1-2-dilauroylphosphatidylcholine and 1-2-oleoylphosphatidylcholine

Transmembrane helices dominate the landscape for many membrane proteins. Often flanked by interfacial aromatic residues, these transmembrane helices also contain loops and interhelix segments, which could help in stabilizing a transmembrane orientation. Using

Topics: Alanine; Amino Acid Sequence; Dimyristoylphosphatidylcholine; Glycine; Hydrogen Bonding; Lipid Bilayers; Membrane Proteins; Peptides; Phosphatidylcholines; Protein Conformation, alpha-Helical; Protein Unfolding; Water

Amyloid-β (Aβ) peptide has been implicated in Alzheimer's disease, which is a leading cause of death worldwide. The interaction of Aβ peptides with the lipid bilayers of neuronal cells is a critical step in disease pathogenesis. Recent evidence indicates that lipid bilayer thickness influences Aβ membrane-associated aggregation, while understanding how Aβ interacts with lipid bilayers remains elusive. To address this question, we employed supported lipid bilayer (SLB) platforms composed of different-length phosphatidylcholine (PC) lipids (C12:0 DLPC, C18:1 DOPC, C18:1-C16:0 POPC), and characterized the resulting interactions with soluble Aβ monomers. Quartz crystal microbalance-dissipation (QCM-D) experiments identified concentration-dependent Aβ peptide adsorption onto all tested SLBs, which was corroborated by fluorescence recovery after photobleaching (FRAP) experiments indicating that higher Aβ concentrations led to decreased membrane fluidity. These commonalities pointed to strong Aβ peptide-membrane interactions in all cases. Notably, time-lapsed fluorescence microscopy revealed major differences in Aβ-induced membrane morphological responses depending on SLB hydrophobic thickness. For thicker DOPC and POPC SLBs, membrane remodeling involved the formation of elongated tubule and globular structures as a passive means to regulate membrane stress depending on Aβ concentration. In marked contrast, thin DLPC SLBs were not able to accommodate extensive membrane remodeling. Taken together, our findings reveal that membrane thickness influences the membrane morphological response triggered upon Aβ adsorption.

Topics: Amyloid beta-Peptides; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Membrane Fluidity; Microscopy, Fluorescence; Peptide Fragments; Phosphatidylcholines; Quartz Crystal Microbalance Techniques

Additive force fields are designed to account for induced electronic polarization in a mean-field average way, using effective empirical fixed charges. The limitation of this approximation is cause for serious concerns, particularly in the case of lipid membranes, where the molecular environment undergoes dramatic variations over microscopic length scales. A polarizable force field based on the classical Drude oscillator offers a practical and computationally efficient framework for an improved representation of electrostatic interactions in molecular simulations. Building on the first-generation Drude polarizable force field for the dipalmitoylphosphatidylcholine 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) molecule, the present effort was undertaken to improve this initial model and expand the force field to a wider range of phospholipid molecules. New lipids parametrized include dimyristoylphosphatidylcholine (DMPC), dilauroylphosphatidylcholine (DLPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoylphosphatidylethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). The iterative optimization protocol employed in this effort led to lipid models that achieve a good balance between reproducing quantum mechanical data on model compound representative of phospholipids and reproducing a range of experimental condensed phase properties of bilayers. A parametrization strategy based on a restrained ensemble-maximum entropy methodology was used to help accurately match the experimental NMR order parameters in the polar headgroup region. All the parameters were developed to be compatible with the remainder of the Drude polarizable force field, which includes water, ions, proteins, DNA, and selected carbohydrates.

Topics: Diffusion; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Quantum Theory; Thermodynamics

The aggregation of amyloid-β (Aβ) on lipid bilayers has been implicated as a mechanism by which Aβ exerts its toxicity in Alzheimer's disease (AD). Lipid bilayer thinning has been observed during both oxidative stress and protein aggregation in AD, but whether these pathological modifications of the bilayer correlate with Aβ misfolding is unclear. Here, we studied peptide-lipid interactions in synthetic bilayers of the short-chain lipid dilauroyl phosphatidylcholine (DLPC) as a simplified model for diseased bilayers to determine their impact on Aβ aggregate, protofibril, and fibril formation. Aβ aggregation and fibril formation in membranes composed of dioleoyl phosphatidylcholine (DOPC) or 1- palmitoyl-2-oleoyl phosphatidylcholine mimicking normal bilayers served as controls. Differences in aggregate formation and stability were monitored by a combination of thioflavin-T fluorescence, circular dichroism, atomic force microscopy, transmission electron microscopy, and NMR. Despite the ability of all three lipid bilayers to catalyze aggregation, DLPC accelerates aggregation at much lower concentrations and prevents the fibrillation of Aβ at low micromolar concentrations. DLPC stabilized globular, membrane-associated oligomers, which could disrupt the bilayer integrity. DLPC bilayers also remodeled preformed amyloid fibrils into a pseudo-unfolded, molten globule state, which resembled on-pathway, protofibrillar aggregates. Whereas the stabilized, membrane-associated oligomers were found to be nontoxic, the remodeled species displayed toxicity similar to that of conventionally prepared aggregates. These results provide mechanistic insights into the roles that pathologically thin bilayers may play in Aβ aggregation on neuronal bilayers, and pathological lipid oxidation may contribute to Aβ misfolding.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Humans; Lipid Bilayers; Phosphatidylcholines; Protein Aggregates; Protein Structure, Secondary

