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

Ion currents through potassium channels are gated. Constriction of the ion conduction pathway at the inner helix bundle, the textbook gate of Kir potassium channels, has been shown to be an ineffective permeation control, creating a rift in our understanding of how these channels are gated. Here we present evidence that anionic lipids act as interactive response elements sufficient to gate potassium conduction. We demonstrate the limiting barrier to K

Topics: Anions; Binding Sites; Crystallography, X-Ray; Humans; Ion Channel Gating; Ion Transport; Liposomes; Membrane Lipids; Molecular Dynamics Simulation; Mutation; Phosphatidylcholines; Phosphatidylserines; Potassium; Potassium Channels, Inwardly Rectifying

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

Previous studies suggested that fibrillar human IAPP (hIAPP) is more likely to deposit in β-cells, resulting in β-cell injury. However, the changes in the conformation of hIAPP in lipid environment and the mechanism involved in β-cell damage are unclear.. Synthetic hIAPP was incubated with five types of free fatty acids and phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), which constitute the cell membrane. Thioflavin-T fluorescence assay was conducted to analyze the degree of hIAPP fibrosis, and circular dichroism spectroscopy was performed to detect the β-fold formation of hIAPP. Furthermore, INS-1 cells were infected with human IAPP delivered by a GV230-EGFP plasmid. The effects of endogenous hIAPP overexpression induced by sodium palmitate on the survival, endoplasmic reticulum (ER) stress, and apoptosis of INS-1 cells were evaluated.. The five types of free fatty acids can accelerate the fibrosis of hIAPP. Sodium palmitate also maintained the stability of fibrillar hIAPP. POPS, not POPC, accelerated hIAPP fibrosis. Treatment of INS-1 cells with sodium palmitate increased the expression of hIAPP, activated ER stress and ER stress-dependent apoptosis signaling pathways, and increased the apoptotic rate.. Free fatty acids and anionic phospholipid can promote β-fold formation and fibrosis in hIAPP. High lipid induced the overexpression of hIAPP and aggravated ER stress and apoptosis in INS-1 cells, which caused β-cell death in high lipid environment.. Our study reveals free fatty acids and hIAPP synergistically implicated in endoplasmic reticulum stress and apoptosis of islet β-cells.

Topics: Amyloid; Apoptosis; Cell Membrane; Endoplasmic Reticulum Stress; Fatty Acids, Nonesterified; Fibrosis; Gene Expression Regulation; Humans; Insulin-Secreting Cells; Islet Amyloid Polypeptide; Lipid Metabolism; Lipids; Palmitic Acid; Phosphatidylcholines; Phosphatidylserines; Protein Conformation, beta-Strand; Protein Folding

In vitro characterization of membrane proteins requires experimental approaches providing mimics of the microenvironment that proteins encounter in native membranes. In this context, supported lipid bilayers provide a suitable platform to investigate membrane proteins by a broad range of surface-sensitive techniques such as neutron reflectometry (NR), quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR), atomic force microscopy (AFM), and fluorescence microscopy. Nevertheless, the successful incorporation of membrane proteins in lipid bilayers with sufficiently high concentration and controlled orientation relative to the bilayer remains challenging. We propose the unconventional use of peptide discs made by phospholipids and amphipathic 18A peptides to mediate the formation of supported phospholipid bilayers with two different types of membrane proteins, CorA and tissue factor (TF). The membrane proteins are reconstituted in peptide discs, deposited on a solid surface, and the peptide molecules are then removed with extensive buffer washes. This leaves a lipid bilayer with a relatively high density of membrane proteins on the support surface. As a very important feature, the strategy allows membrane proteins with one large extramembrane domain to be oriented in the bilayer, thus mimicking the in vivo situation. The method is highly versatile, and we show its general applicability by characterizing with the above-mentioned surface-sensitive techniques two different membrane proteins, which were efficiently loaded in the supported bilayers with ∼0.6% mol/mol (protein/lipid) concentration corresponding to 35% v/v for CorA and 8% v/v for TF. Altogether, the peptide disc mediated formation of supported lipid bilayers with membrane proteins represents an attractive strategy for producing samples for structural and functional investigations of membrane proteins and for preparation of suitable platforms for drug testing or biosensor development.

Topics: Aluminum Silicates; Cation Transport Proteins; Gold; Humans; Lipid Bilayers; Peptides; Phosphatidylcholines; Phosphatidylserines; Recombinant Proteins; Silicon Dioxide; Thromboplastin

Over the last few decades, anticancer peptides (ACPs) have turned into potential warheads against cancer. Apart from small molecules and monoclonal antibodies, ACPs have been proven to be effective against cancer cells. ACPs are small cationic peptides that selectively bind to the negatively charged cancer cell membrane and kill them by various mechanisms. In the present study, we prepared a random scrambled library of 1200 peptides from the 100 known ACPs and virtually screened them for their anticancer properties. From in silico-predicted ACPs, 27 peptides were prioritized based on their support vector machine (SVM) score. Based on the SVM score and properties such as hydrophobicity, size, overall net charge, secondary structure, and synthetic feasibility, finally, four peptides were synthesized and screened for their biological activities. Cancer cell membrane-deforming potential of two most active peptides, peptide1 and peptide2 was assessed with molecular dynamics simulation. We found that peptide1 remains adsorbed to the membrane surface, while peptide2 has membrane penetration capability. The present study will be helpful in the computational design of ACPs and understanding their interaction with the cancerous cell's membrane.

Topics: Antineoplastic Agents; Cell Line, Tumor; Computer Simulation; Drug Design; Humans; Membrane Lipids; Molecular Dynamics Simulation; Peptides; Phosphatidylcholines; Phosphatidylserines

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

Grb7 is an adapter protein, overexpressed in HER2+ve breast and other cancers, and identified as a therapeutic target. Grb7 promotes both proliferative and migratory cellular pathways through interaction of its SH2 domain with upstream binding partners including HER2, SHC, and FAK. Here we present the evaluation of a series of monocyclic and bicyclic peptide inhibitors that have been developed to specifically and potently target the Grb7 SH2-domain. All peptides tested were found to inhibit signaling in both ERK and AKT pathways in SKBR-3 and MDA-MB-231 cell lines. Proliferation, migration, and invasion assays revealed, however, that the second-generation bicyclic peptides were not more bioactive than the first generation G7-18NATE peptide, despite their higher in vitro affinity for the target. This was found not to be due to steric hindrance by the cell-permeability tag, as ascertained by ITC, but to differences in the ability of the bicyclic peptides to interact with and penetrate cellular membranes, as determined using SPR and mass spectrometry. These studies reveal that just small differences to amino acid composition can greatly impact the effectiveness of peptide inhibitors to their intracellular target and demonstrate that G7-18NATE remains the most effective peptide inhibitor of Grb7 developed to date.

Topics: Amino Acid Sequence; Antineoplastic Agents; Binding Sites; Cell Line; Cell Line, Tumor; Cell Membrane Permeability; Cell Movement; Cell Proliferation; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; GRB7 Adaptor Protein; Humans; Lipid Bilayers; Peptides, Cyclic; Phosphatidylcholines; Phosphatidylserines; Protein Binding; Proto-Oncogene Proteins c-akt; Signal Transduction; src Homology Domains; Structure-Activity Relationship

Understanding the toxicity of ionic liquids (ILs) is crucial in the search of greener chemicals. By comparing in vivo toxicity and in vitro interactions determined between compounds and biomimetic lipid membranes, more detailed toxicity vs. structure relation can be obtained. However, determining the interactions between non-surface-active compounds and liposomes has been a challenging task. Organisational changes induced by ILs and IL-like spirocyclic compounds within 1,6-diphenyl-1,3,5-hexatriene-doped biomimetic liposomes was studied by steady-state fluorescence anisotropy technique. The extent of organisational changes detected within the liposome bilayers were compared to the toxicity of the compounds determined using Vibrio Fischeri bacteria. Four liposome compositions made of pure 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocoline (POPC) and mixtures of POPC, 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoserine (POPS), and cholesterol (Chol) were tested as biomimetic models. Changes observed within the POPC/POPS/Chol 55:20:25 bilayers correlated the best with the toxicity results: ten out of twelve compounds followed the trend of increasing bilayer disorder - increasing toxicity. The study suggests that the toxicity of non-surface-active compounds is dependent on their ability to diffuse into the bilayers. The extent of bilayer's organisational changes correlates rather well with the toxicity of the compounds. Highly sensitive technique, such as fluorescence anisotropy measurements, is needed for detecting subtle changes within the bilayer structures.

Topics: Biomimetics; Cholesterol; Diphenylhexatriene; Fluorescence Polarization; Green Chemistry Technology; Ionic Liquids; Lipid Bilayers; Liposomes; Membranes; Phosphatidylcholines; Phosphatidylserines; Surface-Active Agents

Intracellular vesicle fusion is mediated by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18 (SM) proteins. It is generally accepted that membrane fusion occurs when the vesicle and target membranes are brought into close proximity by SNAREs and SM proteins. In this work, we demonstrate that, for fusion to occur, membrane bilayers must be destabilized by a conserved membrane-embedded motif located at the juxtamembrane region of the vesicle-anchored v-SNARE. Comprised of basic and hydrophobic residues, the juxtamembrane motif perturbs the lipid bilayer structure and promotes SNARE-SM-mediated membrane fusion. The juxtamembrane motif can be functionally substituted with an unrelated membrane-disrupting peptide in the membrane fusion reaction. These findings establish the juxtamembrane motif of the v-SNARE as a membrane-destabilizing peptide. Requirement of membrane-destabilizing peptides is likely a common feature of biological membrane fusion.

Topics: Amino Acid Sequence; Animals; Caenorhabditis elegans; Cell Membrane; Drosophila melanogaster; Humans; Lipid Bilayers; Membrane Fusion; Mice; Models, Molecular; Munc18 Proteins; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Sequence Alignment; Sequence Homology, Amino Acid; SNARE Proteins; Synaptosomal-Associated Protein 25; Transport Vesicles; Vesicle-Associated Membrane Protein 2; Xenopus laevis

Extracellular vesicles (ECVs) are heterogeneous membrane-enclosed structures containing proteins, nucleic acids, and lipids that participate in intercellular communication by transferring their contents to recipient cells. Although most of the attention has been directed at the biologic effect of proteins and microRNA, the contribution of phospholipids present in ECVs on cellular activation has not been extensively addressed. We investigated the biologic effect of phosphatidylserine (PS) and phosphatidylcholine (PC), 2 phospholipids highly abundant in ECVs. A transcriptomic analysis revealed that ∼4700 genes were specifically modified by exposing peritoneal macrophages to PS or PC liposomes in vivo. Among them, the expression of several chemokines and cytokines was highly upregulated by PS liposome treatment, translating into a massive neutrophil infiltration of the peritoneum capable of neutralizing a septic polymicrobial insult. Both the l and d stereoisomers of PS induced the same response, suggesting that the effect was related to the negative charge of the phospholipid head. We concluded that an increase in the internal negative charge of the cell triggers a signaling cascade activating an innate immune response capable of controlling infection.-Cauvi, D. M., Hawisher, D., Dores-Silva, P. R., Lizardo, R. E., De Maio, A. Macrophage reprogramming by negatively charged membrane phospholipids controls infection.

Topics: Animals; Cells, Cultured; Cellular Reprogramming; Coinfection; Extracellular Vesicles; Female; Macrophages, Peritoneal; Male; Mice; Mice, Inbred ICR; Phosphatidylcholines; Phosphatidylserines; Sepsis

The understanding of amyloid β-peptide (Aβ) interactions with cellular membranes is a crucial molecular challenge against Alzheimer's disease. Indeed, Aβ prefibrillar oligomeric intermediates are believed to be the most toxic species, able to induce cellular damages directly by membrane damage. We present a neutron-scattering study on the interaction of large unilamellar vesicles (LUV), as cell membrane models, with both freshly dissolved Aβ and early toxic prefibrillar oligomers, intermediate states in the amyloid pathway. In addition, we explore the effect of coincubating the Aβ-peptide with the chaperonin Hsp60, which is known to strongly interact with it in its aggregation pattern. In fact, the interaction of the LUV with coincubated Aβ/Hsp60, right after mixing and after following the aggregation protocol leading to the toxic intermediates in the absence of Hsp60, is studied. Neutron spin echo experiments show that the interaction with both freshly dissolved and aggregate Aβ species brings about an increase in membrane stiffness, whereas the presence of even very low amounts of Hsp60 (ratio Aβ/Hsp60 = 25:1) maintains unaltered the elastic properties of the membrane bilayer. A coherent interpretation of these results, related to previous literature, can be based on the ability of the chaperonin to interfere with Aβ aggregation, by the specific recognition of the Aβ-reactive transient species. In this framework, our results strongly suggest that early in a freshly dissolved Aβ solution are present some species able to modify the bilayer dynamics, and the chaperonin plays the role of an assistant in such stochastic "misfolding events", avoiding the insult on the membrane as well as the onset of the aggregation cascade.

Topics: Amyloid beta-Peptides; Animals; Cattle; Chaperonin 60; Gangliosides; Lipid Bilayers; Peptide Fragments; Phosphatidylcholines; Phosphatidylserines; Protein Multimerization; Unilamellar Liposomes

The spontaneous self-assembly of α-synuclein (α-syn) into aggregates of different morphologies is associated with the development of Parkinson's disease. However, the mechanism behind the spontaneous assembly remains elusive. The current study shows a novel effect of phospholipid bilayers on the assembly of the α-syn aggregates. Using time-lapse atomic force microscopy, it was discovered that α-syn assembles into aggregates on bilayer surfaces, even at the nanomolar concentration range. The efficiency of the aggregation process depends on the membrane composition, with the greatest efficiency observed for of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS). Importantly, assembled aggregates can dissociate from the surface, suggesting that on-surface aggregation is a mechanism by which pathological aggregates may be produced. Computational modeling revealed that dimers of α-syn assembled rapidly, through the membrane-bound monomer on POPS bilayer, due to an aggregation-prone orientation of α-syn. Interaction of α-syn with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) leads to a binding mode that does not induce a fast assembly of the dimer. Based on these findings, we propose a model in which the interaction of α-syn with membranes plays a critical role initiating the formation of α-syn aggregates and the overall aggregation process.

