1-palmitoyl-2-oleoylphosphatidylcholine and 1-palmitoyl-2-oleoylphosphatidylethanolamine

In light of an increasing number of antibiotic-resistant bacterial strains, it is essential to understand an action imposed by various antimicrobial agents on bacteria at the molecular level. One of the leading mechanisms of killing bacteria is related to the alteration of their plasmatic membrane. We study bio-inspired peptides originating from natural antimicrobial proteins colicins, which can disrupt membranes of bacterial cells. Namely, we focus on the α-helix H1 of colicin U, produced by bacterium Shigella boydii, and compare it with analogous peptides derived from two different colicins. To address the behavior of the peptides in biological membranes, we employ a combination of molecular simulations and experiments. We use molecular dynamics simulations to show that all three peptides are stable in model zwitterionic and negatively charged phospholipid membranes. At the molecular level, their embedment leads to the formation of membrane defects, membrane permeation for water, and, for negatively charged lipids, membrane poration. These effects are caused by the presence of polar moieties in the considered peptides. Importantly, simulations demonstrate that even monomeric H1 peptides can form toroidal pores. At the macroscopic level, we employ experimental co-sedimentation and fluorescence leakage assays. We show that the H1 peptide of colicin U incorporates into phospholipid vesicles and disrupts their membranes, causing leakage, in agreement with the molecular simulations. These insights obtained for model systems seem important for understanding the mechanisms of antimicrobial action of natural bacteriocins and for future exploration of small bio-inspired peptides able to disrupt bacterial membranes.

Topics: Amino Acid Sequence; Colicins; Molecular Dynamics Simulation; Permeability; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Protein Conformation, alpha-Helical; Shigella boydii; Unilamellar Liposomes

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

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

Supported lipid bilayers (SLBs) embedded with hydrophobic quantum dots (QDs) undergo temporal structural rearrangement.. Synchrotron X-ray reflectivity (XRR) was applied to monitor the temporal structural changes over a period of 24 h of mixed SLBs of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) / 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine (POPE) intercalated with 4.9 nm hydrophobic cadmium sulphide quantum dots (CdS QDs). The QD-embedded SLBs (QD-SLBs) were formed via rupture of the mixed liposomes on a positively charged polyethylene imine (PEI) monolayer. Atomic force microscopy (AFM) imaging provided complementary characterization of the bilayer morphology.. Our results show time-dependent perturbations in the SLB structure due to the interaction upon QD incorporation. Compared to the SLB without QDs, at 3 h incubation time, there was a measurable decrease in the bilayer thickness and a concurrent increase in the scattering length density (SLD) of the QD-SLB. The QD-SLB then became progressively thicker with increasing incubation time, which - along with the fitted SLD profile - was attributed to the structural rearrangement due to the QDs being expelled from the inner leaflet to the outer leaflet of the bilayer. Our results give unprecedented mechanistic insights into the structural evolution of QD-SLBs on a polymer cushion, important to their potential biomedical and biosensing applications.

Topics: Lipid Bilayers; Models, Chemical; Phosphatidylcholines; Phosphatidylethanolamines; Quantum Dots

Mixed polydiacetylene (PDA) lipid vesicles mimic cell membranes and exhibit a colorimetric response induced by mechanical stress, which can be used to determine the affinity of proteins or molecules for lipid membranes. Due to a simple spectroscopic readout, PDA assays are amenable to high-throughput screens; however, these assays exhibit batch-to-batch variability. Sensitivity of the assay is also influenced by physicochemical properties associated with different lipids. Here, a method of normalizing PDA assays to reduce variability and enable direct comparison across lipid systems is described.

Topics: Amyloid beta-Peptides; Colorimetry; Lipid Bilayers; Phosphatidylcholines; Phosphatidylethanolamines; Polyacetylene Polymer

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

The voltage-dependent anion channel (VDAC) is a mitochondrial outer membrane protein whose fundamental function is to facilitate and regulate the flow of metabolites between the cytosol and the mitochondrial intermembrane space. Using coarse-grained molecular dynamics simulations, we investigated the dependence of VDAC selectivity towards small inorganic anions on two factors: the ionic strength and the lipid composition. In agreement with experimental data we found that VDAC becomes less anion selective with increasing salt concentration due to the screening of a few basic residues that point into the pore lumen. The molecular dynamics simulations provide insight into the regulation mechanism of VDAC selectivity by the composition in the lipid membrane and suggest that the ion distribution is differently modulated by POPE compared to the POPC bilayer. This occurs through the more persistent interactions of acidic residues located at both rims of the β-barrel with head groups of POPE which in turn impact the electrostatic potential and thereby the selectivity of the pore. This mechanism occurs not only in POPE single component membranes but also in a mixed POPE/POPC bilayer by an enrichment of POPE over POPC lipids on the surface of VDAC. Thus we show here that computationally-inexpensive coarse-grained simulations are able to capture, in a semi-quantitative way, essential features of VDAC anion selectivity and could pave the way toward a molecular level understanding of metabolite transport in natural membranes.

Topics: Animals; Mice; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Salts; Static Electricity; Surface Properties; Voltage-Dependent Anion Channels

The μ opioid receptor (μOR), which is part of the G protein-coupled receptors family, is a membrane protein that is modulated by its lipid environment. In the present work, we model μOR in three different membrane systems: POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), and DPPC (1, 2-dipalmitoyl-sn-glycero-3-phosphocholine) through 45 μs molecular dynamics (MD) simulations at the coarse-grained level. Our theoretical studies provide new insights to the lipid-induced modulation of the receptor. Particularly, to characterize how μOR interacts with each lipid, we analyze the tilt of the protein, the number of contacts occurring between the lipids and each amino acid of the receptor, and the μOR-lipid interface described as a network graph. We also analyze the variations in the number and the nature of the protein contacts that are induced by the lipid structure. We show that POPC interacts preferentially with helix 1 (H1) and helices H5-H6, POPE, with H5-H6 and H6-H7, and DPPC, with H4 and H6. We demonstrate how each of the three lipids shape the structure of the μOR.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Lipid Bilayers; Lipids; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Protein Binding; Protein Conformation; Receptors, Opioid, mu