Quantitative characterization of membrane defects (pores) is important for elucidating the molecular basis of many membrane-active peptides. We study kinetic defects induced by melittin in vesicular and planar lipid bilayers. Fluorescence spectroscopy measurements indicate that melittin induces time-dependent calcein leakage. Solution atomic force microscopy (AFM) is used to visualize melittin-induced membrane defects. After initial equilibration, the most probable defect radius is ∼3.8 nm in 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) bilayers. Unexpectedly, defects become larger with longer incubation, accompanied by substantial shape transformation. The initial defect radius is ∼4.7 nm in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers. Addition of 30 mol % cholesterol to DOPC bilayers suppresses defect kinetics, although the inhibitory impact is negated by longer incubation. Overall, the kinetic rate of defect development follows DLPC > DOPC > DOPC/cholesterol. Kinetic defects are also observed when anionic lipids are present. Based on the observation that defects can occupy as large as 40% of the bilayer surface, we propose a kinetic defect growth model. We also study the effect of melittin on the phase behavior of DOPC/egg-sphingomyelin/cholesterol bilayers. We find that melittin initially suppresses or eliminates liquid-ordered (Lo) domains; Lo domains gradually emerge and become the dominant species with longer incubation; and defects in phase-coexisting bilayers have a most probable radius of ∼5 nm and are exclusively localized in the liquid-disordered (Ld) phase. Our experimental data highlight that melittin-induced membrane defects are not static; conversely, spontaneous defect growth is intrinsically associated with membrane permeabilization exerted by melittin.

Topics: Cholesterol; Kinetics; Lipid Bilayers; Melitten; Microscopy, Atomic Force; Phosphatidylcholines; Spectrometry, Fluorescence

Peptides of the "WALP" family, acetyl-GWW(LA)(n)LWWA-[ethanol]amide, have proven to be opportune models for investigating lipid-peptide interactions. Because the average orientations and motional behavior of the N- and C-terminal Trp (W) residues differ, it is of interest to investigate how the positions of the tryptophans influence the properties of the membrane-incorporated peptides. To address this question, we synthesized acetyl-GGWW(LA)(n)-ethanolamide and acetyl-(AL)(n)WWG-ethanolamide, in which n = 4 or 8, which we designate as "N-anchored" and "C-anchored" peptides, respectively. Selected (2)H or (15)N labels were incorporated for solid-state nuclear magnetic resonance (NMR) spectroscopy. These peptides can be considered "half"-anchored WALP peptides, having only one pair of interfacial Trp residues near either the amino or the carboxyl terminus. The hydrophobic lengths of the (n = 8) peptides are similar to that of WALP23. These longer half-anchored WALP peptides incorporate into lipid bilayers as α-helices, as reflected in their circular dichroism spectra. Solid-state NMR experiments indicate that the longer peptide helices assume defined transmembrane orientations with small non-zero average tilt angles and moderate to high dynamic averaging in bilayer membranes of 1,2-dioleoylphosphatidylcholine, 1,2-dimyristoylphosphatidylcholine, and 1,2-dilauroylphosphatidylcholine. The intrinsically small apparent tilt angles suggest that interactions of aromatic residues with lipid headgroups may play an important role in determining the magnitude of the peptide tilt in the bilayer membrane. The shorter (n = 4) peptides, in stark contrast to the longer peptides, display NMR spectra that are characteristic of greatly reduced motional averaging, probably because of peptide aggregation in the bilayer environment, and CD spectra that are characteristic of β-structure.

