1-palmitoyl-2-oleoylglycero-3-phosphoglycerol and 1-palmitoyl-2-oleoylglycero-3-phosphoserine

The neuronal protein α-synuclein, linked to Parkinson's disease, binds to negatively charged vesicles adopting a partial α-helix structure, but helix arrangement at the vesicle surface is not fully understood. Using linear dichroism spectroscopy (LD), we study the interaction of monomeric α-synuclein with large unilamellar vesicles of 1,2-dioleoyl-

Topics: alpha-Synuclein; Amino Acid Sequence; Phosphatidylglycerols; Phosphatidylserines; Protein Binding; Protein Conformation, alpha-Helical; Unilamellar Liposomes

TRAAK is an ion channel from the two-pore domain potassium (K

Topics: Adenosine; Cations, Monovalent; Cloning, Molecular; Gene Expression; Genetic Vectors; Glycerophospholipids; Humans; Ion Channel Gating; Ion Transport; Kinetics; Liposomes; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Phosphatidylserines; Pichia; Potassium; Potassium Channels; Protein Binding; Protein Isoforms; Recombinant Proteins

Penetratin is a cell penetrating peptide (CPP) that can enter cells by direct translocation through the plasma membrane. The molecular mechanism of this translocation still remains poorly understood. Here we provide insights on this mechanism by studying the direct translocation of the peptide across model membranes based on Droplet Interface Bilayers (DIBs), which are bilayers at the interface between two adhering aqueous-in-oil droplets. We first showed with symmetric bilayers made of a mix of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (POPC) that the translocation of penetratin required the presence of at least 40% of POPG on both leaflets. Interestingly when replacing POPG with another anionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), translocation was inefficient. To elucidate the lipid partners required at each step of the CPP translocation process, we then investigated the crossing of asymmetric bilayers. We found that POPG on the proximal leaflet and POPS on the distal leaflet allowed penetratin translocation. Translocation was not observed when POPS was on the proximal leaflet and POPG on the distal leaflet or if POPS on the distal leaflet was replaced with POPC. These observations led us to propose a three-step translocation mechanism: (i) peptide recruitment by anionic lipids, (ii) formation of a transient peptide-lipid structure leading to the initiation of translocation which required specifically POPG on the proximal leaflet, (iii) termination of the translocation process favored by a driving force provided by anionic lipids in the distal leaflet.

Topics: Cell-Penetrating Peptides; Lipid Bilayers; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines

Circulating C-reactive protein (CRP) recognizes altered plasma membranes and activates complements systems in the acute phase of inflammation and infection in human. We have shown previously the calcium-independent adsorption of CRP toward 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and lysophosphatidylcholine (LPC) on supported phospholipid monolayers. Here, we extended our study to other phospholipids and additives to elucidate the pattern recognition of CRP using a surface plasmon resonance biosensor. Surface density and lateral fluidity depended on the type of phospholipids in the monolayers as characterized by SPR and fluorescence recovery after photobleaching measurements. CRP recognized 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) in the supported POPC monolayers without calcium at pH 7.4 and 5.5. As opposed to LPC, CRP did not recognize 3-sn-lysophosphatidylethanolamine in the POPC monolayers in calcium-free conditions. While, the addition of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or sphingomyelin to supported POPC monolayers blocked CRP adsorption. Calcium-dependent CRP binding was observed only at pH 5.5 on supported monolayers of engineered phospholipids with inverted headgroups relative to POPC. The complement 1q (C1q) protein recognized the active form of CRP on the supported phospholipid monolayers. The discovery of CRP recognition with these phospholipids aids our understanding of the activation dynamics of CRP with phospholipid-based biomaterials when used during the acute phase.

Topics: Adsorption; C-Reactive Protein; Calcium; Humans; Hydrogen-Ion Concentration; Lysophosphatidylcholines; Membrane Lipids; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Phospholipids; Protein Binding; Surface Plasmon Resonance; Surface Properties; Unilamellar Liposomes

Lipid membranes interact with and influence the aggregation of many amyloid-forming proteins. Orb2 is a cytoplasmic polyadenylation element-binding protein homolog in Drosophila melanogaster that forms functional amyloids necessary for long-term memory. One isoform, Orb2A, has a unique N-terminus that has been shown to be important for the formation of amyloid-like aggregates and long-term memory in vivo. Orb2A is also found enriched in the synaptic membrane fraction. Our sequence and hydropathy analysis suggests that it can form an amphipathic helix, which is ideal for lipid membrane interaction. We used circular dichroism and site-directed spin labeling coupled with electron paramagnetic resonance to test the first 88 amino acids of Orb2A for lipid interaction. We show that Orb2A1-88 interacts with anionic lipid membranes using an amphipathic helix at its unique N-terminus. This interaction depends on the charge of the lipid membrane and the degree of membrane curvature. We used transmission electron microscopy and electron paramagnetic resonance to show that the presence of anionic small unilamellar vesicles inhibits amyloid fibril formation by Orb2A. This inhibition by anionic membranes could be a potential mechanism regulating Orb2A amyloid formation in vivo.