Topics: alpha-Synuclein; Computer Simulation; Humans; Lipid Bilayers; Models, Chemical; Phosphatidylcholines; Phosphatidylserines; Protein Aggregates

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

Signaling events at membranes are often mediated by membrane lipid composition or membrane physical properties. These membrane properties could act either by favoring the membrane binding of downstream effectors or by modulating their activity. Several proteins can sense/generate membrane physical curvature (

Topics: Animals; Cell Line; Cholesterol; Diacylglycerol Kinase; Diglycerides; Enzyme Assays; Humans; Liposomes; Membrane Fusion; Micelles; Molecular Structure; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylinositols; Phosphatidylserines; Spodoptera; Surface Properties

Ras protein colocalization at the plasma membrane is implicated in the activation of signaling cascades that promote cell growth, survival, and motility. However, the mechanisms that underpin Ras self-association remain unclear. We use molecular dynamics simulations to show how basic and hydrophobic components of the disordered C-terminal membrane tether of K-Ras4B combine to regulate its membrane interactions. Specifically, anionic lipids attract lysine residues to the membrane surface, thereby splitting the peptide population into two states that exchange on the microsecond time scale. These states differ in the membrane insertion of a methionine residue, which is influenced by local membrane composition. As a result, these states may impose context-dependent biases on the disposition of Ras' signaling domain, with possible implications for the accessibility of its effector binding surfaces. We investigate Ras' ability to nanocluster by fly-casting for patches of anionic lipids and find that while anionic lipids promote the intermolecular association of K-Ras4B membrane tethers, at short range this appears to be a passive process in which anionic lipids electrostatically screen these cationic peptides to mitigate their natural repulsion. Together with the sub-microsecond stability of interpeptide contacts, this result suggests that experimentally observed K-Ras4B nanoclustering is not driven by direct intermolecular contact of its membrane tethers.

Topics: Amino Acid Sequence; Humans; Lipid Bilayers; Methionine; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylserines; Protein Domains; Proto-Oncogene Proteins p21(ras); Static Electricity

The small GTPase Rab5 is a key regulator of endosomal trafficking processes and a marker for the early endosome. The C-terminal hypervariable region (HVR) of Rab5 is post-translationally modified at residues Cys

Topics: Amino Acid Sequence; Binding Sites; Cholesterol; Diterpenes; Endosomes; Glycosylation; Humans; Lipid Bilayers; Models, Molecular; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylinositol Phosphates; Phosphatidylserines; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; rab5 GTP-Binding Proteins; Signal Transduction; Sphingomyelins; Thermodynamics

Complexin-1 is a SNARE effector protein that decreases spontaneous neurotransmitter release and enhances evoked release. Complexin binds to the fully assembled four-helical neuronal SNARE core complex as revealed in competing molecular models derived from x-ray crystallography. Presently, it is unclear how complexin binding to the postfusion complex accounts for its effects upon spontaneous and evoked release in vivo. Using a combination of spectroscopic and imaging methods, we characterize in molecular detail how complexin binds to the 1:1 plasma membrane t-SNARE complex of syntaxin-1a and SNAP-25 while simultaneously binding the lipid bilayer at both its N- and C-terminal ends. These interactions are cooperative, and binding to the prefusion acceptor t-SNARE complex is stronger than to the postfusion core complex. This complexin interaction reduces the affinity of synaptobrevin-2 for the 1:1 complex, thereby retarding SNARE assembly and vesicle docking in vitro. The results provide the basis for molecular models that account for the observed clamping effect of complexin beginning with the acceptor t-SNARE complex and the subsequent activation of the clamped complex by Ca

Topics: Adaptor Proteins, Vesicular Transport; Animals; Escherichia coli; Lipid Bilayers; Liposomes; Mutation; Nerve Tissue Proteins; Phosphatidylcholines; Phosphatidylserines; Protein Binding; Rats; Recombinant Proteins; Surface Properties; Synaptosomal-Associated Protein 25; Syntaxin 1; Vesicle-Associated Membrane Protein 2

Topics: Cholesterol; Digitonin; Lipid Bilayers; Liposomes; Micelles; Phosphatidylcholines; Phosphatidylserines; Thermodynamics

Amyloid beta peptide (Aβ) is the major protein component of the amyloid plaques that are present in the brains of Alzheimer's disease (AD) patients. Aβ42 peptide is a known neurotoxic agent that binds to neurons and, under specific aggregation conditions, triggers cell death. Aβ peptide can undergo specific amino acid posttranslational modifications, such as phosphorylation, that are important for modulating its proteolytic degradation, aggregation, binding to lipid membranes and neurotoxic functions. Peptides phosphorylated at serine 8 in full-length Aβ42 (pAβ42) were synthesised and compared to native Aβ42 peptide. Their secondary structures, aggregation properties and interactions with plasma membranes of primary cortical neurons were investigated. The results revealed that pAβ42 has increased β-sheet formation with rapid amyloid formation in a synthetic lipid environment, which was associated with increased cellular binding but concomitant diminished neurotoxicity. Our data support the notion that phosphorylation of Aβ42 promotes the formation of amyloid plaques in the brain, which lack the neurotoxic properties associated with oligomeric species causing pathogenesis in AD.

Topics: Amyloid; Amyloid beta-Peptides; Animals; Cell Membrane; Cerebral Cortex; Cholesterol; Embryo, Mammalian; Humans; Mice; Mice, Inbred C57BL; Neurons; Peptide Fragments; Phosphatidylcholines; Phosphatidylserines; Phosphorylation; Primary Cell Culture; Protein Aggregates; Protein Binding; Protein Processing, Post-Translational; Protein Structure, Secondary; Proteolysis; 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

Interactions of soluble proteins with the cell membrane are critical within the blood coagulation cascade. Of particular interest are the interactions of γ-carboxyglutamic acid-rich domain-containing clotting proteins with lipids. Variability among conventional analytical methods presents challenges for comparing clotting protein-lipid interactions. Most previous studies have investigated only a single clotting protein and lipid composition and have yielded widely different binding constants. Herein, we demonstrate that a combination of lipid bilayer nanodiscs and a multiplexed silicon photonic analysis technology enables high-throughput probing of many protein-lipid interactions among blood-clotting proteins. This approach allowed direct comparison of the binding constants of prothrombin, factor X, activated factor VII, and activated protein C to seven different binary lipid compositions. In a single experiment, the binding constants of one protein interacting with all lipid compositions were simultaneously determined. A simple surface regeneration then facilitated similar binding measurements for three other coagulation proteins. As expected, our results indicated that all proteins exhibit tighter binding (lower

Topics: Factor VIIa; Factor X; High-Throughput Screening Assays; Humans; Kinetics; Lipid Bilayers; Nanostructures; Optical Phenomena; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylserines; Protein Array Analysis; Protein C; Prothrombin; Recombinant Proteins; Silicon

Binding of the retroviral structural protein Gag to the cellular plasma membrane is mediated by the protein's matrix (MA) domain. Prominent among MA-PM interactions is electrostatic attraction between the positively charged MA domain and the negatively charged plasma membrane inner leaflet. Previously, we reported that membrane association of HIV-1 Gag, as well as purified Rous sarcoma virus (RSV) MA and Gag, depends strongly on the presence of acidic lipids and is enhanced by cholesterol (Chol). The mechanism underlying this enhancement was unclear. Here, using a broad set of in vitro and in silico techniques we addressed molecular mechanisms of association between RSV MA and model membranes, and investigated how Chol enhances this association. In neutron scattering experiments with liposomes in the presence or absence of Chol, MA preferentially interacted with preexisting POPS-rich clusters formed by nonideal lipid mixing, binding peripherally to the lipid headgroups with minimal perturbation to the bilayer structure. Molecular dynamics simulations showed a stronger MA-bilayer interaction in the presence of Chol, and a large Chol-driven increase in lipid packing and membrane surface charge density. Although in vitro MA-liposome association is influenced by disparate variables, including ionic strength and concentrations of Chol and charged lipids, continuum electrostatic theory revealed an underlying dependence on membrane surface potential. Together, these results conclusively show that Chol affects RSV MA-membrane association by making the electrostatic potential at the membrane surface more negative, while decreasing the penalty for lipid headgroup desolvation. The presented approach can be applied to other viral and nonviral proteins.

Topics: Animals; Cell Membrane; Cholesterol; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylserines; Protein Binding; Protein Conformation; Protein Domains; Retroviridae Proteins; Rous sarcoma virus; Solvents; Static Electricity

Integrins are heterodimeric (αβ) cell surface receptors that are potential therapeutic targets for a number of diseases. Despite the existence of structural data for all parts of integrins, the structure of the complete integrin receptor is still not available. We have used available structural data to construct a model of the complete integrin receptor in complex with talin F2-F3 domain. It has been shown that the interactions of integrins with their lipid environment are crucial for their function but details of the integrin/lipid interactions remain elusive. In this study an integrin/talin complex was inserted in biologically relevant bilayers that resemble the cell plasma membrane containing zwitterionic and charged phospholipids, cholesterol and sphingolipids to study the dynamics of the integrin receptor and its effect on bilayer structure and dynamics. The results of this study demonstrate the dynamic nature of the integrin receptor and suggest that the presence of the integrin receptor alters the lipid organization between the two leaflets of the bilayer. In particular, our results suggest elevated density of cholesterol and of phosphatidylserine lipids around the integrin/talin complex and a slowing down of lipids in an annulus of ~30 Å around the protein due to interactions between the lipids and the integrin/talin F2-F3 complex. This may in part regulate the interactions of integrins with other related proteins or integrin clustering thus facilitating signal transduction across cell membranes.

Topics: Amino Acid Motifs; Binding Sites; Cholesterol; Humans; Integrin alphaVbeta3; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Multimerization; Protein Structure, Tertiary; Protein Subunits; Sequence Homology, Amino Acid; Talin; Thermodynamics

Tailor-made ionic liquids based on imidazolium salts have recently attracted a large amount of attention because of their extraordinary properties and versatile functionality. An intriguing ability to interact with and stabilize membranes has already been reported for 1,3-dialkylimidazolium compounds. We now reveal further insights into the field by investigating 1,3-dimethyl-4,5-dialkylimidazolium (C

Topics: 2-Naphthylamine; Diphenylhexatriene; Fluorescent Dyes; Imidazoles; Ionic Liquids; Laurates; Lipid Bilayers; Models, Chemical; Molecular Structure; Phosphatidylcholines; Phosphatidylserines; Transition Temperature; Unilamellar Liposomes; Viscoelastic Substances

Kindlins co-activate integrins alongside talin. They possess, like talin, a FERM domain (4.1-erythrin-radixin-moiesin domain) comprising F0-F3 subdomains, but with a pleckstrin homology (PH) domain inserted in the F2 subdomain that enables membrane association. We present the crystal structure of murine kindlin-3 PH domain determined at a resolution of 2.23 Å and characterise its lipid binding using biophysical and computational approaches. Molecular dynamics simulations suggest flexibility in the PH domain loops connecting β-strands forming the putative phosphatidylinositol phosphate (PtdInsP)-binding site. Simulations with PtdInsP-containing bilayers reveal that the PH domain associates with PtdInsP molecules mainly via the positively charged surface presented by the β1-β2 loop and that it binds with somewhat higher affinity to PtdIns(3,4,5)P

Topics: Amino Acid Sequence; Animals; Binding Sites; Cloning, Molecular; Crystallography, X-Ray; Cytoskeletal Proteins; Escherichia coli; Gene Expression; Kinetics; Mice; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylinositols; Phosphatidylserines; Pleckstrin Homology Domains; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Receptors, Cytoplasmic and Nuclear; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid

Amyloid formation by islet amyloid polypeptide (IAPP) contributes to β-cell dysfunction in type 2 diabetes. Perturbation of the β-cell membrane may contribute to IAPP-induced toxicity. We examine the effects of lipid composition, salt, and buffer on IAPP amyloid formation and on the ability of IAPP to induce leakage of model membranes. Even low levels of anionic lipids promote amyloid formation and membrane permeabilization. Increasing the percentage of the anionic lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS) or 1,2-dioleoyl-sn-glycero-3-phospho(1'-rac-glycerol), enhances the rate of amyloid formation and increases the level of membrane permeabilization. The choice of zwitterionic lipid has no noticeable effect on membrane-catalyzed amyloid formation but in most cases affects leakage, which tends to decrease in the following order: 1,2-dioleoyl-sn-glycero-3-phosphocholine > 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine > sphingomyelin. Uncharged lipids that increase the level of membrane order weaken the ability of IAPP to induce leakage. Leakage is due predominately to pore formation rather than complete disruption of the vesicles under the conditions used in these studies. Cholesterol at or below physiological levels significantly reduces the rate of vesicle-catalyzed IAPP amyloid formation and decreases the susceptibility to IAPP-induced leakage. The effects of cholesterol on amyloid formation are masked by 25 mol % POPS. Overall, there is a strong inverse correlation between the time to form amyloid and the extent of vesicle leakage. NaCl reduces the rate of membrane-catalyzed amyloid formation by anionic vesicles, but accelerates amyloid formation in solution. The implications for IAPP membrane interactions are discussed, as is the possibility that the loss of phosphatidylserine asymmetry enhances IAPP amyloid formation and membrane damage in vivo via a positive feedback loop.

Topics: Amino Acid Sequence; Amyloid; Cell Membrane; Cell Membrane Permeability; Cholesterol; Glycerylphosphorylcholine; Humans; Insulin-Secreting Cells; Islet Amyloid Polypeptide; Kinetics; Lipid Bilayers; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Sodium Chloride; Sphingomyelins

This section provides detailed protocols for the analysis of a mammalian diacylglycerol kinase, DGKθ, including an activity assay, a kinetic analysis, preparation of small unilamellar vesicles, and a vesicle pulldown assay. The goal of this section is to provide an overview of the unique challenges inherent in the study of an interfacial enzyme such as DGKθ and to outline methods useful for analysis. We include a short tutorial on selecting lipids for forming the interface since this is critical for a successful in vitro assay, and lipids are important regulators of this enzyme. The general principles can be applied to the study of other interfacial enzymes.