Choline amino acid ([Ch][AA]) based ionic liquids (ILs) are considered to be highly biodegradable and biocompatible solvents. The toxicological scrutiny and environmental fate analysis of these ILs are fundamental requisites to employ these ILs on large scale applications. In the present work, we investigate how the presence of the simplest form of [Ch][AA] ILs, cholinium glycinate ([Ch][Gly]), affects the structure and stability of homogeneous 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine (POPE) lipid bilayers by using atomistic molecular dynamics simulation. The study reveals a considerable compression of the POPC bilayer along with an enhanced ordering of hydrocarbon lipid tails on increasing the concentration of [Ch][Gly] IL. On the other hand, the stability and structure of the POPE bilayer is hardly affected at lower concentration of [Ch][Gly]; however, at higher concentration (20 mol %), the structure of the bilayer is slightly changed. The H-bond analysis reveals that [Ch]

Topics: Choline; Glycine; Hydrogen Bonding; Ionic Liquids; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Water

Coarse grained molecular dynamics of the permeation of the peptide human beta-defensin-3 (HBD3) in two different lung surfactant models (BLES and CUROSURF) at surface tension of 20 mN m

Topics: beta-Defensins; Cholesterol; Drug Compounding; Humans; Lysophosphatidylcholines; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Phosphatidylserines; Pulmonary Surfactant-Associated Protein B; Pulmonary Surfactants; Static Electricity; Thermodynamics; Water

Biological membranes are remarkably heterogeneous, composed of diverse lipid mixtures with distinct chemical structure and composition. By combining molecular dynamics simulations and the newly developed Lipid-Force Distribution Analysis (L-FDA), we explore force transmission in complex multi-component membrane models mimicking eukaryotic organelles. We found that the chemical-moiety based segmentation at membrane interfaces revealed a distinctive distribution of bonded and non-bonded forces in diverse membrane environment. Our molecular stress analysis could have far-reaching implications in describing the relationship between membrane mechanical properties and functional states of chemically distinct lipids.

Topics: Algorithms; Cluster Analysis; Endoplasmic Reticulum; Golgi Apparatus; Lipid Bilayers; Mitochondria; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines

We measured the effect of intrinsic lipid curvature, J

Topics: Lipid Bilayers; Mechanical Phenomena; Phosphatidylcholines; Phosphatidylethanolamines

Phosphatidylinositol (PI) lipids are necessary for many cellular signaling pathways of membrane associated proteins, such as angiomotin (Amot). The Amot family regulates cellular polarity, growth, and migration. Given the low concentration of PI lipids in these membranes, it is likely that such protein-membrane interactions are stabilized by lipid domains or small lipid clusters. By small-angle X-ray scattering, we show that nonphosphorylated PI lipids induce lipid demixing in ternary mixtures of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), likely because of preferential interactions between the head groups of PE and PI. These results were obtained in the presence of buffer containing tris(hydroxymethyl)aminomethane, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, NaCl, ethylenediaminetetraacetic acid, dithiothreitol, and benzamidine at pH 8.0 that in previous work showed an ability to cause PC to phase separate but are necessary to stabilize Amot for in vitro experimentation. Collectively, this provided a framework for determining the effect of Amot on lipid organization. Using fluorescence spectroscopy, we were able to show that the association of Amot with this lipid platform causes significant reorganization of the lipid into a more homogenous structure. This reorganization mechanism could be the basis for Amot membrane association and fusogenic activity previously described in the literature and should be taken into consideration in future protein-membrane interaction studies.

Topics: Escherichia coli; Intercellular Signaling Peptides and Proteins; Liposomes; Membrane Proteins; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylinositols; Protein Domains; Scattering, Small Angle; Temperature; X-Ray Diffraction

In the present work we investigated the differential interactions of the antimicrobial peptides (AMPs) aurein 1.2 and maculatin 1.1 with a bilayer composed of a mixture of the lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE). We carried out molecular dynamics (MD) simulations using a coarse-grained approach within the MARTINI force field. The POPE/POPG mixture was used as a simple model of a bacterial (prokaryotic cell) membrane. The results were compared with our previous findings for structures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a representative lipid of mammalian cells. We started the simulations of the peptide-lipid system from two different initial conditions: peptides in water and peptides inside the hydrophobic core of the membrane, employing a pre-assembled lipid bilayer in both cases. Our results show similarities and differences regarding the molecular behavior of the peptides in POPE/POPG in comparison to their behavior in a POPC membrane. For instance, aurein 1.2 molecules can adopt similar pore-like structures on both POPG/POPE and POPC membranes, but the peptides are found deeper in the hydrophobic core in the former. Maculatin 1.1 molecules, in turn, achieve very similar structures in both kinds of bilayers: they have a strong tendency to form clusters and induce curvature. Therefore, the results of this study provide insight into the mechanisms of action of these two peptides in membrane leakage, which allows organisms to protect themselves against potentially harmful bacteria. Graphical Abstract Aurein pore structure (green) in a lipid bilayer composed by POPE (blue) and POPG (red) mixture. It is possible to see water beads (light blue) inside the pore.

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Anura; Binding Sites; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Binding; Protein Conformation, alpha-Helical; Protein Interaction Domains and Motifs

Understanding interactions between functional nanoparticles and lipid bilayers is important to many emerging biomedical and bioanalytical applications. In this paper, we report incorporation of hydrophobic cadmium sulphide quantum dots (CdS QDs) into mixed 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) liposomes, and into their supported bilayers (SLBs). The QDs were found embedded in the hydrophobic regions of the liposomes and the supported bilayers, which retained the QD fluorescent properties. In particular, we studied the effect of the QD size (2.7-5.4 nm in diameter) on the formation kinetics and structure of the supported POPC/POPE bilayers, monitored in situ using quartz crystal microbalance with dissipation monitoring (QCM-D), as the liposomes ruptured onto the substrate. The morphology of the obtained QD-lipid hybrid bilayers was studied using atomic force microscopy (AFM), and their structure by synchrotron X-ray reflectivity (XRR). It was shown that the incorporation of hydrophobic QDs promoted bilayer formation on the PEI cushion, evident from the rupture and fusion of the QD-endowed liposomes at a lower surface coverage compared to the liposomes without QDs. Furthermore, the degree of disruption in the supported bilayer structure caused by the QDs was found to be correlated with the QD size. Our results provide mechanistic insights into the kinetics of the rupturing and formation process of QD-endowed supported lipid bilayers via liposome fusion on polymer cushions.