Topics: Amino Acid Sequence; Dimyristoylphosphatidylcholine; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Nuclear Magnetic Resonance, Biomolecular; Oligopeptides; Peptides; Phosphatidylcholines; Protein Structure, Secondary; Tryptophan

The partitioning of different cholesterol-modified single-stranded DNA molecules (chol-DNAs) between the domains of phase-separated lipid vesicles is investigated by laser-scanning confocal fluorescence microscopy. All chol-DNAs studied preferentially localized into the fluid phase of giant vesicles in liquid-solid phase coexistence (1:1 DLPC:DPPC, 1:1 DLPC:DMPE). Partitioning behavior of chol-DNAs into liquid-liquid phase-separated vesicles (DOPC/DPPC/cholesterol) was found to be less straightforward. Single-cholesterol-anchored DNA molecules partitioned roughly equally between coexisting domains, whereas chol-DNAs with two cholesterol anchors were seen to be enriched in the liquid-ordered domains with apparent surface concentrations up to double that of the liquid-disordered phase. Quantitative analysis of the fluorescence intensity of DNA between the two phases also revealed a weaker dependence of the apparent partitioning on the initial lipid composition of the vesicles. We rationalize these observations by proposing a simple partitioning model based on the conformational entropy of insertion of a cholesterol anchor into each phase.

Topics: Cholesterol; DNA, Single-Stranded; Fluorescent Dyes; Microscopy, Fluorescence; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Unilamellar Liposomes

Giant vesicles generated from synthetic and natural lipids such as phosphatidylcholines are useful models for understanding mechanical properties of cell membranes. Line tension is the one-dimensional force enabling the closing of transient pores on cell membranes. Transient pores were repeatedly and reproducibly formed on the membrane edge of giant vesicles generated from synthetic and natural phosphatidylcholines employing a nitrogen-pumped coumarin dye laser (440 nm). Line tension was determined at room temperature from closing of these pores that occurred over several seconds when the radius of the vesicle could be considered to be constant. The value of line tension depends on the nature of the lipid for single lipid systems, which, at room temperature, yielded a vesicle bilayer region in the gel, fluid, or mixed gel and fluid phases. The line tension for vesicles generated from phosphatidylcholines with saturated acyl chains of lengths of 12-18 carbon atoms ranges from 1 to 12 pN, exhibiting an increase with chain length. Vesicles generated from the natural Egg-PC, which is a mixture of lipids, are devoid of phase transition and exhibited the largest value of line tension (32 pN). This value is much larger than that estimated from the line tensions of vesicles obtained from lipids with homologous acyl chains. This study, to our knowledge, is the first to employ laser ablation to generate transient pores and determine line tension from the rate of pore closure and demonstrate a relationship between line tension and acyl chain length.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Algorithms; Chemical Phenomena; Chemistry, Physical; Dimyristoylphosphatidylcholine; Elasticity; Kinetics; Lipid Bilayers; Phosphatidylcholines; Surface Tension; Unilamellar Liposomes; Viscosity

Sarcoplasmic reticulum Ca-transporting ATPase (EC 3.6.1.38) was isolated from rabbit white muscle, purified and reconstituted into vesicles of synthetic diacylphosphatidylcholines with monounsaturated acyl chains using the cholate dilution method. In fluid bilayers at 37 degrees C, the specific activity of ATPase displays a maximum (31.5+/-0.8 IU/mg) for dioleoylphosphatidylcholine (diC18:1PC) and decreases progressively for both shorter and longer acyl chain lengths. Besides the hydrophobic mismatch between protein and lipid bilayer, changes in the bilayer hydration and lateral interactions detected by small angle neutron scattering (SANS) can contribute to this acyl chain length dependence. When reconstituted into dierucoylphosphatidylcholine (diC22:1PC), the zwitterionic surfactant N-dodecyl-N,N-dimethylamine N-oxide (C12NO) stimulates the ATPase activity from 14.2+/-0.6 to 32.5+/-0.8 IU/mg in the range of molar ratios C12NO:diC22:1PC=0/1.2. In dilauroylphosphatidylcholines (diC12:0PC) and diC18:1PC, the effect of C12NO is twofold-the ATPase activity is stimulated at low and inhibited at high C12NO concentrations. In diC18:1PC, it is observed an increase of activity induced by C12NO in the range of molar ratios C12NO:diC18:1PC< or =1.3 in bilayers, where the bilayer thickness estimated by SANS decreases by 0.4+/-0.1 nm. In this range, the 31P-NMR chemical shift anisotropy increases indicating an effect of C12NO on the orientation of the phosphatidylcholine dipole N(+)-P- accompanied by a variation of the local membrane dipole potential. A decrease of the ATPase activity is observed in the range of molar ratios C12NO:diC18:1PC=1.3/2.5, where mixed tubular micelles are detected by SANS in C12NO+diC18:1PC mixtures. It is concluded that besides hydrophobic thickness changes, the changes in dipole potential and curvature frustration of the bilayer could contribute as well to C12NO effects on Ca(2+)-ATPase activity.