Topics: Adenosine Triphosphate; Animals; Diacylglycerol Kinase; Diglycerides; Enzyme Assays; Intracellular Membranes; Isoenzymes; Kinetics; Mammals; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phosphorylation; Protein Binding; Saccharomyces cerevisiae; Surface Properties; Unilamellar Liposomes

Topics: Deuterium Exchange Measurement; Marburgvirus; Mass Spectrometry; Models, Chemical; Phosphatidylcholines; Phosphatidylserines; Protein Multimerization; Protein Structure, Quaternary; Viral Matrix Proteins

Understanding the energetics of peripheral protein-membrane interactions is important to many areas of biophysical chemistry and cell biology. Estimating free-energy landscapes by molecular dynamics (MD) simulation is challenging for such systems, especially when membrane recognition involves complex lipids, e.g., phosphatidylinositol phosphates (PIPs). We combined coarse-grained MD simulations with umbrella sampling to quantify the binding of the well-explored GRP1 pleckstrin homology (PH) domain to model membranes containing PIP molecules. The experimentally observed preference of GRP1-PH for PIP3 over PIP2 was reproduced. Mutation of a key residue (K273A) within the canonical PIP-binding site significantly reduced the free energy of PIP binding. The presence of a noncanonical PIP-interaction site, observed experimentally in other PH domains but not previously in GRP1-PH, was also revealed. These studies demonstrate how combining coarse-grained simulations and umbrella sampling can unmask the molecular basis of the energetics of interactions between peripheral membrane proteins and complex cellular membranes.

Topics: Animals; Binding Sites; Humans; Lipid Bilayers; Mice; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylinositol Phosphates; Phosphatidylserines; Pleckstrin Homology Domains; Protein Binding; Receptors, Cytoplasmic and Nuclear; Thermodynamics

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

Dengue virus is a flavivirus responsible for millions of infections per year. Its surface contains a phospholipid bilayer, within which are embedded the envelope (E) and membrane (M) proteins, arranged with icosahedral geometry. Exposure to low pH triggers the E proteins to undergo conformational changes, which precede fusion with the host cell membrane and release of the viral genome. The flavivirus membrane exhibits significant local curvature and deformation, as revealed by cryoelectron microscopy (cryo-EM), but its precise structure and interactions with envelope components remain unclear. We now report simulations of the dengue viral particle that refine its envelope structure in unprecedented detail. Our final models are morphologically consistent with cryo-EM data, and reveal the structural basis for membrane curvature. Electrostatic interactions increased envelope complex stability; this coupling has potential functional significance in the context of the viral fusion mechanism and infective states.

Topics: Binding Sites; Cryoelectron Microscopy; Dengue Virus; Hydrogen-Ion Concentration; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Protein Binding; Protein Conformation, alpha-Helical; Protein Interaction Domains and Motifs; Static Electricity; Viral Envelope Proteins; Virion

Phospholipid vesicles are commonly used to get insights into the mechanism by which oligomers of amyloidogenic proteins damage membranes. Oligomers of the protein α-synuclein (αS) are thought to create pores in phospholipid vesicles containing a high amount of anionic phospholipids but fail to damage vesicle membranes at low surface charge densities. The current understanding of how αS oligomers damage the membranes is thus incomplete. This incomplete understanding may, in part, result from the choice of model membrane systems. The use of free-standing membranes such as vesicles may interfere with the unraveling of some damage mechanisms because the line tension at the edge of a membrane defect or pore ensures defect closure. Here, we have used supported lipid bilayers (SLBs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPC/POPS) to study the membrane damage caused by αS oligomers. Although αS oligomers were not able to initiate the disruption of POPC/POPS vesicles or intact SLBs, oligomers did stabilize and enlarge pre-existing SLB defects. The increased exposure of lipid acyl chains at the edges of defects very likely facilitates membrane-oligomer interactions, resulting in the growth of fractal domains devoid of lipids. Concomitant with the appearance of the fractal membrane damage patterns, lipids appear in solution, directly implicating αS oligomers in the observed lipid extraction. The growth of the membrane damage patterns is not limited by the binding of lipids to the oligomer. The analysis of the shape and growth of the lipid-free domains suggests the involvement of an oligomer-dependent diffusion-limited extraction mechanism. The observed αS oligomer-induced propagation of membrane defects offers new insights into the mechanisms by which αS oligomers can contribute to the loss in membrane integrity.

Topics: alpha-Synuclein; Cell Membrane; Fractals; Lipid Bilayers; Microscopy, Atomic Force; Microscopy, Confocal; Phosphatidylcholines; Phosphatidylserines; Time-Lapse Imaging; Unilamellar Liposomes

Phosphatidylserine (PS) has previously been found to bind Cu

Topics: Cations, Divalent; Copper; Electron Spin Resonance Spectroscopy; Hydrogen-Ion Concentration; Lipid Bilayers; Oxidation-Reduction; Phosphatidylcholines; Phosphatidylserines; Quantum Theory; Stereoisomerism; Thermodynamics

To this day, α-tocopherol's (aToc) role in humans is not well known. In previous studies, we have tried to connect aToc's biological function with its location in a lipid bilayer. In the present study, we have determined, by means of small-angle neutron diffraction, that not only is aToc's hydroxyl group located high in the membrane but its tail also resides far from the center of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers. In addition, we located aToc's hydroxyl group above the lipid backbone in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), and sphingomyelin bilayers, suggesting that aToc's location near the lipid-water interface may be a universal property of vitamin E. In light of these data, how aToc efficiently terminates lipid hydroperoxy radicals at the membrane center remains an open question.

Topics: alpha-Tocopherol; Humans; Lipid Bilayers; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Sphingomyelins; Surface Properties; Temperature; Thermodynamics; Water

Determining the structure of membrane-active peptides inside lipid bilayers is essential to understand their mechanism of action. Molecular dynamics simulations can easily provide atomistic details, but need experimental validation. We assessed the reliability of self-assembling (or "minimum-bias") and potential of mean force (PMF) approaches, using all-atom (AA) and coarse-grained (CG) force-fields. The LAH4 peptide was selected as a stringent test case, since it is known to attain different orientations depending on the protonation state of its four histidine residues. In all simulations the histidine side-chains inserted in the membrane when neutral, while they interacted with phospholipid headgroups in their charged state. This led to transmembrane orientations for neutral-His LAH4 in all minimum-bias AA simulations and in most CG trajectories. By contrast, the charged-His peptide stabilized membrane defects in AA simulations, whereas it was located at the membrane surface in some CG trajectories, and interacted with both lipid leaflets in others. This behavior is consistent with the higher antimicrobial activity and membrane-permeabilizing behavior of the charged-His LAH4. In addition, good agreement with solid-state NMR orientational data was observed in AA simulations. PMF calculations correctly predicted a higher membrane affinity for the neutral-His peptide. Interestingly, the structures and relative populations of PMF local free-energy minima corresponded to those determined in the less computationally demanding minimum-bias simulations. These data provide an indication about the possible membrane-perturbation mechanism of the charged-His LAH4 peptide: by interacting with lipid headgroups of both leaflets through its cationic side-chains, it could favor membrane defects and facilitate translocation across the bilayer.

Topics: Antimicrobial Cationic Peptides; Cell Membrane Permeability; Histidine; Kinetics; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylserines; Static Electricity; Thermodynamics; Water

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

In contrast to the static snapshots provided by protein crystallography, G protein-coupled receptors constitute a group of proteins with highly dynamic properties, which are required in the receptors' function as signaling molecule. Here, the human neuropeptide Y2 receptor was reconstituted into a model membrane composed of monounsaturated phospholipids and solid-state NMR was used to characterize its dynamics. Qualitative static (15)N NMR spectra and quantitative determination of (1)H-(13)C order parameters through measurement of the (1)H-(13)C dipolar couplings of the CH, CH2 and CH3 groups revealed axially symmetric motions of the whole molecule in the membrane and molecular fluctuations of varying amplitude from all molecular segments. The molecular order parameters (S(backbone) = 0.59-0.67, S(CH2) = 0.41-0.51 and S(CH3) = 0.22) obtained in directly polarized (13)C NMR experiments demonstrate that the Y2 receptor is highly mobile in the native-like membrane. Interestingly, according to these results the receptor was found to be slightly more rigid in the membranes formed by the monounsaturated phospholipids than by saturated phospholipids as investigated previously. This could be caused by an increased chain length of the monounsaturated lipids, which may result in a higher helical content of the receptor. Furthermore, the incorporation of cholesterol, phosphatidylethanolamine, or negatively charged phosphatidylserine into the membrane did not have a significant influence on the molecular mobility of the Y2 receptor.

Topics: Carbon-13 Magnetic Resonance Spectroscopy; Cell Membrane; Fatty Acids, Monounsaturated; Humans; Models, Molecular; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipids; Protein Structure, Secondary; Protein Structure, Tertiary; Receptors, Neuropeptide Y

Cationic membrane-proximal amino acids determine the topology of membrane proteins by interacting with anionic lipids that are restricted to the intracellular membrane leaflet. This mechanism implies that anionic lipids interfere with electrostatic interactions of membrane proteins. The integrin αIIbβ3 transmembrane (TM) complex is stabilized by a membrane-proximal αIIb(Arg(995))-β3(Asp(723)) interaction; here, we examine the influence of anionic lipids on this complex. Anionic lipids compete for αIIb(Arg(995)) contacts with β3(Asp(723)) but paradoxically do not diminish the contribution of αIIb(Arg(995))-β3(Asp(723)) to TM complex stability. Overall, anionic lipids in annular positions stabilize the αIIbβ3 TM complex by up to 0.50 ± 0.02 kcal/mol relative to zwitterionic lipids in a headgroup structure-dependent manner. Comparatively, integrin receptor activation requires TM complex destabilization of 1.5 ± 0.2 kcal/mol, revealing a sizeable influence of lipid composition on TM complex stability. We implicate changes in lipid headgroup accessibility to small molecules (physical membrane characteristics) and specific but dynamic protein-lipid contacts in this TM helix-helix stabilization. Thus, anionic lipids in ubiquitous annular positions can benefit the stability of membrane proteins while leaving membrane-proximal electrostatic interactions intact.

Topics: Amino Acid Sequence; Humans; Molecular Dynamics Simulation; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Phosphatidylcholines; Phosphatidylserines; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Stability; Protein Structure, Secondary

Key challenges in the development of drug delivery systems are the prevention of serum compartment interaction and the targeted delivery of the cargo. Layer-by-Layer microcarriers offer many advantages due to various options in drug assembly and multifunctional design. Surface modification with a supported lipid membrane enhances biocompatibility, drug protection ability, and specific functionality. However, the integration of functionalized lipids strongly influences the membrane formation and is often accompanied by submicrometer irregularities: The accessibility of underlying polymers to serum components may change the carrier's properties and enhances the susceptibility to opsonization. Therefore, the formation of a tightly assembled multifunctional lipid membrane has been emphasized. A phosphatidylserine/phosphatidylcholine (POPS/POPC) bilayer equipped with phosphatidylethanolamine-polyethylene glycol-biotin (PE-PEG-Biotin) was used to facilitate a biotin/streptavidin binding site for a variable attachment of an additional function, such as antibodies for specific targeting. Thus, a prefunctionalized carrier where only the outer functionality needs to be replaced without disturbing the underlying structure could be created.

Topics: Biotin; Drug Carriers; Lipid Bilayers; Liposomes; Particle Size; Phosphatidylcholines; Phosphatidylserines; Polyethylene Glycols; Silicon Dioxide; Streptavidin; Surface Properties

Class 1 cytokine receptors regulate essential biological processes through complex intracellular signalling networks. However, the structural platform for understanding their functions is currently incomplete as structure-function studies of the intracellular domains (ICDs) are critically lacking. The present study provides the first comprehensive structural characterization of any cytokine receptor ICD and demonstrates that the human prolactin (PRL) receptor (PRLR) and growth hormone receptor (GHR) ICDs are intrinsically disordered throughout their entire lengths. We show that they interact specifically with hallmark lipids of the inner plasma membrane leaflet through conserved motifs resembling immuno receptor tyrosine-based activation motifs (ITAMs). However, contrary to the observations made for ITAMs, lipid association of the PRLR and GHR ICDs was shown to be unaccompanied by changes in transient secondary structure and independent of tyrosine phosphorylation. The results of the present study provide a new structural platform for studying class 1 cytokine receptors and may implicate the membrane as an active component regulating intracellular signalling.

Topics: Cell Line; Cell Membrane; Circular Dichroism; Humans; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Peptide Fragments; Phosphatidylcholines; Phosphatidylserines; Protein Folding; Protein Structure, Tertiary; Receptors, Prolactin; Receptors, Somatotropin; Recombinant Proteins; Scattering, Small Angle; Signal Transduction; Tyrosine; X-Ray Diffraction

Annexins play critical roles in membrane organization, membrane trafficking and vesicle transport. The family members share the ability to bind to membranes with high affinities, but the interactions between annexins and membranes remain unclear. Here, using long-time molecular dynamics simulations, we provide detailed information for the binding of an annexin V trimer to a POPC/POPS lipid bilayer. Calcium ions function as bridges between several negatively charged residues of annexin V and the oxygen atoms of lipids. The preferred calcium-bridges are those formed via the carboxyl oxygen atoms of POPS lipids. H-bonds and hydrophobic interactions formed by several critical residues have also been observed in the annexin-membrane interface. The annexin-membrane binding causes small changes of annexin trimer structures, while has significant effects on lipid bilayer structures. The lipid bilayer shows a bent shape and forms a concave region in the annexin-membrane interaction interface, which provides an atomic-level evidence to support the view that annexins could disturb the stability of lipids and bend membranes. This study provides insights into the commonly occurring PS-dependent and calcium-dependent binding of proteins to membranes.

Topics: Annexin A5; Calcium; Humans; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylserines; Protein Binding; Protein Stability

We have investigated the interactions between cationic poly(amidoamine) (PAMAM) dendrimers of generation 4 (G4), a potential gene transfection vector, with net-anionic model biomembranes composed of different ratios of zwitterionic phosphocholine (PC) and anionic phospho-L-serine (PS) phospholipids. Two types of model membranes were used: solid-supported bilayers, prepared with lipids carrying palmitoyl-oleoyl (PO) and diphytanoyl (DPh) acyl chains, and free-standing bilayers, formed at the interface between two aqueous droplets in oil (droplet interface bilayers, DIBs) using the DPh-based lipids. G4 dendrimers were found to translocate through POPC:POPS bilayers deposited on silica surfaces. The charge density of the bilayer affects translocation, which is reduced when the ionic strength increases. This shows that the dendrimer-bilayer interactions are largely controlled by their electrostatic attraction. The structure of the solid-supported bilayers remains intact upon translocation of the dendrimer. However, the amount of lipids in the bilayer decreases and dendrimer/lipid aggregates are formed in bulk solution, which can be deposited on the interfacial layers upon dilution of the system with dendrimer-free solvent. Electrophysiology measurements on DIBs confirm that G4 dendrimers cross the lipid membranes containing PS, which then become more permeable to ions. The obtained results have implications for PAMAM dendrimers as delivery vehicles to cells.