Topics: Lipid Bilayers; Liposomes; Microscopy, Atomic Force; Particle Size; Phosphatidylcholines; Phosphatidylethanolamines; Quantum Dots; Quartz Crystal Microbalance Techniques; Synchrotrons

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

Daptomycin is a cyclic lipopeptide of clinical importance in the treatment of multidrug resistant infections, including those caused by methicillin-resistant S. aureus strains. Similar to many other antimicrobial peptides, daptomycin binds with preference to anionic membranes such as those typically found in prokaryotes. However, in contrast to most linear α-helical peptides, daptomycin binds to lipid bilayers only in the presence of calcium ions, and its activity in vivo is absolutely Ca

Topics: Anti-Bacterial Agents; Calcium; Daptomycin; Kinetics; Lipid Bilayers; Models, Chemical; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Unilamellar Liposomes

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

Interaction of human islet amyloid polypeptide (hIAPP) peptides with cell membrane is crucial for the understanding of amyloid toxicity associated with Type II diabetes (T2D). While it is known that the hIAPP-membrane interactions are considered to promote hIAPP aggregation into fibrils and induce membrane disruption, the membrane-induced conformation, orientation, aggregation, and adsorption behaviors of hIAPP peptides have not been well understood at the atomic level. Herein, we perform all-atom explicit-water molecular dynamics (MD) simulations to study the adsorption, orientation, and surface interaction of hIAPP aggregates with different sizes (monomer to tetramer) and conformations (monomer with α-helix and tetramer with β-sheet-rich U-turn) upon adsorption on the lipid bilayers composed of both pure zwitterionic POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and mixed anionic POPC/POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) (3:1) lipids. MD simulation results show that hIAPP monomer with α-helical conformation and hIAPP pentamer with β-sheet conformation can adsorb on both POPC and POPC/POPE bilayers via a preferential orientation of N-terminal residues facing toward the bilayer surface. The hIAPP aggregates show stronger interactions with mixed POPC/POPE lipids than pure POPC lipids, consistent with experimental observation that hIAPP adsorption and fibrililation are enhanced on mixed lipid bilayers. While electrostatic interactions are main attractive forces to drive the hIAPP aggregates to adsorb on both bilayers, the introduction of the more hydrophilic head groups of POPE lipids further promote the formation of the interfacial hydrogen bonds. Complement to our previous studies of hIAPP aggregates in bulk solution, this computational work increases our knowledge about the mechanism of amyloid peptide-membrane interactions that is central to the understanding the progression of all amyloid diseases.

Topics: Humans; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Islet Amyloid Polypeptide; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Protein Aggregates; Protein Conformation; Static Electricity; Water

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

The present study aimed to explore the underlying mechanisms of epicatechin-3-gallate-(4β→8, 2β→O→7)-epicatechin-3-gallate (A-type ECG dimer) and epigallocatechin-3-gallate-(4β→8, 2β→O→7)-epigallocatechin-3-gallate (A-type EGCG dimer) involved in their strong inhibitory effects on 3T3-L1 preadipocytes differentiation. In the synthetic "lipid raft-like" liposome, A-type ECG and EGCG dimers incorporated into the liposome with high affinity and decreased the fluidity of the liposome. In 3T3-L1 preadipocytes, A-type ECG and EGCG dimers possibly bonded to lipid rafts cholesterol and disrupted the integrity of lipid rafts, thus exerting their notable inhibitory effects on 3T3-L1 preadipocytes differentiation by suppressing mitotic clonal expansion process and mRNA levels of PPARγ, C/EBPα and SREBP1C. A highly positive correlation between the cholesterol binding capacity of the two dimers and their inhibitory effect on 3T3-L1 preadipocytes differentiation (R

Topics: 3T3-L1 Cells; Adipocytes; Animals; Catechin; Cell Differentiation; Cholesterol; Dimerization; Lipid Bilayers; Membrane Microdomains; Mice; Microscopy, Fluorescence; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines

The tear film is a thin multilayered structure covering the cornea. Its outermost layer is a lipid film underneath of which resides on an aqueous layer. This tear film lipid layer (TFLL) is itself a complex structure, formed by both polar and nonpolar lipids. It was recently suggested that due to tear film dynamics, TFLL contains inhomogeneities in the form of polar lipid aggregates. The aqueous phase of tear film contains lachrymal-origin proteins, whereby lysozyme is the most abundant. These proteins can alter TFLL properties, mainly by reducing its surface tension. However, a detailed nature of protein-lipid interactions in tear film is not known. We investigate the interactions of lysozyme with TFLL in molecular details by employing coarse-grained molecular dynamics simulations. We demonstrate that lysozyme, due to lateral restructuring of TFLL, is able to penetrate the tear lipid film embedded in inverse micellar aggregates.

Topics: Adsorption; Cholesterol Esters; Humans; Kinetics; Molecular Dynamics Simulation; Muramidase; Phosphatidylcholines; Phosphatidylethanolamines; Sphingomyelins; Sulfoglycosphingolipids; Surface Tension; Tears; Thermodynamics; Triolein; Water

Antimicrobial peptides (AMPs) represent new alternatives to cope with the increasing number of multi-drug resistant microbial infections. Recently, a derivative of the frog-skin AMP esculentin-1a, Esc(1-21), was found to rapidly kill both the planktonic and biofilm forms of the Gram-negative bacterium Pseudomonas aeruginosa with a membrane-perturbing activity as a plausible mode of action. Lately, its diastereomer Esc(1-21)-1c containing two d-amino acids i.e.

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Biofilms; Cholesterol; Cytotoxins; Kinetics; Leucine; Lipid Bilayers; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Plankton; Protein Conformation, alpha-Helical; Pseudomonas aeruginosa; Ranidae; Serine; Skin; Spheroplasts; Stereoisomerism; Structure-Activity Relationship

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

Cardiotoxins (CTXs) are single polypeptide chain consisting of 59-62 amino acids with four disulfide bridges and globular proteins of simple β-sheet folds. The CTXs are one of principal toxic components causing haemolysis and damaging various cells and belong to three-finger toxin (TFT) superfamily of snake venoms. However, there is no natural or synthetic small molecular inhibitor to the protein toxins to date. In the present study, modes of interaction of cardiotoxin 1 (CTX1) from Indian cobra (Naja naja) with heterogeneous erythrocyte membrane (EM) model system have been extensively examined by using all-atom molecular dynamics (MD) simulations in near physiological conditions and comprehensive analyses of the MD data revealed two distinct principal regions ('head groove' and 'loop groove') of the protein toxin for establishing structural interactions with the EM system. Moreover, combined analyses of data from high-throughput virtual screening of NCI small molecular database, in vitro haemolytic assays for top-hits of the chemical compounds against crude venom of Naja naja and as well CTXs purified from the venom and pharmacokinetic examinations on the chemical compounds retarding haemolytic activities of CTXs suggested that Etidronic acid and Zoledronic acid are promising prototypic chemical inhibitors to CTXs of snake venoms.

