forces is obtained. The forces calculated are in the range of published data using sorption isotherms and osmotic stress technique in combination with SAXS. A simple interaction potential which includes orientational order of water adsorbed on surfaces gives a physical base for these findings. Therefore, deuterium NMR may become a powerful tool for hydration force analysis complementing well-known methods.

Anisotropic Incorporation of Functional Molecules and Synthesis of Low – Dimensional Clusters in Cast Multibilayer Films

Abstract The phospholipid molecules, the major component of biomembrane bilagers, usually contain two fatty acid chains as the hydrophobic moiety and a phosphate derivative like phosphocholine AS the hydrophilic moiety. Double-chain ammonium salts were found to be a simple synthetic analogue of phosphocholine lipids. These ammonium salts form bilayer membranes spontaneously by dispersion in water, when the alkyl chain length is C10 to C20. Subsequent studies demonstrated that the ammonium head group may be replaced with other hydrophilic units. Anionic double-chain amphiphiles(sulfonate, phosphate, carboxylate) have been shown to produce bilayer membranes. Nonionic(e.g.,polyoxyethylene) and zwitterionic counter-parts also form bilayers.

Formation and Properties of a Network Gel Formed from Mixtures of Diacetylenic and Short-Chain Phosphocholine Lipids

A polymerizable gel superaggreagate has been formed from low concentrations (6 mM) of phospholipid mixtures of polymerizable 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine and 1,2-bis(dinonoyl)-sn-glycero-3-phosphocholine. Transmission electron microscopy (TEM) revealed that the superaggregate structure consisted of a network of twisted, braided fibers and that the pore size of the gel ranged from 0.1 to 1.0 μm. TEM of gel plated with Ni revealed that the width of the fibers was 280 A. Optical microscopy demonstrated that the onset of the gel phase occurred at mole fraction 0.43 DNPC in the absence of salt and 0.36 in the presence of 0.25 M NaCl. Polymerization did not affect the morphology of the gel but did increase its temperature stability.

Synthesis of phosphocholine and quaternary amine ether lipids and evaluation of in vitro antineoplastic activity.

The in vitro antineoplastic activity of many phosphorus-containing (e.g., phosphocholines) and non-phosphorus-containing (e.g., quaternary ammonium salts) ether lipids has been evaluated in the HL-60 promyelocytic cell line. These compounds are analogues of ET-18-OMe (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine). Structural modification of 1-(alkylamido)-, -(alkylthio)-, and -(alkyloxy)propyl backbones has provided further insight into the structure-activity relationships of these lipids. In this study, a long saturated C-1 chain and a three-carbon backbone with a single short C-2 substituent were preferred. At the positively charged nitrogen of phosphocholines, fewer than three substituents caused a significant loss of activity, and substituents larger than methyl decreased activity slightly. In the nonphosphorus compounds, many nitrogen heterocycles and also a sulfonium moiety were incorporated without changing the degree of activity; however, a thiazolium group decreased activity. The most active compound, 29 [N-[3-(hexadecyloxy)-2-methoxypropyl]-3-(hydroxymethyl)pyridinium bromide], was approximately twice as active as the reference standard, ET-18-OMe, in a trypan blue dye exclusion assay.

Liquid Crystalline Phases of a Diacetylenic Phosphocholine Lipid

Abstract We have studied the liquid crystalline behavior of a polymerizable diacetylenic phosphocholine lipid l,2-bis(10,12-tricosadiynoyl-sn-glycero-3-phosphocholine (DC8,9PC). Optical polarized microscopy reveals several types of textures corresponding to normal as well as tilted smectic phases for the dry sample while only the oily streak texture of the Lα phase is seen for the sample with 5% water. X-ray diffraction data show that the lamellar order in the hydrated sample is more pronounced than that in the dry sample. Also, a pronounced change in the layer structure occurs for both the dry and 5% hydrated samples at the chain melting temperature. These structural changes are discussed in relation to the optical microscopic observations.

Biological thermodynamic data for the calibration of differential scanning calorimeters: dynamic temperature data on the gel to liquid crystal phase transition of dialkylphosphatidylcholine in water suspensions

Heat capacity measurements of the gel to liquid crystalline transition of phosphocholine lipid suspensions in buffer solutions were performed using a differential scanning calorimeter (DSC) over a range of experimental conditions. Since the temperatures at the transition peak maxima, i.e. transition temperatures, were observed to increase linearly with scan rate due to the response time of the DSC, they were corrected to zero scan rate and found to agree closely with the literature values. The transition temperatures and enthalpies of the seven dialkylphosphatidylcholine lipids investigated ranged from 286.6 ± 0.1 K and 15.9 ±0.6 kJ mol −1 for 1,2-ditridecanoyl- sn-glycero-3-phosphocholine to 353.7 ± 0.1 K and 69.1 ±3.3 kJ mol −1 for l,2-ditetracosanoyl- sn-glycero-3-phosphocholine. At the slowest scan rates of 3–5 K h −1, the transition widths at half the peak maximum were close to 0.2 K. The transition temperatures were found to be independent of the following: the ionic strength of the sodium phosphate buffer from 0.01–0.02 M, the pH of the buffer from 6 to 8, the concentration of the suspension from 0.1–1.0 mass%, the commercial source of the lipid, and storage of the lipid in a freezer for 4 years. Suspensions of the dialkylphosphatidylcholine lipids in sodium phosphate buffer solutions can be used as sources of dynamic temperature data for the calibration of DSCs.

X-Ray Diffraction Studies of Tubules Formed from a Diacetylenic Phosphocholine Lipid

AbstractThe diacetylenic phosphocholine lipid 1,2-bis(10, 12-tricosadiyonol)-sn-glycerol- 3-phosphocholine forms, upon cooling below the chain melting temperature, hollow cylindrical structures known as ‘tubules’. These tubules are approximately one micron in diameter and may range from tens to hundreds of microns in length. We have carried out x-ray diffraction studies of tubules (1) in 75/25 ethanol/water by volume and (2) in a dessicated state. We present results which show marked differences in the nature of both the in-plane and inter-layer correlations associated with the wrapped multilayer structure of the tubules in the two states.