Topics: Amino Acid Sequence; Amyloid; Animals; Binding Sites; Circular Dichroism; Drosophila melanogaster; Drosophila Proteins; Electron Spin Resonance Spectroscopy; Escherichia coli; Microscopy, Electron, Transmission; mRNA Cleavage and Polyadenylation Factors; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Protein Isoforms; Protein Structure, Secondary; Surface Properties; Transcription Factors; Unilamellar Liposomes

Multidrug resistance against the existing antibiotics is one of the most challenging threats across the globe. Antimicrobial peptides (AMPs), in this regard, are considered to be one of the effective alternatives that can overcome bacterial resistance. MSI-594, a 24-residue linear alpha-helical cationic AMP, has been shown to function via the carpet mechanism to disrupt bacterial membrane systems. To better understand the role of lipid composition in the function of MSI-594, in the present study, eight different model membrane systems have been studied using accelerated molecular dynamics (aMD) simulations. The simulated results are helpful in discriminating the particular effects of cationic MSI-594 against zwitterionic POPC, anionic POPG and POPS, and neutral POPE lipid moieties. Additionally, the effects of various heterogeneous POPC/POPG (7 : 3), POPC/POPS (7 : 3), and POPG/POPE (1 : 3 and 3 : 1) bilayer systems on the dynamic interaction of MSI-594 have also been investigated. The effect on the lipid bilayer due to the interaction with the peptide is characterized by lipid acyl-chain order, membrane thickness, and acyl-chain dynamics. Our simulation results show that the lipid composition affects the membrane interaction of MSI-594, suggesting that membrane selectivity is crucial to its mechanism of action. The results reported in this study are helpful to obtain accurate atomistic-level information governing MSI-594 and its membrane disruptive antimicrobial mechanism of action, and to design next generation potent antimicrobial peptides.

Topics: Amino Acid Sequence; Antimicrobial Cationic Peptides; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Molecular Dynamics Simulation; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Phosphatidylserines; Protein Structure, Secondary

Recently, several peptides have been studied regarding the defence process against pathogenic microorganisms, which are able to act against different targets, with the purpose of developing novel bioactive compounds. The present work focuses on the structural and functional evaluation of the palindromic antimicrobial peptide Pa-MAP2, designed based on the peptide Pa-MAP from Pleuronectes americanus. For a better structural understanding, molecular modelling analyses were carried out, together with molecular dynamics and circular dichroism, in different media. Antibacterial activity against Gram-negative and positive bacteria was evaluated, as well as cytotoxicity against human erythrocytes, RAW 264.7, Vero and L6 cells. In silico docking experiments, lipid vesicle studies, and atomic force microscopy (AFM) imaging were carried out to explore the activity of the peptide. In vivo studies on infected mice were also done. The palindromic primary sequence favoured an α-helix structure that was pH dependent, only present on alkaline environment, with dynamic N- and C-terminals that are stabilized in anionic media. Pa-MAP2 only showed activity against Gram-negative bacteria, with a MIC of 3.2 μM, and without any cytotoxic effect. In silico, lipid vesicles and AFM studies confirm the preference for anionic lipids (POPG, POPS, DPPE, DPPG and LPS), with the positively charged lysine residues being essential for the initial electrostatic interaction. In vivo studies showed that Pa-MAP2 increases to 100% the survival rate of mice infected with Escherichia coli. Data here reported indicated that palindromic Pa-MAP2 could be an alternative candidate for use in therapeutics against Gram-negative bacterial infections.