Topics: Amino Acid Sequence; Animals; Antidotes; Cholesterol; Cobra Cardiotoxin Proteins; Diphosphonates; Disulfides; Elapid Venoms; Elapidae; Erythrocyte Membrane; Etidronic Acid; Hemolysis; High-Throughput Screening Assays; Humans; Imidazoles; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Protein Domains; Protein Structure, Secondary; Small Molecule Libraries; Structure-Activity Relationship; User-Computer Interface; Zoledronic Acid

Naturally occurring antimicrobial peptides important for innate immunity are widely studied for their antimicrobial and anticancer activity. The primary target of these AMPs is believed to be the bacterial cytoplasmic membrane. However, the interaction between cytoplasmic membrane and the antimicrobial peptides remains poorly understood. Therefore to focus on the target membrane composition that is required by AMPs to interact with membranes, we have examined the interaction of the antimicrobial and anticancer active 11-residue GA-K4 (FLKWLFKWAKK) peptide with model and intact cell membranes. Effect on the structural conformational properties of GA-K4 peptide was investigated by means of far-UV CD and fluorescence spectroscopic methods. The different conformation of GA-K4 peptide in large unilamellar vesicles (LUV) bilayer and micelle environment suggest that the curvature has an influence on the secondary structure acquired by the peptide. Furthermore, the leakage experiment result confirmed that GA-K4 induced the leakage of cytoplasmic membrane in Staphylococcus аureus bacterial cells. Fluorescence data revealed the interfacial location of GA-K4 peptide in the model membranes. The blue-shift in emission wavelength by tryptophan residues in fluorescence data indicated the penetration of GA-K4 peptide in micelles and phospholipid bilayers. These results showed that the GA-K4 peptide is a membrane-active peptide and its activity depends on membrane curvature and lipid composition. Although further studies are required to confirm the mechanism of action, the data suggest mechanism of toroidal pore formation for the interaction of GA-K4 peptide with membranes. Our studies will be helpful in better understanding of the membrane requirment of peptides to express their therapeutic effects.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Antineoplastic Agents; Benzothiazoles; Carbocyanines; Cell Membrane; Cell Membrane Permeability; Fluorescent Dyes; Kinetics; Lipid Bilayers; Lysophosphatidylcholines; Micelles; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Structure, Secondary; Spectrometry, Fluorescence; Staphylococcus aureus; Unilamellar Liposomes

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

The SNAREs SNAP25 and SNAP23 are proteins that are initially cytosolic after translation, but then become stably attached to the cell membrane through palmitoylation of cysteine residues. For palmitoylation to occur, membrane association is a prerequisite, but it is unclear which motif may increase the affinities of the proteins for the target membrane. In experiments with rat neuroendocrine cells, we find that a few basic amino acids in the cysteine-rich region of SNAP25 and SNAP23 are essential for plasma membrane targeting. Reconstitution of membrane-protein binding in a liposome assay shows that the mechanism involves protein electrostatics between basic amino acid residues and acidic lipids such as phosphoinositides that play a primary role in these interactions. Hence, we identify an electrostatic anchoring mechanism underlying initial plasma membrane contact by SNARE proteins, which subsequently become palmitoylated at the plasma membrane.

Topics: Amino Acid Motifs; Animals; Binding Sites; Cell Membrane; Cloning, Molecular; Escherichia coli; Gene Expression; Liposomes; Lipoylation; PC12 Cells; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Plasmids; Protein Binding; Protein Processing, Post-Translational; Protein Transport; Rats; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Static Electricity; Synaptosomal-Associated Protein 25; Vesicular Transport Proteins

Adequate membrane fluidity is required for a variety of key cellular processes and in particular for proper function of membrane proteins. In most eukaryotic cells, membrane fluidity is known to be regulated by fatty acid desaturation and cholesterol, although some cells, such as insect cells, are almost devoid of sterol synthesis. We show here that insect and mammalian cells present similar microviscosity at their respective physiological temperature. To investigate how both sterols and phospholipids control fluidity homeostasis, we quantified the lipidic composition of insect SF9 and mammalian HEK 293T cells under normal or sterol-modified condition. As expected, insect cells show minimal sterols compared with mammalian cells. A major difference is also observed in phospholipid content as the ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) is inverted (4 times higher in SF9 cells). In vitro studies in liposomes confirm that both cholesterol and PE can increase rigidity of the bilayer, suggesting that both can be used by cells to maintain membrane fluidity. We then show that exogenously increasing the cholesterol amount in SF9 membranes leads to a significant decrease in PE:PC ratio whereas decreasing cholesterol in HEK 293T cells using statin treatment leads to an increase in the PE:PC ratio. In all cases, the membrane fluidity is maintained, indicating that both cell types combine regulation by sterols and phospholipids to control proper membrane fluidity.

Topics: Animals; Cell Membrane; Cholesterol; Fluorescence Polarization; HEK293 Cells; Humans; Lipid Bilayers; Liposomes; Membrane Fluidity; Models, Biological; Particle Size; Phosphatidylcholines; Phosphatidylethanolamines; Sf9 Cells; Species Specificity; Spodoptera; Temperature

Mitotane (o,p'.-DDD) is an orphan drug approved for the treatment of adrenocortical carcinoma. The mechanisms, which are responsible for this activity of the drug, are not completely understood. It can be hypothesized that an impact of mitotane is mediated by the interaction with cellular membranes. However, an interaction of mitotane with (lipid) membranes has not yet been investigated in detail. Here, we characterized the interaction of mitotane and its main metabolite o,p'-dichlorodiphenyldichloroacetic acid (o,p'-DDA) with lipid membranes by applying a variety of biophysical approaches of nuclear magnetic resonance, electron spin resonance, and fluorescence spectroscopy. We found that mitotane and o,p'-DDA bind to lipid membranes by inserting into the lipid-water interface of the bilayer. Mitotane but not o,p'-DDA directly causes a disturbance of bilayer structure leading to an increased permeability of the membrane for polar molecules. Mitotane induced alterations of the membrane integrity required the presence of phosphatidylethanolamine and/or cholesterol. Collectively, our data for the first time characterize the impact of mitotane on the lipid membrane structure and dynamics, which may contribute to a better understanding of specific mitotane effects and side effects.