Topics: Dendrimers; Light; Lipid Bilayers; Neutron Diffraction; Phosphatidylcholines; Phosphatidylserines; Quartz Crystal Microbalance Techniques; Scattering, Radiation

The solubility limits of cholesterol in small unilamellar vesicles made of POPS and POPC were probed using Small Angle Neutron Scattering (SANS) and coarse grained (CG) molecular dynamics (MD) simulations. SANS, being non-invasive, allowed the direct and quantitative measurement of cholesterol in intact vesicles. Our experimental measurements reveal a 61% mole fraction solubility limit of cholesterol in POPC, consistent with previous studies. However, in POPS the solubility limit of cholesterol is found to be 73% mole fraction. Previous work reports solubility limits of cholesterol in POPS varying significantly, ranging from 36% up to 66%. The CG MD simulations are in remarkable quantitative agreement with our experimental results showing similar solubility limits. Further, neither experiments nor simulations show evidence of stable nanodomains of cholesterol in POPS membranes as suggested in some previous reports.

Topics: Cholesterol; Molecular Dynamics Simulation; Neutron Diffraction; Phosphatidylcholines; Phosphatidylserines; Scattering, Small Angle; Solubility; Unilamellar Liposomes

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

Cellular pathways involving α-synuclein (αS) seem to be causative for development of Parkinson's disease. Interactions between αS and lipid membranes appear to be important for the physiological function of the protein and influence the pathological aggregation of αS leading to the formation of amyloid plaques. Upon membrane binding the unstructured αS folds into amphipathic helices. In our work we characterized the penetration depth and probed the local environment of Trp-residues introduced along the αS sequence. We could show that while the entire helix is well embedded in the lipid bilayer, segments with a shallower penetration and supposable higher flexibility exist.

Topics: Acrylamide; alpha-Synuclein; Amino Acid Sequence; Cell Membrane; Humans; Lipid Bilayers; Lipids; Micelles; Molecular Sequence Data; Parkinson Disease; Phosphatidylcholines; Phosphatidylserines; Protein Binding; Protein Structure, Tertiary; Tryptophan

Beta-amyloid (1-40) is one of the two most abundant species of amyloid-beta peptides present as fibrils in the extracellular senile plaques in the brain of Alzheimer's patients. Recently, the molecular aggregates constituting the early stage of fibril formation, i.e., oligomers and protofibrils, have been investigated as the main responsible for amyloid-beta cytotoxic effect. The molecular mechanism leading to neurodegeneration is still under debate, and it is common opinion that it may reside in the interaction between amyloid species and the neural membrane. In this investigation Atomic Force Microscopy and spectroscopy have been used to understand how structural (and mechanical) properties of POPC/POPS lipid bilayers, simulating the phospholipid composition and negative net charge of neuritic cell membranes, are influenced by the interaction with Aβ(1-40), in different stages of the peptide aggregation. Substantial differences in the damage caused to the lipid bilayers have been observed, confirming the toxic effect exerted especially by Aβ(1-40) prefibrillar oligomers.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Cell Membrane; Humans; Lipid Bilayers; Microscopy, Atomic Force; Neurons; Peptide Fragments; Phosphatidylcholines; Phosphatidylserines

A time-dependent fluorescence shift method, biomimetic colorimetric assays, and molecular dynamics simulations have been performed in search of explanations why arginine rich peptides with intermediate lengths of about 10 amino acids translocate well through cellular membranes, while analogous lysine rich peptides do not. First, we demonstrate that an important factor for efficient peptide adsorption, as the first prerequisite for translocation across the membrane, is the presence of negatively charged phospholipids in the bilayer. Second, we observe a strong tendency of adsorbed arginine (but not lysine) containing peptides to aggregate at the bilayer surface. We suggest that this aggregation of oligoarginines leads to partial disruption of the bilayer integrity due to the accumulated large positive charge at its surface, which increases membrane-surface interactions due to the increased effective charge of the aggregates. As a result, membrane penetration and translocation of medium length oligoarginines becomes facilitated in comparison to single arginine and very long polyarginines, as well as to lysine containing peptides.

Topics: 2-Naphthylamine; Adsorption; Arginine; Biomimetics; Colorimetry; Dextrans; Fluorescence; Fluorescent Dyes; Laurates; Lipid Bilayers; Lysine; Membrane Potentials; Molecular Dynamics Simulation; Mycophenolic Acid; Peptides; Phosphatidylcholines; Phosphatidylserines; Phospholipids; Polyacetylene Polymer; Polymers; Polyynes

The bovine seminal plasma contains phosphocholine-binding proteins, which associate to sperm membranes upon ejaculation. These binder-of-sperm (BSP) proteins then induce a phospholipid and cholesterol efflux from these membranes. In this work, we determined physical and chemical parameters controlling this efflux by characterizing the lipid extraction induced by BSP1, the most abundant of BSP protein in bull seminal plasma, from model membranes with different composition. The model membranes were formed from binary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (Lyso-PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) or cholesterol. The modulation of BSP1-induced lipid extraction from membranes by their chemical composition and their physical properties brings us to propose a 3-step extraction mechanism. First, the protein associates with membranes via specific binding to phosphocholine groups. Second, BSP1 penetrates in the membrane, essentially in the external lipid leaflet. Third, BSP1 molecules solubilize a lipid patch coming essentially from the outer lipid leaflet, without any lipid specificity, to ultimately form small lipid/protein auto-assemblies. The stoichiometry of these complexes corresponds to 10-15 lipids per protein. It is also shown that fluid-phase membranes are more prone to BSP1-induced lipid extraction than gel-phase ones. The inhibition of the lipid extraction in this case appears to be related to the inhibition of the protein penetration in the membrane (step 2) and not to the protein association with PC head groups (step 1). These findings contribute to our understanding of the mechanism by which BSP1 modify the lipid composition of sperm membranes, a key event in sperm capacitation.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; Cattle; Cell Membrane; Dose-Response Relationship, Drug; Lipids; Liposomes; Lysophosphatidylcholines; Male; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phosphorylcholine; Protein Binding; Seminal Vesicle Secretory Proteins; Sperm Capacitation; Spermatozoa; Temperature

The amino-terminal domain of huntingtin (Htt17), located immediately upstream of the decisive polyglutamine tract, strongly influences important properties of this large protein and thereby the development of Huntington's disease. Htt17 markedly increases polyglutamine aggregation rates and the level of huntingtin's interactions with biological membranes. Htt17 adopts a largely helical conformation in the presence of membranes, and this structural transition was used to quantitatively analyze membrane association as a function of lipid composition. The apparent membrane partitioning constants increased in the presence of anionic lipids but decreased with increasing amounts of cholesterol. When membrane permeabilization was tested, a pronounced dye release was observed from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles and 75:25 (molar ratio) POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine vesicles but not across bilayers that better mimic cellular membranes. Solid-state nuclear magnetic resonance structural investigations indicated that the Htt17 α-helix adopts an alignment parallel to the membrane surface, and that the tilt angle (∼75°) was nearly constant in all of the membranes that were investigated. Furthermore, the addition of Htt17 resulted in a decrease in the lipid order parameter in all of the membranes that were investigated. The lipid interactions of Htt17 have pivotal implications for membrane anchoring and functional properties of huntingtin and concomitantly the development of the disease.

Topics: Amino Acid Sequence; Cell Membrane; Humans; Huntingtin Protein; Lipid Bilayers; Liposomes; Models, Molecular; Molecular Sequence Data; Nerve Tissue Proteins; Permeability; Phosphatidylcholines; Phosphatidylserines; Protein Structure, Secondary; Protein Structure, Tertiary

Disruption of the integrity of the plasma membrane by amyloidogenic proteins is linked to the pathogenesis of a number of common age-related diseases. Although accumulating evidence suggests that adverse environmental stressors such as unbalanced levels of metal ions may trigger amyloid-mediated membrane damage, many features of the molecular mechanisms underlying these events are unknown. Using human islet amyloid polypeptide (hIAPP, aka amylin), an amyloidogenic peptide associated with β-cell death in type 2 diabetes, we demonstrate that the presence of Ca(2+) ions inhibits membrane damage occurring immediately after the interaction of freshly dissolved hIAPP with the membrane, but significantly enhances fiber-dependent membrane disruption. In particular, dye leakage, quartz crystal microbalance, atomic force microscopy, and NMR experiments show that Ca(2+) ions promote a shallow membrane insertion of hIAPP, which leads to the removal of lipids from the bilayer through a detergent-like mechanism triggered by fiber growth. Because both types of membrane-damage mechanisms are common to amyloid toxicity by most amyloidogenic proteins, it is likely that unregulated ion homeostasis, amyloid aggregation, and membrane disruption are all parts of a self-perpetuating cycle that fuels amyloid cytotoxicity.

Topics: Amyloid; Animals; Calcium; Cations; Cell Membrane; Coloring Agents; Detergents; Humans; Hydrophobic and Hydrophilic Interactions; Islet Amyloid Polypeptide; Lipid Bilayers; Microscopy, Atomic Force; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Quartz Crystal Microbalance Techniques; Rats

Discoidal high-density lipoprotein (HDL) particles are known to fractionalize into several discrete populations. Factors regulating their size are, however, less understood. To reveal the effect of lipid composition on their formation and characteristics, we prepared several reconstituted HDLs (rHDLs) with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and sphingomyelin at phospholipid to apolipoprotein A-I ratios of 100 and 25. When reconstitution was conducted at 37°C, the efficiency of rHDL formation from POPC was decreased as compared with that conducted at 4°C. Moreover, large rHDLs with a Stokes diameter of 9.6nm became dominant over small rHDL with a diameter of 7.9nm, which was distinctly observed at 4°C. The aminophospholipids POPS and POPE promoted the formation of small rHDLs at 37°C, but fluorescence experiments revealed that they did so in a different fashion: Fluorescence lifetime data suggested that the head group of POPS reduces hydrophobic hydration, especially in small rHDLs, suggesting that this lipid stabilizes the saddle-shaped bilayer structure in small rHDLs. Fluorescence lifetime and anisotropy data showed that incorporation of POPE increases acyl chain order and water penetration into the head group region in large rHDLs, suggesting that POPE destabilizes the planar bilayer structure. These results imply that these aminophospholipids contribute to the formation of small rHDLs under biological conditions.

Topics: Algorithms; Anisotropy; Apolipoprotein A-I; Kinetics; Lipid Bilayers; Lipoproteins, HDL; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipids; Spectrometry, Fluorescence; Sphingomyelins; Temperature

Effects of alkali metal chlorides on the properties of mixed negatively charged lipid bilayers are experimentally measured and numerically simulated. Addition of 20mol% of negatively charged phosphatidylserine to zwitterionic phosphatidylcholine strengthens adsorption of monovalent cations revealing their specificity, in the following order: Cs(+)

Topics: Cesium; Chlorides; Lipid Bilayers; Phosphatidylcholines; Phosphatidylserines; Potassium Chloride; Sodium Chloride

Anticancer peptides are polycationic amphiphiles capable of preferentially killing a wide spectrum of cancer cells relative to noncancerous cells. Their primary mode of action is an interaction with the cell membrane and subsequent activation of lytic effects; however, the exact mechanism responsible for this mode of action remains controversial. Using zeta potential analyses we demonstrate the interaction of a small anticancer peptide with membrane model systems and cancer cells. Electrostatic interactions have a pivotal role in the cell killing process, and in contrast to the antimicrobial peptides action cell death occurs without achieving full neutralization of the membrane charge.

Topics: Antineoplastic Agents; Cell Death; Cell Line, Tumor; Humans; Inhibitory Concentration 50; Membranes, Artificial; Peptides; Phosphatidylcholines; Phosphatidylserines; Static Electricity; Stereoisomerism

Protein kinase Cα (PKCα) is activated by its translocation to the membrane. Activity assays show the importance of PIP(2) in determining the specific activity of this enzyme. A FRET stopped flow fluorescence study was carried out to monitor the rapid kinetics of protein binding to model membranes containing POPC/POPS/DOG and eventually PIP(2). The results best fitted a binding mechanism in which protein bound to the membrane following a two-phase mechanism with a first bimolecular reaction followed by a slow unimolecular reaction. In the absence of PIP(2), the rapid protein binding rate was especially dependent on POPS concentration. Formation of the slow high affinity complex during the second phase seems to involve specific interactions with POPS and DOG since it is only sensitive to changes within relatively low concentration ranges of these lipids. Both the association and dissociation rate constants fell in the presence of PIP(2). We propose a model in which PKCα binds to the membranes via a two-step mechanism consisting of the rapid membrane initial recruitment of PKCα driven by interactions with POPS and/or PIP(2) although interactions with DOG are involved too. PKCα searches on the lipid bilayer in two dimensions to establish interactions with its specific ligands.

Topics: Animals; Cell Membrane; Diglycerides; Fluorescence Resonance Energy Transfer; Kinetics; Lipid Bilayers; Membrane Lipids; Models, Molecular; Phosphatidylcholines; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylserines; Protein Binding; Protein Kinase C-alpha; Protein Structure, Tertiary; Spectrometry, Fluorescence; Swine

Topics: Calcium; Catalytic Domain; Cell Membrane; Computer Simulation; Factor VIIa; Humans; Hydrophobic and Hydrophilic Interactions; Ions; Lipid Bilayers; Lipids; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylserines; Phospholipids; Protein Binding; Thromboplastin

We have used attenuated total internal reflection infrared spectroscopy (ATR-IR) spectroscopy to study the association of the C2 domain from protein kinase Cα (PKCα) with different phospholipid membranes, so as to characterise the mode of membrane docking and its modulation by the second-messenger lipid PIP₂. In parallel, we have also examined the membrane interaction of the C2 domain from cytosolic phospholipase A₂. PIP₂ did not induce significant changes in secondary structure of the membrane-bound PKCα-C2 domain, nor did binding of the PKCα-C2 domain change the dichroic ratios of the lipid chains, whereas the C2 domain from phospholipase A₂ did perturb the lipid chain orientation. Measurements of the dichroic ratios for the amide I and amide II protein bands were combined so as to distinguish the tilt of the β-sheets from that of the β-strands within the sheet. When associated with POPC/POPS membranes, the β-sandwich of the PKCα-C2 domain is inclined at an angle α=35° to the membrane normal, i.e., is oriented more nearly perpendicular than parallel to the membrane. In the process of membrane docking, the tilt angle increases to α=44° in the presence of PIP₂, indicating that the β-sandwich comes closer to the membrane surface, so confirming the importance of this lipid in determining docking of the C2 domain and consequent activation of PKCα.