Topics: Alanine; Amino Acid Sequence; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Cell Survival; Chlorocebus aethiops; Cholesterol; Erythrocytes; Escherichia coli; Escherichia coli Infections; Flounder; Humans; Lipopolysaccharides; Mice; Molecular Dynamics Simulation; Molecular Sequence Data; Peptidomimetics; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Protein Structure, Secondary; Protein Structure, Tertiary; Survival Analysis; Unilamellar Liposomes; Vero Cells

Lipids and proteins are organized in cellular membranes in clusters, often called 'lipid rafts'. Although raft-constituent ordered lipid domains are thought to be energetically unfavourable for membrane fusion, rafts have long been implicated in many biological fusion processes. For the case of HIV gp41-mediated membrane fusion, this apparent contradiction can be resolved by recognizing that the interfaces between ordered and disordered lipid domains are the predominant sites of fusion. Here we show that line tension at lipid domain boundaries contributes significant energy to drive gp41-fusion peptide-mediated fusion. This energy, which depends on the hydrophobic mismatch between ordered and disordered lipid domains, may contribute tens of kBT to fusion, that is, it is comparable to the energy required to form a lipid stalk intermediate. Line-active compounds such as vitamin E lower line tension in inhomogeneous membranes, thereby inhibit membrane fusion, and thus may be useful natural viral entry inhibitors.

Topics: Cholesterol; HIV Envelope Protein gp41; HIV-1; Humans; Lipid Bilayers; Membrane Fusion; Membrane Microdomains; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Phosphatidylserines; Thermodynamics; Virus Internalization; Vitamin E

We present a new atom density profile (ADP) model and a statistical approach for extracting structural characteristics of lipid bilayers from X-ray and neutron scattering data. Models for five lipids with varying head and tail chemical composition in the fluid phase, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), are optimized using a simplex based method to simultaneously reproduce both neutron and X-ray scattering data. Structural properties are determined using statistical analysis of multiple optimal model structures. The method and models presented make minimal assumptions regarding the atomic configuration, while taking into account the underlying physical properties of the system. The more general model and statistical approach yield data with well defined uncertainties, indicating the precision in determining density profiles, atomic locations, and bilayer structural characteristics. Resulting bilayer structures include regions exhibiting large conformational variation. Due to the increased detail in the model, the results demonstrate the possibility of a distinct hydration layer within the interfacial (backbone) region.

Topics: Lipid Bilayers; Models, Chemical; Neutron Diffraction; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Quantum Theory; Scattering, Radiation; X-Ray Diffraction

α-Synuclein (αS) is a membrane-binding protein with sequence similarity to apolipoproteins and other lipid-carrying proteins, which are capable of forming lipid-containing nanoparticles, sometimes referred to as "discs." Previously, it has been unclear whether αS also possesses this property. Using cryo-electron microscopy and light scattering, we found that αS can remodel phosphatidylglycerol vesicles into nanoparticles whose shape (ellipsoidal) and dimensions (in the 7-10-nm range) resemble those formed by apolipoproteins. The molar ratio of αS to lipid in nanoparticles is ∼1:20, and αS is oligomeric (including trimers and tetramers). Similar nanoparticles form when αS is added to vesicles of mitochondrial lipids. This observation suggests a mechanism for the previously reported disruption of mitochondrial membranes by αS. Circular dichroism and four-pulse double electron electron resonance experiments revealed that in nanoparticles αS assumes a broken helical conformation distinct from the extended helical conformation adopted when αS is bound to intact vesicles or membrane tubules. We also observed αS-dependent tubule and nanoparticle formation in the presence of oleic acid, implying that αS can interact with fatty acids and lipids in a similar manner. αS-related nanoparticles might play a role in lipid and fatty acid transport functions previously attributed to this protein.

Topics: alpha-Synuclein; Cholesterol; Chromatography, Gel; Cryoelectron Microscopy; Fluorescence Resonance Energy Transfer; Humans; Lipoproteins; Membranes, Artificial; Mitochondrial Membranes; Nanoparticles; Particle Size; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Protein Structure, Quaternary; Protein Structure, Secondary

The assembly of electrically addressable, planar supported bilayers composed of biologically relevant lipids, such as those used in vesicular systems, will greatly enhance the experimental capabilities in membrane and membrane protein research. Here we assess the electrical properties of bilayers composed of a wide range of physiologically relevant lipids and lipid combinations. We demonstrate that robust, biologically relevant, planar supported bilayers with high resistance composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 25 mol % cholesterol can be constructed with high reproducibility. Furthermore, to enable studies of pore-forming peptides, which are commonly cationic, we demonstrate the construction of bilayers with biologically relevant outer leaflets incorporating up to 10 mol % negatively charged lipids. Unique features of the platform are that (1) the substrate is commercially available, atomically smooth, single-crystal silicon, (2) the polymer cushion allows for the natural incorporation of membrane proteins, and (3) the platform is highly reproducible.