Topics: Adrenal Glands; Ascorbic Acid; Biological Assay; Electron Spin Resonance Spectroscopy; Fluorescence; Lipid Bilayers; Lipids; Mitotane; Organ Specificity; Phosphatidylcholines; Phosphatidylethanolamines; Proton Magnetic Resonance Spectroscopy; Unilamellar Liposomes

Atomistic molecular dynamics simulations have become an important source of information for the structure and dynamics of biomembranes at molecular detail difficult to access in experiments. A number of force fields for lipid membrane simulations have been derived in the past; the choice of the most suitable force field is, however, frequently hampered by the availability of parameters for specific lipids. Additionally, the comparison of different quantities among force fields is often aggravated by varying simulation parameters. Here, we compare four atomistic lipid force fields, namely, the united-atom GROMOS54a7 and the all-atom force fields CHARMM36, Slipids, and Lipid14, for a broad range of structural and dynamical properties of saturated and monounsaturated phosphatidylcholine bilayers (DMPC and POPC) as well as for monounsaturated phosphatidylethanolamine bilayers (POPE). Additionally, the ability of the different force fields to describe the gel-liquid crystalline phase transition is compared and their computational efficiency estimated. Moreover, membrane properties like the water flux across the lipid bilayer and lipid acyl chain protrusion probabilities are compared.

Topics: Dimyristoylphosphatidylcholine; Lipid Bilayers; Molecular Dynamics Simulation; Molecular Structure; Phosphatidylcholines; Phosphatidylethanolamines

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

Sorafenib and regorafenib are small-molecule kinase inhibitors approved for the treatment of locally recurrent or metastatic, progressive, differentiated thyroid carcinoma, renal cell carcinoma, and hepatocellular carcinoma (sorafenib) and of colorectal cancer (regorafenib). As of now, the mechanisms, which are responsible for their antitumor activities, are not completely understood. Given the lipophilic nature of the molecules, it can be hypothesized that the pharmacological impact is mediated by the interaction with cellular membranes as it is true for many pharmacologically active molecules. However, an interaction of sorafenib or regorafenib with lipid membranes has not yet been investigated in detail. Here, we characterized the interaction of both drugs with lipid membranes by applying a variety of biophysical approaches including nuclear magnetic resonance, electron spin resonance, and fluorescence spectroscopy. We found that sorafenib and regorafenib bind to lipid membranes by inserting into the lipid-water interface of the bilayer. This membrane embedding causes a disturbance of bilayer structure leading to an increased permeability of the membrane for polar molecules. One approach shows that the extent of the effects depends on the membrane lipid composition underlining a particular role of phosphatidylcholine and cholesterol. Our data for the first time characterize the impact of sorafenib and regorafenib on the lipid membrane structure and dynamics, which may contribute to a better understanding of their effectiveness in the treatment of malignancies as well as of their side effects.

Topics: Antineoplastic Agents; Ascorbic Acid; Cell Membrane; Cell Membrane Permeability; Cholesterol; Dithionite; Kinetics; Niacinamide; Oxidation-Reduction; Phenylurea Compounds; Phosphatidylcholines; Phosphatidylethanolamines; Pyridines; Sorafenib; Spin Labels; Staining and Labeling; Unilamellar Liposomes

The initial steps of membrane disruption by antimicrobial peptides (AMPs) involve binding to bacterial membranes in a surface-bound (S) orientation. To evaluate the effects of lipid composition on the S state, molecular dynamics simulations of the AMPs piscidin 1 (p1) and piscidin 3 (p3) were carried out in four different bilayers: 3:1 DMPC/DMPG, 3:1 POPC/POPG, 1:1 POPE/POPG, and 4:1 POPC/cholesterol. In all cases, the addition of 1:40 piscidin caused thinning of the bilayer, though thinning was least for DMPC/DMPG. The peptides also insert most deeply into DMPC/DMPG, spanning the region from the bilayer midplane to the headgroups, and thereby only mildly disrupting the acyl chains. In contrast, the peptides insert less deeply in the palmitoyl-oleoyl containing membranes, do not reach the midplane, and substantially disrupt the chains, i.e., the neighboring acyl chains bend under the peptide, forming a basket-like conformation. Curvature free energy derivatives calculated from the simulation pressure profiles reveal that the peptides generate positive curvature in membranes with palmitoyl and oleoyl chains but negative curvature in those with myristoyl chains. Curvature inductions predicted with a continuum elastic model follow the same trends, though the effect is weaker, and a small negative curvature induction is obtained in POPC/POPG. These results do not directly speak to the relative stability of the inserted (I) states or ease of pore formation, which requires the free energy pathway between the S and I states. Nevertheless, they do highlight the importance of lipid composition and acyl chain packing.

Topics: Antimicrobial Cationic Peptides; Dimyristoylphosphatidylcholine; Fish Proteins; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Structure, Secondary; 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

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

The molecular pathogenesis of Alzheimer's disease (AD) is complex and sparsely understood. The relationship between AD's amyloid β (Aβ) peptides and neuronal membranes is central to Aβ's cytotoxicity and is directly modulated by the composition of the lipid headgroups. Molecular studies of the insertion of model Aβ40 protofilaments in lipid bilayers revealed strong interactions that affect the structural integrity of both the membranes and the ordered amyloid aggregates. In particular, electrostatics plays a crucial role in the interaction between Aβ protofilaments and palmytoil-oleoyl-phosphatidylethanolamine (POPE) lipids, a common component of neuronal plasma membranes. Here, we use all-atom molecular dynamics and steered molecular dynamics simulations to systematically compare the effects that POPE and palmytoil-oleoyl-phosphatidylcholine (POPC) headgroups have on the Aβ-lipid interactions. We find that Aβ protofilaments exhibit weaker electrostatic interactions with POPC headgroups and establish significantly shorter-lived contacts with the POPC bilayer. This illustrates the crucial yet complex role of electrostatic and hydrogen bonding interactions in modulating the anchoring and insertion of Aβ peptides into lipid bilayers. Our study reveals the atomistic details behind the barrier created by the lipid headgroup region in impeding solution-aggregated fibrillar oligomers to spontaneously insert into POPC bilayers, in contrast to the POPE case. While the biological reality is notoriously more complex (e.g., including other factors such as cholesterol), our results evidence a simple experimentally and computationally testable case for probing the factors that control the insertion of Aβ oligomeric aggregates in neuronal cell membranes--a process central to their neurotoxicity.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Computer Simulation; Humans; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Static Electricity; Statistics, Nonparametric; Structure-Activity Relationship