Topics: Cell Membrane; Cytosol; Humans; Models, Molecular; Phosphatidylcholines; Phosphatidylserines; Phospholipases A2; Protein Kinase C-alpha; Protein Structure, Tertiary; Spectrophotometry, Infrared; Spectroscopy, Fourier Transform Infrared

Vesicular stomatitis virus (VSV) is a prototypic virus commonly used in studies of endocytosis and membrane trafficking. One proposed mechanism for VSV entry involves initial fusion with internal vesicles of multivesicular endosomes followed by back-fusion of these vesicles into the cytoplasm. One feature of endosomal internal vesicles is that they contain the lipid bis(monoacylglycero)phosphate (BMP). Here, we show that the presence of BMP significantly increases the rate of VSV G-mediated membrane fusion. The increased fusion was selective for VSV and was not evident for another enveloped virus, influenza virus. Our data provide a biological rationale for a two-step infection reaction during VSV entry, and suggest that BMP preferentially affects the ability of VSV G to mediate lipid mixing during membrane fusion.

Topics: Animals; Cell Line; Cholesterol; Endosomes; Hydrogen-Ion Concentration; Lipids; Liposomes; Lysophospholipids; Membrane Fusion; Membrane Glycoproteins; Monoglycerides; Phosphatidylcholines; Phosphatidylserines; Vesicular stomatitis Indiana virus; Viral Envelope Proteins; Virus Internalization

PMP1, a regulatory subunit of the yeast plasma membrane H(+)-ATPase, is a single transmembrane helix protein. Its cytoplasmic C-terminus possesses several positively charged residues and interacts with phosphatidylserine lipids as shown through both (1)H- and (2)H-NMR experiments. We used all-atom molecular dynamics simulations to obtain atomic-scale data on the effects of membrane interface lipid composition on PMP1 structure and tilt. PMP1 was embedded in two hydrated bilayers, differing in the composition of the interfacial region. The neutral bilayer is composed of POPC (1-palmitoyl-2-oleoyl-3-glycero-phosphatidylcholine) lipids and the negatively charged bilayer is composed of POPC and anionic POPS (1-palmitoyl-2-oleoyl-3-glycero-phosphatidylserine) lipids. Our results were consistent with NMR data obtained previously, such as a lipid sn-2 chain lying on the W28 aromatic ring and in the groove formed on one side of the PMP1 helix. In pure POPC, the transmembrane helix is two residues longer than the initial structure and the helix tilt remains constant at 6 ± 3°. By contrast, in mixed POPC-POPS, the initial helical structure of PMP1 is stable throughout the simulation time even though the C-terminal residues interact strongly with POPS headgroups, leading to a significant increase of the helix tilt within the membrane to 20 ± 5°.

Topics: Cell Membrane; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Membrane Proteins; Membranes, Artificial; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylserines; Protein Structure, Secondary; Proteolipids; Proton-Translocating ATPases; Saccharomyces cerevisiae Proteins; Time Factors

Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus. It has been suggested that the N-terminal part, which contains a conserved intramolecular disulfide bond between residues 2 and 7, interacts with membranes, ultimately leading to membrane damage and beta-cell death. Here, we used variants of the hIAPP(1-19) fragment and model membranes of phosphatidylcholine and phosphatidylserine (7:3, molar ratio) to examine the role of this disulfide in membrane interactions. We found that the disulfide bond has a minor effect on membrane insertion properties and peptide conformational behavior, as studied by monolayer techniques, (2)H NMR, ThT-fluorescence, membrane leakage, and CD spectroscopy. The results suggest that the disulfide bond does not play a significant role in hIAPP-membrane interactions. Hence, the fact that this bond is conserved is most likely related exclusively to the biological activity of IAPP as a hormone.

Topics: Amino Acid Sequence; Amyloid; Cell Membrane; Disulfides; Humans; Islets of Langerhans; Molecular Sequence Data; Oxidation-Reduction; Peptide Fragments; Phosphatidylcholines; Phosphatidylserines; Protein Binding; Protein Structure, Secondary

Prion diseases are neurodegenerative disorders of the central nervous system that are associated with the misfolding of the prion protein (PrP). PrP is glycosylphosphatidylinositol-anchored, and therefore the hydrophobic membrane environment may influence the process of prion conversion. This study investigates how the morphology and mechanism of growth of prion aggregates on membranes are influenced by lipid composition. Atomic force microscopy is used to image the aggregation of prions on supported lipid bilayers composed of mixtures of the zwitterionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the anionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS). Circular dichroism shows that PrP interactions with POPS membranes result in an increase in beta-sheet structure, whereas interactions with POPC do not influence PrP structure. Prion aggregation is observed on both zwitterionic and anionic membranes, and the morphology of the aggregates formed is dependent on the anionic phospholipid content of the membrane. The aggregates that form on POPC membranes have uniform dimensions and do not disrupt the lipid bilayer. The presence of POPS results in larger aggregates with a distinctive sponge-like morphology that are disruptive to membranes. These data provide detailed information on the aggregation mechanism of PrP on membranes, which can be described by classic models of growth.

Topics: Centrifugation, Density Gradient; Lipid Bilayers; Models, Biological; Particle Size; Phosphatidylcholines; Phosphatidylserines; Phospholipids; Prions; Protein Structure, Quaternary; Protein Structure, Secondary; Solutions

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

We report on the reversible association of anionic liposomes induced by an antimicrobial peptide (LAH4). The process has been characterized for mixed membranes of POPC and POPS at molar ratios of 1:1, 3:1, and 9:1. Although the vesicles remain in suspension in the presence of excess amounts of peptide, the addition of more lipids results in surface charge neutralization, aggregation of the liposomes, and formation of micrometer-sized structures that coexist in equilibrium with vesicles in suspension. At low ratios of anionic lipids, vesicle aggregation is a reversible process, and vesicle disassembly is observed upon inversion of the surface charge by further supplementation with anionic vesicles. In contrast, a different process, membrane fusion, occurs in the presence of high phosphatidylserine concentrations. Upon binding to membranes containing low POPS concentrations, the peptide adopts an in-plane alpha-helical structure, a secondary structure that is conserved during vesicle association and dissociation. Our finding that peptides are essential for vesicle aggregation contributes to a better understanding of the activity of antimicrobial peptides, and suggests an additional layer of complexity in membrane-protein lipid interactions.

Topics: Antimicrobial Cationic Peptides; Circular Dichroism; Light; Lipid Bilayers; Liposomes; Peptides; Phosphatidylcholines; Phosphatidylserines; Protein Structure, Secondary; Scattering, Radiation; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis

The Ca2+-independent membrane interactions of the soluble C2 domains from synaptotagmin 1 (syt1) were characterized using a combination of site-directed spin labeling and vesicle sedimentation. The second C2 domain of syt1, C2B, binds to membranes containing phosphatidylserine and phosphatidylcholine in a Ca2+-independent manner with a lipid partition coefficient of approximately 3.0 x 10(2) M(-1). A soluble fragment containing the first and second C2 domains of syt1, C2A and C2B, has a similar affinity, but C2A alone has no detectable affinity to phosphatidylcholine/phosphatidylserine bilayers in the absence of Ca2+. Although the Ca2+-independent membrane affinity of C2B is modest, it indicates that this domain will never be free in solution within the cell. Site-directed spin labeling was used to obtain bilayer depth restraints, and a simulated annealing routine was used to generate a model for the membrane docking of C2B in the absence of Ca2+. In this model, the polybasic strand of C2B forms the membrane binding surface for the domain; however, this face of C2B does not penetrate the bilayer but is localized within the aqueous double layer when C2B is bound. This double-layer location indicates that C2B interacts in a purely electrostatic manner with the bilayer interface. In the presence of Ca2+, the membrane affinity of C2B is increased approximately 20-fold, and the domain rotates so that the Ca2+-binding loops of C2B insert into the bilayer. This Ca2+-triggered conformational change may act as a switch to modulate the accessibility of the polybasic face of C2B and control interactions of syt1 with other components of the fusion machinery.

Topics: Animals; Calcium; Computer Simulation; Electron Spin Resonance Spectroscopy; Kinetics; Lipid Bilayers; Membranes, Artificial; Models, Molecular; Phosphatidylcholines; Phosphatidylserines; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Rats; Synaptotagmin I; Water

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

We present molecular dynamics simulations of a multicomponent, asymmetric bilayer in mixed aqueous solutions of sodium and potassium chloride. Because of the geometry of the system, there are two aqueous solution regions in our simulations: one mimics the intracellular region, and one mimics the extracellular region. Ion-specific effects are evident at the membrane/aqueous solution interface. Namely, at equal concentrations of sodium and potassium, sodium ions are more strongly adsorbed to carbonyl groups of the lipid headgroups. A significant concentration excess of potassium is needed for this ion to overwhelm the sodium abundance at the membrane. Ion-membrane interactions also lead to concentration-dependent and cation-specific behavior of the electrostatic potential in the intracellular region because of the negative charge on the inner leaflet. In addition, water permeation across the membrane was observed on a timescale of approximately 100 ns. This study represents a step toward the modeling of realistic biological membranes at physiological conditions in intracellular and extracellular environments.

Topics: Cell Membrane; Cell Membrane Permeability; Computer Simulation; Membranes, Artificial; Models, Chemical; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Potassium; Potassium Chloride; Sodium; Sodium Chloride; Sphingomyelins; Static Electricity; Time Factors; Water

The alpha-synuclein (alpha-syn) protein is clearly implicated in Parkinson's disease (PD). Mutations or triplication of the alpha-syn gene leads to early onset PD, possibly by accelerating alpha-syn oligomerization. alpha-syn interacts with lipids, and this membrane binding activity may relate to its toxic activity. To understand how the alpha-syn aggregation state affects its lipid binding activity we used surface plasmon resonance to study the interaction of wild-type and mutant alpha-syn with a charged phospholipid membrane, as a function of its aggregation state. Apparent dissociation constants for alpha-syn indicated that an intermediate species, present during the lag phase of amyloid formation, binds with an increased affinity to the membrane surface. Formation of this species was dependent upon the rate of fibril formation. Fluorescence anisotropy studies indicate that only upon the formation of amyloid material can alpha-syn perturb the acyl-chain region of the lipid bilayer. Circular dichroism spectroscopy showed that upon aging, both wild-type and mutant alpha-syn lose their ability to form lipid-bound alpha-helical species once they become fibrillar. These results indicate that alpha-syn forms a high affinity lipid binding intermediate species during fibril formation. Oligomeric alpha-syn is known to be toxic, and it is feasible that the high affinity binding species described here may correspond to a toxic species involved in PD.

Topics: alpha-Synuclein; Circular Dichroism; Humans; Lipids; Microscopy, Electron; Phosphatidylcholines; Phosphatidylserines; Protein Structure, Quaternary; Protein Structure, Secondary; Surface Plasmon Resonance; Unilamellar Liposomes

Cationic amphiphiles used for transfection can be incorporated into biological membranes. By differential scanning calorimetry (DSC), cholesterol solubilization in phospholipid membranes, in the absence and presence of cationic amphiphiles, was determined. Two different systems were studied: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)+cholesterol (1:3, POPC:Chol, molar ratio) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (POPS)+cholesterol (3:2, POPS:Chol, molar ratio), which contain cholesterol in crystallite form. For the zwitterionic lipid POPC, cationic amphiphiles were tested, up to 7 mol%, while for anionic POPS bilayers, which possibly incorporate more positive amphiphiles, the fractions used were higher, up to 23 mol%. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and DOTAP in methyl sulfate salt form (DOTAPmss) were found to cause a small decrease on the enthalpy of the cholesterol transition of pure cholesterol aggregates, possibly indicating a slight increase on the cholesterol solubilization in POPC vesicles. With the anionic system POPS:Chol, the cationic amphiphiles dramatically change the cholesterol crystal thermal transition, indicating significant changes in the cholesterol aggregates. For structural studies, phospholipids spin labeled at the 5th or 16th carbon atoms were incorporated. In POPC, at the bilayer core, the cationic amphiphiles significantly increase the bilayer packing, decreasing the membrane polarity, with the cholesterol derivative 3 beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol (DC-chol) displaying a stronger effect. In POPS and POPS:Chol, DC-chol was also found to considerably increase the bilayer packing. Hence, exogenous cationic amphiphiles used to deliver nucleic acids to cells can change the bilayer packing of biological membranes and alter the structure of cholesterol crystals, which are believed to be the precursors to atherosclerotic lesions.

Topics: Calorimetry, Differential Scanning; Cholesterol; Electron Spin Resonance Spectroscopy; Fatty Acids, Monounsaturated; Lipid Bilayers; Phosphatidylcholines; Phosphatidylserines; Quaternary Ammonium Compounds; Solubility; Temperature; Thermodynamics

Molecular partitioning into biomembranes is of fundamental importance in diverse biochemical processes and reactions. The majority of aqueous/membrane partition data using model membrane systems, is obtained with pure phosphatidylcholine bilayers, being lipid mixtures less used, while studies involving bilayers containing zwitterionic/anionic mixtures of phospholipids are even more scarce. In this study, the solvatochromic effects of 1-pyrenesulfonate observed at 375 nm in aqueous liposome suspensions, and monitored by second derivative absorption spectrophotometry, enabled the determination of its partition constants into defined phospholipid bilayers. We compare, under cautiously settled experimental conditions, the partition of the anionic amphiphile PSA into fluid zwitterionic bilayers of POPC (Kp=6.7 x 10(3), at 25 degrees C), and into fluid mixed zwitterionic/anionic bilayers containing small proportions of anionic phospholipids. At the same temperature, we found increasing K(p) values in parallel with the proportion of POPS mixed with POPC (Kp=3.4 x 10(4) and Kp=7.3 x 10(4), with 5 and 10 mol% of POPS, respectively). Our interpretation is based on the interfacial properties of fluid and flexible mixed zwitterionic/anionic phospholipid bilayers.