Topics: Animals; Biological Transport; Calcium-Transporting ATPases; Cholates; Dimethylamines; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Magnetic Resonance Spectroscopy; Neutron Diffraction; Oxides; Phosphatidylcholines; Phosphorylcholine; Rabbits; Sarcoplasmic Reticulum; Surface-Active Agents

Critical to biological processes such as membrane fusion and secretion, ion-lipid interactions at the membrane-water interface still raise many unanswered questions. Using reconstituted phosphatidylcholine membranes, we confirm here that multilamellar vesicles swell in salt solutions, a direct indication that salt modifies the interactions between neighboring membranes. By varying sample histories, and by comparing with data from ion carrier-containing bilayers, we eliminate the possibility that swelling is an equilibration artifact. Although both attractive and repulsive forces could be modified by salt, we show experimentally that swelling is driven primarily by weakening of the van der Waals attraction. To isolate the effect of salt on van der Waals interactions, we focus on high salt concentrations at which any possible electrostatic interactions are screened. By analysis of X-ray diffraction data, we show that salt does not alter membrane structure or bending rigidity, eliminating the possibility that repulsive fluctuation forces change with salt. By measuring changes in interbilayer separation with applied osmotic stress, we have determined, using the standard paradigm for bilayer interactions, that 1 M concentrations of KBr or KCl decrease the van der Waals strength by 50%. By weakening van der Waals attractions, salt increases energy barriers to membrane contact, possibly affecting cellular communication and biological signaling.

Topics: Algorithms; Anions; Bromides; Chlorides; Dimyristoylphosphatidylcholine; Lipid Bilayers; Liposomes; Models, Chemical; Osmotic Pressure; Phosphatidylcholines; Phospholipids; Static Electricity; Thermodynamics; Water; X-Ray Diffraction

Quantitative structures of the fully hydrated fluid phases of dimyristoylphosphatidylcholine (DMPC) and dilauroylphosphatidylcholine (DLPC) were obtained at 30 degrees C. Data for the relative form factors F(q(z)) for DMPC were obtained using a combination of four methods. 1), Volumetric data provided F(0). 2), Diffuse x-ray scattering from oriented stacks of bilayers provided relative form factors |F(q(z))| for high q(z), 0.22 < q(z) < 0.8 A(-1). 3), X-ray scattering from extruded unilamellar vesicles with diameter 600 A provided |F(q(z))| for low q(z), 0.1 < q(z) < 0.3 A(-1). 4), Previous measurements using a liquid crystallographic x-ray method provided |F(2 pi h/D)| for h = 1 and 2 for a range of nearly fully hydrated D-spacings. The data from method 4 overlap and validate the new unilamellar vesicles data for DMPC, so method 4 is not required for DLPC or future studies. We used hybrid electron density models to obtain structural results from these form factors. Comparison of the model electron density profiles with that of gel phase DMPC provides areas per lipid A, 60.6 +/- 0.5 A(2) for DMPC and 63.2 +/- 0.5 A(2) for DLPC. Constraints on the model provided by volume measurements and component volumes obtained from simulations put the electron density profiles rho(z) and the corresponding form factors F(q(z)) on absolute scales. Various thicknesses, such as the hydrophobic thickness and the steric thickness, are obtained and compared to literature values.

Topics: Biophysics; Carbon; Dimyristoylphosphatidylcholine; Electrons; Fourier Analysis; Glycerol; Lipid A; Lipid Bilayers; Molecular Conformation; Normal Distribution; Phosphatidylcholines; Scattering, Radiation; Temperature; Water; X-Rays