The widespread environmental presence and commercial use of nanoparticles have raised significant health concerns as a result of many in vitro and in vivo assays indicating toxicity of a wide range of nanoparticle species. Many of these assays have identified the ability of nanoparticles to damage cell membranes. These interactions can be studied in detail using artificial lipid bilayers, which can provide insight into the nature of the particle-membrane interaction through variation of membrane and solution properties not possible with cell-based assays. However, the scope of these studies can be limited because of the low throughput characteristic of lipid bilayer platforms. We have recently described an easy to use, parallel lipid bilayer platform which we have used to electrically investigate the activity of 60 nm diameter amine and carboxyl modified polystyrene nanoparticles (NH2-NP and COOH-NP) with over 1000 lipid bilayers while varying lipid composition, bilayer charge, ionic strength, pH, voltage, serum, particle concentration, and particle charge. Our results confirm recent studies finding activity of NH2-NP but not COOH-NP. Detailed analysis shows that NH2-NP formed pores 0.3-2.3 nm in radius, dependent on bilayer and solution composition. These interactions appear to be electrostatic, as they are regulated by NH2-NP surface charge, solution ionic strength, and bilayer charge. The ability to rapidly measure a large number of nanoparticle and membrane parameters indicates strong potential of this bilayer array platform for additional nanoparticle bilayer studies.

Topics: Animals; Blood Proteins; Cattle; Cholesterol; Lipid Bilayers; Microarray Analysis; Nanoparticles; Osmolar Concentration; Phosphatidylcholines; Phosphatidylethanolamines

Single-amino acid mutations in the human α-synuclein (αS) protein are related to early onset Parkinson's disease (PD). In addition to the well-known A30P, A53T, and E46K mutants, recently a number of new familial disease-related αS mutations have been discovered. How these mutations affect the putative physiological function of αS and the disease pathology is still unknown. Here we focus on the H50Q and G51D familial mutants and show that like wild-type αS, H50Q and G51D monomers bind to negatively charged membranes, form soluble partially folded oligomers with an aggregation number of ~30 monomers under specific conditions, and can aggregate into amyloid fibrils. We systematically studied the ability of these isolated oligomers to permeabilize membranes composed of anionic phospholipids (DOPG) and membranes mimicking the mitochondrial phospholipid composition (CL:POPE:POPC) using a calcein release assay. Small-angle X-ray scattering studies of isolated oligomers show that oligomers formed from wild-type αS and the A30P, E46K, H50Q, G51D, and A53T disease-related mutants are composed of a similar number of monomers. However, although the binding affinity of the monomeric protein and the aggregation number of the oligomers formed under our specific protocol are comparable for wild-type αS and H50Q and G51D αS, G51D oligomers cannot disrupt negatively charged and physiologically relevant model membranes. Replacement of the membrane-immersed glycine with a negatively charged aspartic acid at position 51 apparently abrogates membrane destabilization, whereas a mutation in the proximal but solvent-exposed part of the membrane-bound α-helix such as that found in the H50Q mutant has little effect on the bilayer disrupting properties of oligomers.

Topics: alpha-Synuclein; Cell Membrane Permeability; Fluoresceins; Humans; Membranes, Artificial; Multiprotein Complexes; Mutation, Missense; Parkinson Disease; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Binding; Scattering, Small Angle; X-Ray Diffraction

Maculatin 1.1 (Mac1) is an antimicrobial peptide from the skin of Australian tree frogs and is known to possess selectivity toward Gram-positive bacteria. Although Mac1 has membrane disrupting activity, it is not known how Mac1 selectively targets Gram-positive over Gram-negative bacteria. The interaction of Mac1 with Escherichia coli, Staphylococcus aureus, and human red blood cells (hRBC) and with their mimetic model membranes is here reported. The peptide showed a 16-fold greater growth inhibition activity against S. aureus (4 μM) than against E. coli (64 μM) and an intermediate cytotoxicity against hRBC (30 μM). Surprisingly, Sytox Green uptake monitored by flow cytometry showed that Mac1 compromised both bacterial membranes with similar efficiency at ∼20-fold lower concentration than the reported minimum inhibition concentration against S. aureus. Mac1 also reduced the negative potential of S. aureus and E. coli membrane with similar efficacy. Furthermore, liposomes mimicking the cell membrane of S. aureus (POPG/TOCL) and E. coli (POPE/POPG) were lysed at similar concentrations, whereas hRBC-like vesicles (POPC/SM/Chol) remained mostly intact in the presence of Mac1. Remarkably, when POPG/TOCL and POPE/POPG liposomes were co-incubated, Mac1 did not induce leakage from POPE/POPG liposomes, suggesting a preference toward POPG/TOCL membranes that was supported by surface plasma resonance assays. Interestingly, circular dichroism spectroscopy showed a similar helical conformation in the presence of the anionic liposomes but not the hRBC mimics. Overall, the study showed that Mac1 disrupts bacterial membranes in a similar fashion before cell death events and would preferentially target S. aureus over E. coli or hRBC membranes.

Topics: Amphibian Proteins; Animals; Antimicrobial Cationic Peptides; Anura; Cardiolipins; Cell Membrane; Cholesterol; Dose-Response Relationship, Drug; Erythrocytes; Escherichia coli; Hemolysis; Humans; Liposomes; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Species Specificity; Sphingomyelins; Staphylococcus aureus

Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactions in vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particles in vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interaction in vitro, either by directly contacting acidic lipids or by promoting Gag multimerization.. Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt a folded-over conformation on a lipid bilayer, implicating both the N and C termini in membrane binding. In addition, binding of Gag to membranes is diminished when either terminal domain is truncated. RSV Gag membrane association is significantly less sensitive than HIV Gag membrane association to lipid acyl chain saturation. These findings shed light on Gag assembly and membrane binding, critical steps in the viral life cycle and an untapped target for antiretroviral drugs.