Topics: Lipid Bilayers; Liposomes; Osmolar Concentration; Phosphatidylcholines; Phosphatidylserines; Pyrenes; Spectrophotometry, Ultraviolet; Sulfonic Acids

In the current understanding of exocytosis at the nerve terminal, the C2 domain of synaptotagmin (C2A) is presumed to bind Ca2+ and the membrane in a stepwise fashion: cation then membrane as cation increases the affinity of protein for membrane. Fluorescence spectroscopy data were gathered over a variety of lipid and Ca2+ concentrations, enabling the rigorous application of microscopic binding models derived from partition functions to differentiate between Ca2+ and phosphatidylserine contributions to binding. The data presented here are in variance with previously published models, which were based on the Hill approximation. Rather, the data are consistent with two forms of cooperativity that modulate the responsiveness of C2A: in Ca2+ binding to a network of three cation sites and in interaction with the membrane surface. We suggest synaptotagmin I C2A is preassociated with the synaptic vesicle membrane or nerve terminal. In this state, upon Ca2+ influx the protein will bind the three Ca2+ ions immediately and with high cooperativity. Thus, membrane association creates a high-affinity Ca2+ switch that is the basis for the role of synaptotagmin I in Ca2+-regulated exocytosis. Based on this model, we discuss the implications of protein-induced phosphatidylserine demixing to the exocytotic process.

Topics: Calcium; Exocytosis; Membranes, Artificial; Models, Biological; Phosphatidylcholines; Phosphatidylserines; Protein Structure, Tertiary; Recombinant Proteins; Synaptic Vesicles; Synaptotagmin I

A novel method for the quantitative assessment of the membrane partitioning of a ligand from the aqueous phase is described, demonstrated here with the thoroughly studied antipsychotic chlorpromazine (CPZ). More specifically, collisional quenching of the fluorescence of a pyrene labeled fluorescent lipid analog 1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) by CPZ was utilized, using 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine and -serine (POPC and POPS) liposomes as model membranes. The molar partition coefficient is obtained from two series of titrations, one with constant [phospholipid] and increasing [drug] and the other with constant [drug] and varying total [phospholipid], the latter further comprising of large unilamellar vesicles (LUVs) of POPC/POPS/PPDPC at a constant concentration of 10 microM and indicated concentrations of POPC/POPS LUVs. Notably, the approach described is generic and can be employed in screening for the membrane partitioning of compounds, providing that a suitable fluorescence parameter can be incorporated into one population of liposomes utilized as model membranes.

Topics: Antipsychotic Agents; Chlorpromazine; Fluorescence; Fluorescent Dyes; Ligands; Liposomes; Membrane Lipids; Molecular Structure; Phosphatidylcholines; Phosphatidylserines; Spectrometry, Fluorescence; Water

We recently showed that the antibacterial histidine rich amphipathic peptide LAH4 has significant DNA transfection capabilities in the absence of serum. To further understand the transfection process and to develop the peptides for future applications, we have combined a range of biochemical and biophysical techniques, including fluorescence assisted cell sorting and (2)H solid-state NMR, to characterise the initial binding of the peptide/DNA complexes to the cell surface and the subsequent release of the complexes from the endosome in the presence of serum. Our results show that both primary and secondary peptide structure play important roles in both of these processes. Specifically, we show that an ideal helix length and positioning of the histidine residues should be maintained to obtain optimal resistance to serum effects and release of DNA from the endosome. Inclusion of d-amino acids at the peptide termini does not reduce serum effects however further enrichment of the peptides with histidine residues can enhance transfection efficiency in the presence of serum. The detailed understanding of these two key stages in the transfection process shows that LAH4-L1 and its derivatives are likely to be highly efficient and robust vectors for a range of applications.

Topics: Cell Line, Transformed; Cell Line, Tumor; Cell Transformation, Viral; Cells, Cultured; Cholesterol; DNA; Drug Carriers; Flow Cytometry; Histidine; Humans; Lipids; Luciferases; Models, Chemical; Nuclear Magnetic Resonance, Biomolecular; Peptides; Phosphatidylcholines; Phosphatidylserines; Serum; Transfection

The interaction of Saposin C (Sap C) with negatively charged phospholipids such as phosphatidylserine (PS) is essential for its biological function. In this study, Sap C (initially protonated in a weak acid) was inserted into multilamellar vesicles (MLVs) consisting of either 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (negatively charged, POPS) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (neutrally charged, POPC). The MLVs were then investigated using solid-state NMR spectroscopy under neutral pH (7.0) conditions. The (2)H and (31)P solid-state NMR spectroscopic data of Sap C-POPS and Sap C-POPC MLVs (prepared under the same conditions) were compared using the (2)H order parameter profiles of the POPC-d(31) or POPS-d(31) acyl chains as well as the (31)P chemical shift anisotropy width and (31)P T(1) relaxation times of the phospholipids headgroups. All those solid-state NMR spectroscopic approaches indicate that protonated Sap C disturbs the POPS bilayers and not the POPC lipid bilayers. These observations suggest for the first time that protonated Sap C inserts into PS bilayers and forms a stable complex with the lipids even after resuspension under neutral buffer conditions. Additionally, (31)P solid-state NMR spectroscopic studies of mechanically oriented phospholipids on glass plates were conducted and perturbation effect of Sap C on both POPS and POPC bilayers was compared. Unlike POPC bilayers, the data indicates that protonated Sap C (initially protonated in a weak acid) was unable to produce well-oriented POPS bilayers on glass plates at neutral pH. Conversely, unprotonated Sap C (initially dissolved in a neutral buffer) did not interact significantly with POPS phospholipids allowing them to produce well-oriented bilayers at neutral pH.

Topics: Amino Acid Sequence; Carbon; Catalysis; Escherichia coli; Hydrogen-Ion Concentration; Hydrolysis; Lipid Bilayers; Magnetic Resonance Spectroscopy; Molecular Conformation; Molecular Sequence Data; Phosphatidylcholines; Phosphatidylserines; Phospholipids; Protein Binding; Saposins; Temperature

The kinetics of annexin A1 binding to solid-supported lipid bilayers consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS; 4:1) has been investigated as a function of the calcium ion concentration in the bulk phase. Quartz crystal microbalance measurements in conjunction with scanning force microscopy, fluorescence microscopy, and computer simulations indicate that at a given Ca2+ concentration annexin A1 adsorbs irreversibly on membrane domains enriched in POPS. By contrast, annexin A1 adsorbs reversibly on the POPC-enriched phase, which is composed of single POPS molecules embedded within a POPC matrix. The overall area occupied by the POPS-enriched phase is controlled by the CaCl2 concentration. Monte Carlo simulations suggest that the area of the POPS-enriched phase increases by a factor of 7 when the Ca2+ concentration is changed from 0.01 to 1 mM.

Topics: Adsorption; Annexin A1; Biosensing Techniques; Computer Simulation; Lipid Bilayers; Membranes, Artificial; Microscopy, Atomic Force; Microscopy, Fluorescence; Monte Carlo Method; Particle Size; Phosphatidylcholines; Phosphatidylserines; Stress, Mechanical; Surface Properties; Time Factors

Beta-amyloid peptide (Abeta), which is cleaved from the larger trans-membrane amyloid precursor protein, is found deposited in the brain of patients suffering from Alzheimer's disease and is linked with neurotoxicity. We report the results of studies of Abeta1-42 and the effect of metal ions (Cu2+ and Zn2+) on model membranes using 31P and 2H solid-state NMR, fluorescence and Langmuir Blodgett monolayer methods. Both the peptide and metal ions interact with the phospholipid headgroups and the effects on the lipid bilayer and the peptide structure were different for membrane incorporated or associated peptides. Copper ions alone destabilise the lipid bilayer and induced formation of smaller vesicles but when Abeta1-42 was associated with the bilayer membrane copper did not have this effect. Circular dichroism spectroscopy indicated that Abeta1-42 adopted more beta-sheet structure when incorporated in a lipid bilayer in comparison to the associated peptide, which was largely unstructured. Incorporated peptides appear to disrupt the membrane more severely than associated peptides, which may have implications for the role of Abeta in disease states.

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Peptides; Cations, Divalent; Circular Dichroism; Copper; Humans; Lipid Bilayers; Magnetic Resonance Spectroscopy; Membrane Lipids; Membrane Microdomains; Molecular Sequence Data; Peptide Fragments; Phosphatidylcholines; Phosphatidylserines; Spectrometry, Fluorescence; Zinc

Synaptotagmin I is a synaptic vesicle associated membrane protein that appears to regulate Ca(2+)-mediated exocytosis. Here, the Ca(2+)-dependent membrane interactions of a water soluble fragment of synaptotagmin I (C2AB) that contains its two C2 domains (C2A and C2B) were determined using site-directed spin labeling. Membrane depth parameters were obtained for 19 spin-labeled mutants of C2AB when bound to phosphatidylcholine and phosphatidylserine membranes, and these distance constraints were used in combination with the high-resolution structures of C2A and C2B to generate a model for the membrane orientation and position of synaptotagmin at the bilayer interface. Both C2A and C2B bind to the membrane interface with their first and third Ca(2+) binding loops penetrating the membrane interface. The polybasic face of C2B does not interact with the membrane lipid but is available for electrostatic interaction with other components of the fusion machinery. When compared to positions determined previously for the isolated domains, both C2A and C2B have similar orientations; however, the two domains are positioned deeper into the bilayer interior when present in the tandem construct. These data indicate that C2A and C2B do not act independently but influence their mutual membrane penetration. This may explain the occurrence of multiple C2 domains in proteins that function in membrane trafficking and repair.

Topics: Animals; Cell Membrane; Electron Spin Resonance Spectroscopy; Lipid Bilayers; Phosphatidylcholines; Phosphatidylserines; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Rats; Synaptotagmin I; Terbium

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

(31)P solid-state NMR spectroscopy has been used to investigate the macroscopic phase behavior of phospholipid bilayers in the presence of increasing amounts of magainin antibiotic peptides. Addition of >1 mol% magainin 2 to gel-phase DMPC or liquid crystalline POPC membranes respectively, results in (31)P NMR spectra that are characterized by the coexistence of isotropic signals and line shapes typical for phospholipid bilayers. The isotropic signal intensity is a function of temperature and peptide concentration. At peptide concentrations >4 mol% of the resulting phospholipid (31)P NMR spectra are characteristic of magnetically oriented POPC bilayers suggesting the formation of small disk-like micelles or perforated sheets. In contrast, addition of magainin to acidic phospholipids results in homogenous bilayer-type (31)P NMR spectra with reduced chemical shift anisotropies. The results presented are in good agreement with the interfacial insertion of magainin helices with an alignment parallel to the surface of the phospholipid bilayers. The resulting curvature strain results in detergent-like properties of the amphipathic helical peptides.

Topics: Anisotropy; Circular Dichroism; Detergents; Dimyristoylphosphatidylcholine; Lipid Bilayers; Magnetic Resonance Spectroscopy; Micelles; Models, Biological; Peptides; Phosphatidylcholines; Phosphatidylinositols; Phosphatidylserines; Phosphorus Isotopes; Spectroscopy, Fourier Transform Infrared; Temperature

The membrane interaction of tuberoinfundibular peptide of 39 residues (TIP39), which selectively activates the parathyroid hormone 2 (PTH2) receptor (PTH2-R), has been studied by fluorescence and NMR spectroscopic techniques. Membrane binding would be the first step of a potential membrane-bound activation pathway which has been discussed for a number of neuropeptides and G-protein coupled receptors (GPCRs). Here, the orientation of TIP39 on the surface of membrane mimicking dodecyl-phosphocholine (DPC) micelles was monitored by Photo-CIDNP (chemically-induced dynamic nuclear polarization) NMR which indicates that both Trp25 and Tyr29 face the membrane surface. However, the PTH2 receptor is located in the hypothalamus membrane, for which a more realistic model is required. Therefore, liposomes containing different mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) and cholesterol were used for fluorescence and solid-state NMR spectroscopy. Fluorescence spectroscopy showed that a large proportion of TIP39 added to these liposomes binds to the membrane surface. Proton-decoupled 31P-MAS NMR is used to investigate the potential role of individual lipid headgroups in peptide binding. Significant line-broadening in POPC/cholesterol and POPC/POPS liposomes upon TIP39 association supports a surface binding model and indicates an interaction which is slightly mediated by the presence of POPS and cholesterol. Furthermore, smoothed order parameter profiles obtained from 2H powder spectra of liposomes containing POPC-d31 as bulk lipid in addition to POPS and cholesterol show that TIP39 does not penetrate beyond the headgroup region. Spectra of similar bilayers with POPS-d31 show a small increase in segmental chain order parameters which is interpreted as a small but specific interaction between the peptide and POPS. Our data demonstrate that TIP39 belongs to a class of signaling peptides that associate weakly with the membrane surface but do not proceed to insert into the membrane hydrophobic compartment.

Topics: Animals; Cattle; Cholesterol; Membranes, Artificial; Micelles; Neuropeptides; Nuclear Magnetic Resonance, Biomolecular; Phosphatidylcholines; Phosphatidylserines; Receptor, Parathyroid Hormone, Type 2; Spectrometry, Fluorescence

By means of scanning force and fluorescence microscopy of artificial membranes immobilized on mica surfaces, the lateral organization of the annexin A2/S100A10 heterotetramer (annexin A2t) and its influence on the lateral organization of the lipids within the membrane have been elucidated. Planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) were prepared on atomically flat mica surfaces by the spreading of unilamellar vesicles. Fluorescence images of fluorescently labeled annexin A2t and scanning force microscopy images of nonlabeled protein bound to POPC/POPS bilayers show the formation of micrometer-sized lateral protein domains in the presence of 1 mM CaCl2. By means of scanning force microscopy, not only protein domains became discernible but also small membrane domains, which were attributed to POPS-enriched areas. A depletion of these POPS domains was observed in the vicinity of annexin A2t protein domains. These results indicate that annexin A2t is a peripheral membrane-binding complex capable of inducing lipid segregation.