Topics: Cholesterol; Electricity; Lipid Bilayers; Membrane Proteins; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Reproducibility of Results; Silicon; Surface Properties

The C1 domains of classical and novel PKCs mediate their diacylglycerol-dependent translocation. Using fluorescence resonance energy transfer, we studied the contribution of different negatively charged phospholipids and diacylglycerols to membrane binding. Three different C1B domains of PKCs were studied (the classical gamma, and the novel delta and epsilon), together with different lipid mixtures containing three types of acidic phospholipids and three types of activating diacylglycerols. The results show that C1Bgamma and C1Bepsilon exhibit a higher affinity to bind to vesicles containing 1-palmitoyl-2-oleoyl-sn-phosphatidic acid, 1-palmitoyl-2-oleoyl-sn-phoshatidylserine, or 1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol, with C1Bepsilon being the most relevant case because its affinity for POPA-containing vesicles increased by almost two orders of magnitude. When the effect of the diacylglycerol fatty acid composition on membrane binding was studied, the C1Bepsilon domain showed the highest binding affinity to membranes containing 1-stearoyl-oleoyl-sn-glycerol or 1,2-sn-dioleoylglycerol with POPA as the acidic phospholipid. Of the three diacylglycerols used in this study, 1,2-sn-dioleoylglycerol and 1-stearoyl-oleoyl-sn-glycerol showed the highest affinities for each isoenzyme, whereas 1,2-sn-dipalmitoylglycerol; showed the lowest affinity. DSC experiments showed this to be a consequence of the nonfluid conditions of 1,2-sn-dipalmitoylglycerol;-containing systems.

Topics: Adenosine; Calorimetry, Differential Scanning; Cell Line; Diglycerides; Fluorescence Resonance Energy Transfer; Glycerophospholipids; Humans; Models, Molecular; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Protein Binding; Protein Kinase C; Protein Kinase C-delta; Protein Kinase C-epsilon; Protein Structure, Tertiary; Temperature; Unilamellar Liposomes

The binding of the positively charged antimicrobial peptide cyclo[VKLdKVdYPLKVKLdYP] (GS14dK4) to various lipid bilayer model membranes was investigated using isothermal titration calorimetry. GS14dK4 is a diastereomeric lysine ring-size analogue of the naturally occurring antimicrobial peptide gramicidin S which exhibits enhanced antimicrobial and markedly reduced hemolytic activities compared with GS itself. Large unilamellar vesicles composed of various zwitterionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine [POPC]) and anionic phospholipids {1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(glycerol)] [POPG] and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phosphoserine] [POPS]}, with or without cholesterol, were used as model membrane systems. Dynamic light scattering results indicate the absence of any peptide-induced major alteration in vesicle size or vesicle fusion under our experimental conditions. The binding of GS14dK4 is significantly influenced by the surface charge density of the phospholipid bilayer and by the presence of cholesterol. Specifically, a significant reduction in the degree of binding occurs when three-fourths of the anionic lipid molecules are replaced with zwitterionic POPC molecules. No measurable binding occurs to cholesterol-containing zwitterionic vesicles, and a dramatic drop in binding is observed in the cholesterol-containing anionic POPG and POPS membranes, indicating that the presence of cholesterol markedly reduces the affinity of this peptide for phospholipid bilayers. The binding isotherms can be described quantitatively by a one-site binding model. The measured endothermic binding enthalpy (DeltaH) varies dramatically (+6.3 to +26.5 kcal/mol) and appears to be inversely related to the order of the phospholipid bilayer system. However, the negative free energy (DeltaG) of binding remains relatively constant (-8.5 to -11.5 kcal/mol) for all lipid membranes examined. The relatively small variation of negative free energy of peptide binding together with a pronounced variation of positive enthalpy produces an equally strong variation of TDeltaS (+16.2 to +35.0 kcal/mol), indicating that GS14dK4 binding to phospholipids bilayers is primarily entropy driven.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Calorimetry; Cholesterol; Drug Design; Gramicidin; Lipid Bilayers; Models, Chemical; Molecular Sequence Data; Peptides, Cyclic; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Phospholipids; Protein Binding; Static Electricity; Stereoisomerism; Thermodynamics; Titrimetry