Topics: Cell Membrane; Cholesterol; Escherichia coli; Gene Expression; Gene Products, gag; HIV-1; Hydrodynamics; Lipid Bilayers; Osmolar Concentration; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylinositol 4,5-Diphosphate; Protein Binding; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins; Rous sarcoma virus; Virion

Secretory phospholipase A2 (sPLA2) catalyzes the hydrolysis of sn-2 linkage in the glycerophospholipid, thereby releasing fatty acid and 1-acyl lysophospholipid. Among sPLA2s from various organisms and tissues, group XIV fungal/bacterial sPLA2s are relatively less characterized compared to their mammalian counterparts. Here we report cloning, recombinant expression, refolding, and enzymatic characterization of two sPLA2s, NCU06650 and NCU09423, from the filamentous fungus Neurospora crassa. The hexahistidine-tagged putative mature region of both proteins was expressed in Escherichia coli. Inclusion bodies were solubilized using a high hydrostatic pressure refolding technique. NCU06650 was solubilized without any additives at alkaline pH, and the addition of arginine or non-detergent sulfobetain (NDSB) significantly improved the process at acidic pH. In contrast, NCU09423 was solubilized only when NDSB was added at alkaline pH. Both enzymes displayed a Ca(2+)-dependent lipolytic activity toward E. coli membrane. Mass spectrometry analysis using the synthetic phospholipids as substrates demonstrated that both enzymes preferentially cleaved the sn-2 ester linkage of substrates and generated 1-acyl lysophospholipids, demonstrating that they are bona fide PLA2.

Topics: Amino Acid Sequence; Escherichia coli; Hydrogen-Ion Concentration; Micelles; Molecular Sequence Data; Neurospora crassa; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipases A2, Secretory; Protein Refolding; Recombinant Proteins; Sequence Alignment

Acyl-Coenzyme A is made in the cytosol. Certain enzymes using acyl-CoA seem to operate in the lumen of the ER but no corresponding flippases for acyl-CoA or an activated acyl have been described. In order to test the ability of purified candidate flippases to operate the transport of acyl-CoA through lipid bilayers in vitro we developed three enzyme-coupled assays using large unilamellar vesicles (LUVs) obtained by detergent removal. The first assay uses liposomes encapsulating a water-soluble acyl-CoA:glycerol-3-phosphate acyl transferase plus glycerol-3-phosphate (G3P). It measures formation of [(3)H]lyso-phosphatidic acid inside liposomes after [(3)H]palmitoyl-CoA has been added from outside. Two other tests use empty liposomes containing [(3)H]palmitoyl-CoA in the inner membrane leaflet, to which either soluble acyl-CoA:glycerol-3-phosphate acyl transferase plus glycerol-3-phosphate or alkaline phosphatase are added from outside. Here one can follow the appearance of [(3)H]lyso-phosphatidic acid or of dephosphorylated [(3)H]acyl-CoA, respectively, both being made outside the liposomes. Although the liposomes may retain small amounts of detergent, all these tests show that palmitoyl-CoA crosses the lipid bilayer only very slowly and that the lipid composition of liposomes barely affects the flip-flop rate. Thus, palmitoyl-CoA cannot cross the membrane spontaneously implying that in vivo some transport mechanism is required.

Topics: Acyl Coenzyme A; Alkaline Phosphatase; Biological Transport; Chemistry Techniques, Analytical; Glycerol-3-Phosphate O-Acyltransferase; Glycerophosphates; Lipid Bilayers; Liposomes; Phosphatidylcholines; Phosphatidylethanolamines; Reproducibility of Results; Serum Albumin, Bovine; Unilamellar Liposomes

Analysis of 300 ns (ns) molecular dynamics (MD) simulations of an adenosine A2a receptor (A2a AR) model, conducted in triplicate, in 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) bilayers reveals significantly different protein dynamical behavior. Principal component analysis (PCA) shows that the dissimilarities stem from interhelical rather than intrahelical motions. The difference in the hydrophobic thicknesses of these simulated lipid bilayers is potentially a significant reason for the observed difference in results. The distinct lipid headgroups might also lead to different molecular interactions and hence different protein loop motions. Overall, the A2a AR shows higher mobility and flexibility in POPC as compared to POPE.

Topics: Cell Membrane; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Principal Component Analysis; Protein Conformation; Receptor, Adenosine A2A

A novel strategy for selectively adsorbing phospholipids (PLs) on titania-coated silica core-shell microspheres (TiO2/SiO2) was developed. The TiO2/SiO2 microspheres were prepared through water-vapor-induced internal hydrolysis and then characterized by SEM, UV-vis spectroscopy, X-ray diffraction, and measurements of Brunauer-Emmett-Teller surface area. Analyses showed that the titania layer was uniformly distributed onto the surface of silica particles. The TiO2/SiO2 microspheres were employed as sorbent in solid-phase extraction (SPE), and their absorptive ability was investigated by reversed-phase liquid chromatography-evaporative light scattering detection (RPLC-ELSD). Important factors that affect the extraction, such as loading buffer, eluting buffer, and elution volume, were investigated in detail and optimized by using standard samples. Results reveal that the developed SPE approach had higher recoveries for PLs than that based on pure TiO2 particles. The proposed SPE method was used for extraction of PLs from serum and showed great potential for identifying more kinds of endogenous PL metabolites by ultra performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF MS). The proposed SPE method with the composite sorbent was used to screen PLs from a biological matrix with high selectivity and efficiency. This approach is a promising method for selective extraction of PLs in lipidomics or phospholipidomics.

Topics: Adsorption; Chromatography, Reverse-Phase; Dimyristoylphosphatidylcholine; Humans; Lysophospholipids; Microscopy, Electron, Scanning; Microspheres; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Reproducibility of Results; Silicon Dioxide; Solid Phase Extraction; Spectrophotometry; Tandem Mass Spectrometry; Thermodynamics; Titanium; X-Ray Diffraction

The C2 domain of PKCα (C2α) induces fluorescence self-quenching of NBD-PS in the presence of Ca2+, which is interpreted as the demixing of phosphatidylserine from a mixture of this phospholipid with phosphatidylcholine. Self-quenching of NBD-PS was considerably increased when phosphatidylinositol-4,5-bisphosphate (PIP2) was present in the membrane. When PIP2 was the labeled phospholipid, in the form of TopFluor-PIP2, fluorescence self-quenching induced by the C2 domain was also observed, but this was dependent on the presence of phosphatidylserine. An independent indication of the phospholipid demixing effect given by the C2α domain was obtained by using 2H-NMR, since a shift of the transition temperature of deuterated phosphatidylcholine was observed as a consequence of the addition of the C2α domain, but only in the presence of PIP2. The demixing induced by the C2α domain may have a physiological significance since it means that the binding of PKCα to membranes is accompanied by the formation of domains enriched in activating lipids, like phosphatidylserine and PIP2. The formation of these domains may enhance the activation of the enzyme when it binds to membranes containing phosphatidylserine and PIP2.