Topics: Annexin A2; Lipid Bilayers; Microscopy, Atomic Force; Microscopy, Fluorescence; Phosphatidylcholines; Phosphatidylserines; Protein Structure, Quaternary; Protein Structure, Tertiary; S100 Proteins

The dissipational quartz crystal microbalance (D-QCM) technology was applied to monitor the adsorption of vesicles to membrane-bound annexin A1 by simultaneously reading out the shifts in resonance frequency and dissipation. Solid-supported membranes (SSMs) composed of a chemisorbed octanethiol monolayer and a physisorbed 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine monolayer were immobilized on the gold electrode of a 5 MHz quartz plate. Adsorption and desorption of annexin A1 to the SSM was followed by means of the QCM technique. After nonbound annexin A1 was removed from solution, the second membrane binding was monitored by the D-QCM technique, which allowed distinguishing between adsorbed and ruptured vesicles. The results show that vesicles stay always intact independent of the amount of bound annexin and the vesicle and buffer composition. It was shown that the vesicle adsorption process to membrane-bound annexin A1 is fully irreversible and is mediated by two-dimensional annexin clusters. For N-terminally truncated annexin A1, a decrease in the amount of bound vesicles was observed, which might be the result of fewer binding sites presented by the annexin A1 core. Supported by computer simulations, the results demonstrate that the vesicle adsorption process is electrostatically driven, but compared to those of sole electrostatic binding, the rate constants of adsorption are 1-2 orders of magnitude smaller, indicating the presence of a potential barrier.

Topics: Adsorption; Annexin A1; Biosensing Techniques; Calcium; Computer Simulation; Gold; Membranes, Artificial; Phosphatidylcholines; Phosphatidylserines; Quartz; Sulfhydryl Compounds; Surface Properties; Time Factors

Site directed spin labeling is used to investigate the protein annexin 12 absorbed on a single planar phospholipid bilayer of approximately 2-3 cm(2). Electron paramagnetic resonance spectra of nitroxide side chain at several topological sites reveal a conserved tertiary fold of the protein in the absorbed state, in agreement with earlier diffraction results. The angular dependent spectra of the two-dimensional microcrystals are shown to provide information on the degree of ordering of spin labels in a alpha-helix and in turn on the orientation of the alpha-helix with respect to the surface.

Topics: Annexins; Electron Spin Resonance Spectroscopy; Lipid Bilayers; Models, Molecular; Phosphatidylcholines; Phosphatidylserines; Spin Labels

Lipid activation of protein kinase C alpha (PKC alpha) was studied using a model mixture containing POPC/POPS (molar ratio 4:1) and different proportions of either DPG or POG. The lipid mixtures containing DPG were physically characterized by using different physical techniques, and a phase diagram was constructed by keeping a constant POPC/POPS molar ratio of 4:1 and changing the concentration of 1,2-DPG. The phase diagram displayed three regions delimited by two compounds: compound 1 (CO(1)) with 35 mol % of 1,2-DPG and compound 2 (CO(2)) with 65 mol % of 1,2-DPG. PKC alpha activity was assayed at increasing concentrations of 1,2-DPG, maximum activity being reached at 30 mol % 1,2-DPG, which decreased at higher concentrations. Maximum activity occurred, then, at concentrations of 1,2-DPG which corresponded to the transition from region 1 to region 2 of the phase diagram. It was interesting that this protein was maximally bound to the membrane at all DPG concentrations. Similar results were observed when the enzyme was activated by POG, when a maximum was reached at about 10 mol %. This remained practically constant up to 50 mol %, about which it decreased, the binding level remaining maximal and constant at all POG concentrations. The fact that in the assay conditions used maximal binding was already reached even in the absence of diacylglycerol was attributed to the interaction of the C2 domain with the POPS present in the membrane through the Ca(2+) ions also present. To confirm this, the isolated C2 domain was used, and it was also found to be maximally bound at all DPG concentrations and even in its absence. Since the intriguing interaction patterns observed seemed to be due then to the C1 domain, the PKC alpha mutant D246/248N was used. This mutant has a decreased Ca(2+)-binding capacity through the C2 domain and was not activated nor bound to membranes by increasing concentrations of DPG. However, POG was able to activate the mutant, which showed a similar dependence on POG concentration with respect to activity and binding to membranes. These data underline the importance of unsaturation in one of the fatty acyl chains of the diacylglycerol.

Topics: Calorimetry, Differential Scanning; Diglycerides; Isoenzymes; Magnetic Resonance Spectroscopy; Membrane Lipids; Phosphatidylcholines; Phosphatidylserines; Protein Binding; Protein Kinase C; Protein Kinase C-alpha; Temperature

Human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) are closely related lentiviruses that infect immune cells, but their pathogenesis differ. Localization to the cytosolic leaflet of the plasma membrane is critical for replication of both viruses. This localization is accomplished through the matrix (MA) domain of the Gag precursor protein. In HIV-1, association of MA to anionic membranes appears to be primarily driven by a linear cluster of basic residues in the MA domain and an N-myristoylation signal. Interestingly, the MA protein of EIAV does not contain either of these signals. To understand which factors could promote EIAV assembly we characterized the membrane binding properties of its MA protein using fluorescence and biochemical methods. We find that EIAV MA exists as a multimer in solution whose protein-protein interactions are destabilized by membrane binding. EIAV MA binds strongly to electrically neutral membranes as well as to negatively charged membranes. Fluorescence quenching and chemical modification techniques, as well as trypsin proteolysis, indicate a different exposure of the EIAV MA Trp residues when bound to the two types of membranes, and EIAV MA proteolysis by trypsin differs when bound to the two types of membranes. Based on these data and the known structures of closely related matrix proteins, we constructed a structural model. This model predicts that EIAV MA binds to negatively charged membranes, but EIAV MA has an additional membrane binding region rich in residues that partition favorably into the membrane headgroup region. This secondary site may play a role in early events of viral infection.

Topics: Amino Acid Sequence; Cell Membrane; Fluorescence; Fluorescence Polarization; Infectious Anemia Virus, Equine; Lipid Bilayers; Models, Molecular; Molecular Sequence Data; Phosphatidylcholines; Phosphatidylserines; Protein Binding; Recombinant Fusion Proteins; Sequence Alignment; Solutions; Static Electricity; Thermodynamics; Trypsin; Viral Matrix Proteins; Virus Assembly

It has been proposed that annexin I has two separate interaction sites that are involved in membrane binding and aggregation, respectively. To better understand the mechanism of annexin I-mediated membrane aggregation, we investigated the properties of the inducible secondary interaction site implicated in membrane aggregation. X-ray specular reflectivity measurements showed that the thickness of annexin I layer bound to the phospholipid monolayer was 31 +/- 2 A, indicating that annexin I binds membranes as a protein monomer or monolayer. Surface plasmon resonance measurements of annexin I, V, and mutants, which allowed evaluation of membrane aggregation activity of annexin I separately from its membrane binding, revealed direct correlation between the relative membrane aggregation activity and the relative affinity of the secondary interaction site for the secondary membrane. The secondary binding was driven primarily by hydrophobic interactions, unlike calcium-mediated electrostatic primary membrane binding. Chemical cross-linking of membrane-bound annexin I showed that a significant degree of lateral association of annexin I molecules precedes its membrane aggregation. Taken together, these results support a hypothetical model of annexin I-mediated membrane aggregation, in which a laterally aggregated monolayer of membrane-bound annexin I directly interacts with a secondary membrane via its induced hydrophobic interaction site.

Topics: Annexin A1; Cross-Linking Reagents; Formaldehyde; Humans; Membrane Lipids; Models, Biological; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipids; Protein Binding; Spectrum Analysis; Surface Plasmon Resonance; X-Rays

The lipid activation of protein kinase C alpha (PKC alpha) has been studied by comparing the activation capacity of different 1, 2-diacylglycerols and 1,3-diacylglycerols incorporated into mixed micelles or vesicles. Unsaturated 1,2-diacylglycerols were, in general, more potent activators than saturated ones when 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS)/Triton X-100 mixed micelles and pure POPS vesicles were used. In contrast, these differences were not observed when 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/POPS (4:1, molar ratio) vesicles were used. Diacylglycerols bearing short fatty acyl chains showed a very high activation capacity, however, the capacity was less in mixed micelles. Furthermore, 1, 2-diacylglycerols had a considerably higher activating capacity than 1,3-diacylglycerols in POPS/Triton X-100 mixed micelles and in POPC/POPS vesicles. However, the differences between the two types of diacylglycerols were smaller when pure POPS vesicles were used. Differential scanning calorimetry (DSC) showed that POPC/POPS membrane samples containing diacylglycerols had endothermic transitions in the presence of 200 microM Ca2+ and 5 mM Mg2+. Transitions were not detected when using pure POPS vesicles due to the formation of dehydrated phases as demonstrated by FTIR (Fourier-transform infrared) spectroscopy. PKC alpha binding studies, performed by differential centrifugation in the presence of 200 microM Ca2+ and 5 mM Mg2+, showed that 1,2-sn-dioleoylglycerol (1, 2-DOG) was more effective than 1,3-dioleoylglycerol (1,3-DOG) in promoting binding to POPC/POPS vesicles. However, when pure POPS vesicles were used, PKC alpha was able to bind to membranes containing either 1,2-DOG or 1,3-DOG to the same extent.

Topics: Calcium; Enzyme Activation; Fatty Acids; Glycerol; Isoenzymes; Magnesium; Membranes, Artificial; Octoxynol; Phosphatidylcholines; Phosphatidylserines; Protein Kinase C; Protein Kinase C-alpha; Spectroscopy, Fourier Transform Infrared; Structure-Activity Relationship

Spread phospholipid monolayers are particularly useful as model membranes in that changes in surface pressure (Deltapi) can be monitored in response to protein adsorption to the monolayer, thus providing a unique manner of assessing protein-membrane contact. In the present study, spread monolayers below their collapse pressures have been utilized to evaluate Ca2+-specific adsorption of several vitamin K-dependent coagulation proteins to monolayers that contain negatively charged phospholipid. From combined measurements of Deltapi and Gamma (the surface excess protein concentration), values of dGamma/dpi have been evaluated for different proteins with varying lipid composition of the monolayers. Using mixed, liquid-expanded monolayers at equivalent initial surface pressures (pii) and which contain different amounts of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine, the dGamma/dpi of bovine prothrombin was shown to decrease monotonically with increasing protein affinity for the monolayer. For example, KD values of 7, 20, and 60 nM produced dGamma/dpi values of 14, 17, and 21 nmol m-1 mN-1, respectively. However, the trend in dGamma/dpi appears to originate from characteristics of the monolayer and not from those of the protein, since a much different adsorbate (i.e., a positively charged pyrene derivative) exhibited a similar trend in dGamma/dpi with monolayer composition. On the other hand, dGamma/dpi values of bovine prothrombin, human factor IX, human protein S, bovine protein C, and human protein C, determined using liquid-expanded phosphatidylserine monolayers, were essentially equivalent. Therefore, the five vitamin K-dependent proteins that were examined were equivalent in terms of the manner in which the gamma-carboxyglutamic acid (Gla) domain of each protein perturbed the surface pressure. This study shows that Ca2+-specific membrane contact sites in the Gla domain of the five proteins tested are similar despite the naturally occurring differences in the normal Gla domain sequence of these proteins.

Topics: Adsorption; Animals; Blood Coagulation Factors; Calcium-Binding Proteins; Cattle; Extracellular Matrix Proteins; Factor IX; Humans; Matrix Gla Protein; Membrane Proteins; Membranes, Artificial; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipids; Pressure; Protein C; Prothrombin; Surface Properties; Vitamin K

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

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

The interaction of bovine prothrombin with phospholipids was measured, using as the lipid source monolayers spread at the air-buffer interface. Fluorescence spectroscopy was implemented to determine the equilibrium concentration of free prothrombin in the aqueous subphase of the protein-monolayer suspensions, in a continuous assay system. The increase in surface pressure (pi) from the protein-monolayer adsorption was also measured and, with values of the adsorbed protein concentration (c[s]), was used to calculate dc(s)/d(pi). At a particular phosphatidylserine (PS) content of liquid-expanded (LE) phosphatidylcholine (PC)/PS monolayers, dc(s)/d(pi) was independent of the initial surface pressure (pi[i]), when this latter value exceeded 30 mN/m. However, dc(s)/d(pi) varied significantly with the relative PS content of the monolayer. Values of the equilibrium dissociation constants calculated from the concentration dependence of delta(pi) indicated that the affinity of prothrombin for LE monolayers was higher at higher PS contents and lower packing densities. The affinity of prothrombin for liquid-condensed (LC) PC/PS monolayers was found to be much weaker relative to LE monolayers of similar phospholipid composition. This approach, employing spread monolayers to study prothrombin-phospholipid binding, coupled with a simple and accurate method to determine the free protein concentration in protein-monolayer suspensions, offers significant advantages for the investigation of protein-membrane interaction. The equilibrium characteristics that describe the interaction of prothrombin with the different phospholipid monolayers under various conditions also provide support for previous results which indicated that hydrophobic interactions are involved in the adsorption of vitamin K-dependent coagulation and anticoagulation proteins to model membrane systems.

Topics: Adsorption; Animals; Biophysical Phenomena; Biophysics; Calcium; Cattle; In Vitro Techniques; Membranes, Artificial; Microscopy, Fluorescence; Models, Chemical; Phosphatidylcholines; Phosphatidylserines; Phospholipids; Pressure; Prothrombin; Surface Properties

We describe a puffing method for changing solutions near one surface of lipid bilayers that allows simultaneous measurement of channel activity and extent of solution change at the bilayer surface. Ion adsorption to the lipid headgroups and screening of the bilayer surface charge by mobile ions provided a convenient probe for the ionic composition of the solution at the bilayer surface. Rapid ionic changes induced a shift in bilayer surface potential that generated a capacitive transient current under voltage-clamp conditions. This depended on the ion species and bilayer composition and was accurately described by the Stern-Gouy-Chapman theory. The time course of solute concentrations during solution changes could also be modeled by an exponential exchange of bath and puffing solutions with time constants ranging from 20 to 110 ms depending on the flow pressure. During changes in [Cs+] and [Ca2+] (applied separately or together) both the mixing model and capacitive currents predicted [Cs+] and [Ca2+] transients consistent with those determined experimentally from: 1) the known Cs(+)-dependent conductance of open ryanodine receptor channels and 2) the Ca(2+)-dependent gating of ryanodine receptor Ca2+ channels from cardiac and skeletal muscle.