The nanomechanical response of supported lipid bilayers has been studied by force spectroscopy with atomic force microscopy. We have experimentally proved that the amount of ions present in the measuring system has a strong effect on the force needed to puncture a 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer with an atomic force microscope tip, thus highlighting the role that monovalent cations (so far underestimated, e.g., Na(+)) play upon membrane stability. The increase in the yield threshold force has been related to the increase in lateral interactions (higher phospholipid-phospholipid interaction, decrease in area per lipid) promoted by ions bound into the membrane. The same tendency has also been observed for other phosphatidylcholine bilayers, namely, 2-dilauroyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1,2-dioleoyl-sn-3-phosphocholine, and also for phosphatidylethanolamine bilayers such as 1-palmitoyl-2-oleoyl-sn-3-phosphoethanolamine. Finally, this effect has been also tested on a natural lipid bilayer (Escherichia coli lipid extract), showing the same overall tendency. The kinetics of the process has also been studied, together with the role of water upon membrane stability and its effect on membrane nanomechanics. Finally, the effect of the chemical structure of the phospholipid molecule on the nanomechanical response of the membrane has also been discussed.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Biophysical Phenomena; Biophysics; Dimyristoylphosphatidylcholine; Dose-Response Relationship, Drug; Escherichia coli; Ethanolamines; Ions; Kinetics; Lipid Bilayers; Lipids; Microscopy, Atomic Force; Nanotechnology; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Phosphorylcholine; Silicon Compounds; Sodium; Sodium Chloride; Spectrophotometry

The structure and changes in environment of the M13 major coat protein were studied in model systems, mimicking the initial molecular process of the phage disassembly. For this purpose we have systematically studied protein associations with various detergents and lipids in two different coat protein assemblies: phage particles and S-forms. It is remarkable that the major coat protein can change its conformation to accommodate three distinctly different environments: phage filament, S-form, and membrane-bound form. The structural and environmental changes during this protein transformations were studied by site-directed spin labeling, fluorescence labeling, and CD spectroscopy in different membrane model systems. The phage particles were disrupted only by strong ionic detergents [sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide and (CTAB)] but were not affected by sodium cholate and sodium deoxycholate, nonionic detergents, and dilauroyl-l-alpha-phosphatidylcholine (DLPC) lipid bilayers. Conversion of the phage particles into S-forms by addition of chloroform rendered the coat protein accessible for the association with different ionic and nonionic detergents, as well as DLPC lipids. The disruption of the S-form by all detergents studied was instantaneous but was slower with DLPC vesicles. Only small unilamellar vesicles effectively solubilized the S-form. The data suggest that the viral protein coat is inherently unstable when the major coat protein is exposed to amphiphilic molecules. During conversion from the phage to the S-form, and subsequently to the membrane-bound form, the coat protein undergoes pronounced changes in environment, and in response the alpha-helix content decreases and the local protein structure changes dramatically. This adaptation of the protein conformation enables a stable association of the protein with the membrane.

Topics: Bacteriophage M13; Capsid; Capsid Proteins; Circular Dichroism; Electron Spin Resonance Spectroscopy; Lipid Bilayers; Membrane Proteins; Models, Chemical; Naphthalenesulfonates; Phosphatidylcholines; Spectrometry, Fluorescence; Spin Trapping

Cytochrome P-450 coded for by the 3A gene family requires specific conditions in a reconstituted system, if its catalytic activity is to be efficient. We investigated the mechanism of activation of the catalytic activity of cytochrome P450 3A by phospholipids. Rat P450 PB-1 (3A2), human P450NF (3A4), and rabbit P450 3c (3A6) were used. They had low activity in a reconstituted system (system I) with dilauroylphosphatidylcholine (DLPC) but had high activity with a mixture of phospholipids (DLPC, dioleoylphosphatidylcholine, and phosphatidylserine) and sodium cholate (system II). P450 3A forms are cationic (having a high content of lysine residues) and needed the anionic phospholipid phosphatidylserine to have sufficient activity. Double-reciprocal plots of the metabolic rate of cytochrome P-450 versus the concentration of NADPH-cytochrome P-450 reductase showed that cytochrome P-450 and the reductase interacted more in system II than in system I. P450 PB-1 did not absorb at 450 nm in the presence of reductase, CO, DLPC, and NADPH, although other cytochrome P-450s absorbed at around 450 nm in such a mixture. However, P450 PB-1 was reduced in the presence of the phospholipid mixture and sodium cholate instead of DLPC. These results suggested that the stimulation of catalytic activity by phospholipids involved increased interaction between cytochrome P-450 and the reductase. Studies of proteolytic digestion and chemical cross-linking in systems I and II showed that a P450 3A form needed disaggregation of cytochrome P-450 and/or the reductase, not the formation of an aggregated complex necessary for the catalytic activity of other cytochrome P-450s.

Topics: Animals; Benzene Derivatives; Benzoflavones; Catalysis; Cholic Acid; Cholic Acids; Cytochrome P-450 Enzyme System; Enzyme Activation; Humans; Lidocaine; NADPH-Ferrihemoprotein Reductase; Nifedipine; Oxidation-Reduction; Phosphatidylcholines; Phosphatidylserines; Phospholipids; Rabbits; Rats; Testosterone