Topics: Animals; Calcium; Calcium Channels; Cesium; Heart; Kinetics; Lipid Bilayers; Membrane Potentials; Models, Biological; Muscle Proteins; Muscle, Skeletal; Patch-Clamp Techniques; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Rabbits; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sheep; Solutions; Surface Properties

Magainin 2 is a 23-residue antibiotic peptide found in the skin of Xeonpus laevis (African clawed frog). It belongs to a broad class of alpha-helical peptides which interact directly with the lipid bilayer. Very little is presently known about the nature of this peptide/lipid interaction on the molecular level. We have performed a sequence of lamellar X-ray diffraction experiments to provide some insight into the nature of this interaction. We have found that, at concentrations below the critical concentration for lysis, the peptide causes the membrane thickness to decrease roughly in proportion to the peptide concentration. We further show that this thinning is consistent with a model where the peptide adsorbs within the headgroup region of the lipid bilayer at these concentrations. The energy cost of this thinning may also explain why the peptide inserts at high concentrations. We have already shown that a similar interaction exists for alamethicin interacting with diphytanoylphosphatidylcholine, and it should hold for a wide variety of peptide/lipid systems.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Circular Dichroism; Crystallography, X-Ray; In Vitro Techniques; Lipid Bilayers; Magainins; Membrane Lipids; Molecular Sequence Data; Phosphatidylcholines; Phosphatidylserines; Xenopus laevis; Xenopus Proteins

The isothermal phase behavior of three gramicidin A'/phospholipid mixtures was investigated by an equilibrium Ca(2+)-binding technique. The phospholipid component was 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), or POPS/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at a constant mole ratio of 1/4. The bulk aqueous free Ca2+ concentration, [Ca2+]*f, in equilibrium with one or two gramicidin A'/phospholipid fluid phases and a small amount of the Ca (phosphatidylserine)2 gel phase, was measured as a function of composition at 20 degrees C by use of chromophoric high-affinity Ca2+ chelators. The coexistence of two gramicidin A'/phospholipid fluid phases was detected by an invariance in [Ca2+]*f over the range of compositions throughout which the two phases coexist. The compositions of the two coexisting phases are determined by the compositions at which the invariance in [Ca2+]*f begins and ends. With each of the gramicidin A'/phospholipid mixtures, we estimate that the composition of the gramicidin-poor phase is 0.03-0.04 mole fraction gramicidin A' and the composition of the gramicidin-rich phase is 0.13-0.14 mole fraction gramicidin A'. Characterization of these phases by low-angle X-ray diffraction revealed that, in each case, the gramicidin-poor phase is an L alpha phase and the gramicidin-rich phase is an HII phase. The isothermal phase behavior of gramicidin A'/POPC mixtures at approximately 23 degrees C, as determined by low-angle X-ray diffraction, was found to be similar to that of the other gramicidin A'/phospholipid mixtures.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Binding Sites; Calcium; Chelating Agents; Gramicidin; Kinetics; Magnetic Resonance Spectroscopy; Phosphatidylcholines; Phosphatidylserines; Phosphorus Radioisotopes; Reference Standards; X-Ray Diffraction

The lipid requirement for the binding of wild-type Pseudomonas aeruginosa exotoxin A (ETA) to model endosomal membrane vesicles was evaluated using a fluorescence quenching technique. The binding of toxin to monodisperse model membrane vesicles (0.1 micron diameter) composed of various molar ratios of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) prepared by an extrusion method [Hope, M. J., et al. (1986) Chem. Phys. Lipids 40 89-107] was pH-dependent, with maximal binding observed at pH 4.0. Analysis of the binding curves indicated that the interaction of ETA with the membrane bilayer is dominated by a set of high-affinity binding sites (Kd = 2-8 microM; 60:40 (mol:mol) POPC/POPS large unilamellar vesicles (LUV)). The binding of toxin to membrane vesicles was highly pH-dependent, but was ionic strength-independent. Toxin-induced pore formation in the lipid bilayer, as measured by the release of the fluorescent dye, calcein, from LUV was pH-dependent, with optimal dye release occurring at pH 4.0. The rate of dye release from membrane vesicles decreased rapidly with increasing pH and approached zero at pH 6.0 and higher. The pKa for this process ranged over 4.3-4.5. Calcein release from LUV was also sensitive to changes in the ionic strength of the assay buffer, with maximal release occurring at 50 mM NaCl. Higher ionic strength medium resulted in a dramatic reduction in the rate of dye release from vesicles, indicating that the toxin-induced pore is modulated by ionic interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: ADP Ribose Transferases; Animals; Bacterial Toxins; Binding Sites; Cell Membrane Permeability; Coated Vesicles; Escherichia coli; Exotoxins; Fluoresceins; Fluorescent Antibody Technique; Hydrogen-Ion Concentration; Indicators and Reagents; Kinetics; Lipid Bilayers; Mice; Models, Biological; Osmolar Concentration; Phosphatidylcholines; Phosphatidylserines; Porosity; Pseudomonas aeruginosa; Pseudomonas aeruginosa Exotoxin A; Virulence Factors

A detailed electron spin resonance (ESR) study of mixtures of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and phosphatidylserine (POPS) in oriented multilayers in the liquid crystalline phase is reported with the purpose of characterizing the effects of headgroup mixing on the structural and dynamical properties of the acyl chains. These studies were performed over a range of blends of POPC and POPS and temperatures, utilizing the spin-labeled lipids 16-phosphatidylcholine and 5-phosphatidylcholine as well as cholestane (CSL). The ESR spectra were analyzed by nonlinear least-squares fitting using detailed spectral simulations. Whereas CSL shows almost no variation in ordering and rotational dynamics versus mole fraction POPS, (i.e. XPS), and 5-PC shows small effects, the weakly ordered end-chain labeled 16-PC shows large relative effects, such that the orientational order parameter, S is at a minimum for XPS = 0.5 where it is about one-third the value observed for XPS = 0 and 1. This is directly reflected in the ESR spectrum as a substantial variation in the hyperfine splitting with XPS. The least-squares analysis also shows a reduction in rotational diffusion coefficient, R perpendicular by a fractor of 2 for XPS = 0.5 and permits the estimation of S2, the ordering parameter representing deviations from cylindrically symmetric alignment. These results are contrasted with 2H NMR studies which were insensitive to effects of mixing headgroups on the acyl chains. The ESR results are consistent with a somewhat increased disorder in the end-chain region as well as a small amount of chain tilting upon mixing POPC and POPS. They demonstrate the high sensitivity of ESR to subtle effects in chain ordering and dynamics.

Topics: Biophysical Phenomena; Biophysics; Electron Spin Resonance Spectroscopy; In Vitro Techniques; Lipid Bilayers; Membrane Lipids; Molecular Conformation; Molecular Structure; Phosphatidylcholines; Phosphatidylserines; Spin Labels; Thermodynamics

Deuterium (2H) and phosphorus (31P) nuclear magnetic resonance (NMR) and freeze-fracture electron microscopy were used to study spontaneous vesiculation in model membranes composed of POPC:POPS with or without cholesterol. The NMR spectra indicated the presence of a central isotropic line, the intensity of which is reversibly and linearly dependent upon temperature in the L alpha phase, with no hysteresis when cycling between higher and lower temperatures. Freeze-fracture microscopy showed small, apparently connected vesicles that were only present when the samples were frozen (for freeze-fracture) from an initial temperature of 40-60 degrees C, and absent when the samples are frozen from an initial temperature of 20 degrees C. Analysis of motional narrowing was consistent with the isotropic lines being due to lateral diffusion in (and tumbling of) small vesicles (diameters approximately 50 nm). These results were interpreted in terms of current theories of shape fluctuations in large unilamellar vesicles which predict that small daughter vesicles may spontaneously "erupt" from larger parent vesicles in order to expel the excess area created by thermal expansion of the bilayer surface at constant volume. Assuming that all the increased area due to increasing temperature is associated with the isotropic lines, the NMR results allowed a novel estimate of the coefficient of area expansion alpha A in multilamellar vesicles (MLVs) which is in good agreement with micromechanical measurements upon giant unilamellar vesicles of similar composition. Experiments performed on unilamellar vesicles, which had been placed upon glass beads, confirmed that alpha A determined in this way is unchanged compared with the MLV case. Addition of the highly positively charged (extrinsic) myelin basic protein (MBP) to a POPC:POPS system showed that membrane eruptions of the type described here occur in response to the presence of this protein.

Topics: Biophysical Phenomena; Biophysics; Cholesterol; Freeze Fracturing; Magnetic Resonance Spectroscopy; Membranes, Artificial; Microscopy, Electron; Models, Biological; Phosphatidylcholines; Phosphatidylserines; Temperature

The binding of calcium to headgroup deuterated 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphoserine (POPS) was investigated by using deuterium magnetic resonance in pure POPS membranes and in mixed 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine (POPC)/POPS 5:1 (m:m) bilayers. Addition of CaCl2 to pure POPS bilayers led to two component spectra attributed, respectively, to liquid-crystallin POPS (less than 15 kHz) and POPS molecules in the calcium-induced dehydrated phase (cochleate) (approximately 120 kHz). The liquid-crystalline component has nearly disappeared at a Ca2+ to POPS ratio of 0.5, indicating that, under such conditions, most of the POPS molecules are in the precipitated cochleate phase. After dilution of the POPS molecules in zwitterionic POPC membranes (POPC/POPS 5:1 m:m), single component spectra characteristic of POPS in the liquid-crystalline state were observed in the presence of Molar concentrations of calcium ions (Ca2+ to POPS ratio greater than 50), showing that the amount of dehydrated cochleate PS-Ca2+ phase, if any, was low (less than 5%) under such conditions. Deuterium NMR data obtained in the 15-50 degrees C temperature range with the mixed PC/PS membranes, either in the absence or the presence of Ca2+ ions, indicate that the serine headgroup undergoes a temperature-induced conformational change, independent of the presence of Ca2+. This is discussed in relation to other headgroup perturbations such as that observed upon change of the membrane surface charge density.

Topics: Calcium; Deuterium; Liposomes; Magnetic Resonance Spectroscopy; Models, Biological; Molecular Conformation; Phosphatidylcholines; Phosphatidylserines; Thermodynamics

The binding of calcium, magnesium, lithium, potassium, and sodium to membrane bilayers of 5 to 1 (M/M) 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and 1-palmitoyl- 2-oleoylphosphatidylserine (POPS) was investigated by using deuterium nuclear magnetic resonance (2H NMR). Both lipids were deuteriated on their polar headgroups, and spectra were obtained at 25 degrees C in the liquid-crystalline phase as a function of salt concentration. The spectra obtained with calcium were correlated with 45CaCl2 binding studies to determine the effective membrane-bound calcium at low calcium binding, up to 0.78 calcium per POPS. Deuterium quadrupolar splittings of both POPC and POPS headgroups were shown to be very sensitive to calcium binding. The behavior of these two headgroups over a wide range of CaCl2 concentrations suggests that Ca2+ binding occurs in at least two steps, the first step being achieved with 0.5 M CaCl2, with a stoichiometry of 0.5 Ca2+ per POPS. Correlations of the deuterium Ca2+ binding data with related data obtained after incorporation of a cationic integral peptide showed that the effects of these two cationic molecules of the POPS headgroup are qualitatively similar, and provided further support for two-step Ca2+ binding to the POPC/POPS 5:1 membranes. The corresponding data obtained with magnesium, lithium, and potassium indicate that these cations interact with both the choline and serine headgroups. The amplitudes of headgroup perturbations could be partly correlated to the relative affinities of the metallic cations for the lipid membrane. The two-step binding described with Ca2+ appears to be relevant to the Mg2+ data, and in certain limits to the Li+ data. The data were interpreted in terms of conformational changes of the lipid headgroups induced by an electric field due to the charges of the membrane-bound metallic cations. A conformational change of the serine headgroup induced by the membrane-bound charges is proposed. We propose that the metallic cations can be differentiated on the basis of their respective spatial distribution functions relative to the choline and serine headgroups. According to this interpretation, the divalent cations Ca2+ and Mg2+ are more deeply buried in the membrane than monovalent Na+ and K+, the case of Li+ being intermediate of the latter two. This conclusion is discussed in relation to fundamental theories of the spatial distribution of ions near the interface between water and smooth charged solid surf

Topics: Binding Sites; Calcium; Cations, Divalent; Cations, Monovalent; Deuterium; Lipid Bilayers; Magnetic Resonance Spectroscopy; Molecular Conformation; Peptides; Phosphatidylcholines; Phosphatidylserines

Ca2+ binding between lamellae of phosphatidylserine (PS) and phosphatidylcholine (PC) gives rise to a rigid phase of Ca(PS)2. When aqueous Ca2+, hydrated PS/PC, and Ca(PS)2 coexist at equilibrium, the aqueous Ca2+ concentration is invariant and is characteristic of the PS/PC ratio. This characteristic Ca2+ concentration is 0.040 microM for palmitoyloleoylphosphatidylserine without PC and increases as the inverse square of the PS mole fraction at high PS concentration (Raoult's law) and as the inverse square of the PS mole fraction multiplied by a constant at low PS concentration (Henry's law). For example, for palmitoyloleoylphosphatidylserine/palmitoyloleoylphosphatidylcholi ne = 0.6/0.4 or 0.2/0.8, this characteristic Ca2+ concentration is about 0.1 or about 6 microM, respectively. These observations at constant temperature are summarized in a quaternary phase diagram for the four-component system CaCl2/PS/PC/water.

Topics: Calcium Chloride; Electron Spin Resonance Spectroscopy; Kinetics; Lipid Bilayers; Models, Theoretical; Molecular Conformation; Phosphatidylcholines; Phosphatidylserines; Water; X-Ray Diffraction

The phase behaviour of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) was studied by differential scanning calorimetry and 31P-NMR spectroscopy. Modulation of the phase behaviour of POPE by 1-palmitoyl-2-oleoylphosphatidylserine (POPS). 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1,2-di-olein (DOG), CaCl2, MgCl2, and combinations of these substances was studied. The bilayer-forming lipids, POPS and POPC, raise the bilayer-to-hexagonal phase-transition temperature of POPE. The POPC has a greater effect than POPS, probably because the former lipid is more miscible with POPE. Addition of 10 mM CaCl2 has little effect on the phase-transitions of POPE/POPC mixtures, but it greatly decreases the effectiveness of POPS in raising the bilayer-to-hexagonal phase-transition temperature of POPE. The effectiveness of DOG in lowering the phase-transition temperature of POPE is also greatly reduced in the presence of 10 mM CaCl2. This phenomenon may play a role in the negative feedback regulation of protein kinase C.

Topics: Calcium Chloride; Calorimetry, Differential Scanning; Diglycerides; Magnetic Resonance Spectroscopy; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Protein Kinase C