Topics: Calcium; Cations, Divalent; Fluorescence; Membranes, Artificial; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylinositol 4,5-Diphosphate; Protein Kinase C-alpha; Protein Structure, Tertiary

The use of proteoliposomes as affinity elements in conjunction with a surface plasmon resonance sensor is a high-sensitivity alternative for the detection of multiple analytes. However, one of the most important aspects of these conformations is maintaining the functionality of the immobilized protein, which is determined by the choice of lipids and surfactants employed in the reconstitutions. Previously, we demonstrated the functionality of TLR5-proteoliposomes as screening affinity elements of bacterial flagellin. In this new study we change the conditions of immobilization of TLR5 and evaluate how the fluidity of the membrane and the final size of the liposomes affect the functionality of the construct and thus increase their utility as an affinity element for design of new biosensors. In particular, we used reconstructions into preformed liposomes composed of the lipids POPC, POPC-DMPC and POPC-POPE mediated by the use of surfactants OG, Triton X100, and DDM, respectively. The affinity results were evaluated by SPR technology proteoliposomes and were correlated with the anisotropic change in the membrane status; the final sizes of the proteoliposomes were estimated. Our results clearly show the dependence of fluidity and final size of the proteoliposomes with surface plasmon resonance affinity measurements.

Topics: Biosensing Techniques; Dimyristoylphosphatidylcholine; Escherichia coli; Escherichia coli Proteins; Flagellin; Fluorescence Polarization; Humans; Immobilized Proteins; Kinetics; Lipid Bilayers; Lipids; Membrane Fluidity; Octoxynol; Phosphatidylcholines; Phosphatidylethanolamines; Protein Binding; Proteolipids; Reproducibility of Results; Surface Plasmon Resonance; Surface-Active Agents; Time Factors; Toll-Like Receptor 5

Lipid droplets (LDs) are primary repositories of esterified fatty acids and sterols in animal cells. These organelles originate on the lumenal or cytoplasmic side of endoplasmic reticulum (ER) membrane and are released to the cytosol. In contrast to other intracellular organelles, LDs are composed of a mass of hydrophobic lipid esters coved by phospholipid monolayer. The small size and unique architecture of LDs makes it complicated to study LD structure by modern experimental methods. We discuss coarse-grained molecular dynamics (MD) simulations of LD formation in systems containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), triolein (TO), cholesterol (CHOL), and water. We find that (1) there is more cholesterol in the LD core, than at the interface. (2) No crystallization occurs inside the LD core. (3) According to coarse-grained simulations, the presence of PE lipids at the interface has a little impact on distribution of components and on the overall LD structure. (4) The thickness of the lipid monolayer at the surface of the droplet is similar to the thickness of one leaflet of a bilayer. Computer simulations are shown to be a mighty tool to provide molecular-level insights, which are not available to the experimental techniques.

Topics: Cholesterol; Dimerization; Lipid Bilayers; Lipid Droplets; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Solvents; Triolein; Water

Cholesterol is important for the formation of microdomains in supported lipid bilayers and is enriched in the liquid-ordered phase. To understand the interactions leading to this enrichment, we developed an AFM-based single-lipid-extraction (SLX) approach that enables us to determine the anchoring strength of cholesterol in the two phases of a phase-separated lipid membrane. As expected, the forces necessary for extracting a single cholesterol molecule from liquid-ordered phases are significantly higher than for extracting it from the liquid-disordered phases. Interestingly, application of the Bell model shows two energy barriers that correlate with the head and full length of the cholesterol molecule. The resulting lifetimes for complete extraction are 90 s and 11 s in the liquid-ordered and liquid-disordered phases, respectively. Molecular dynamics simulations of the very same experiment show similar force profiles and indicate that the stabilization of cholesterol in the liquid-ordered phase is mainly due to nonpolar contacts.

Topics: Cholesterol; Hydrogen Bonding; Lipid Bilayers; Microscopy, Atomic Force; Molecular Dynamics Simulation; Phase Transition; Phosphatidylcholines; Phosphatidylethanolamines; Spectrum Analysis; Unilamellar Liposomes

Molecular dynamics (MD) simulations of membranes are often hindered by the slow lateral diffusion of lipids and the limited time scale of MD. In order to study the dynamics of mixing and characterize the lateral distribution of lipids in converged mixtures, we report microsecond-long all-atom MD simulations performed on the special-purpose machine Anton. Two types of mixed bilayers, POPE:POPG (3:1) and POPC:cholesterol (2:1), as well as a pure POPC bilayer, were each simulated for up to 2 μs. These simulations show that POPE:POPG and POPC:cholesterol are each fully miscible at the simulated conditions, with the final states of the mixed bilayers similar to a random mixture. By simulating three POPE:POPG bilayers at different NaCl concentrations (0, 0.15, and 1 M), we also examined the effect of salt concentration on lipid mixing. While an increase in NaCl concentration is shown to affect the area per lipid, tail order, and lipid lateral diffusion, the final states of mixing remain unaltered, which is explained by the largely uniform increase in Na(+) ions around POPE and POPG. Direct measurement of water permeation reveals that the POPE:POPG bilayer with 1 M NaCl has reduced water permeability compared with those at zero or low salt concentration. Our calculations provide a benchmark to estimate the convergence time scale of all-atom MD simulations of lipid mixing. Additionally, equilibrated structures of POPE:POPG and POPC:cholesterol, which are frequently used to mimic bacterial and mammalian membranes, respectively, can be used as starting points of simulations involving these membranes.

Topics: Cholesterol; Lipid Bilayers; Molecular Conformation; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Sodium Chloride; Thermodynamics; Time Factors; Water

Three representatives of naturally occurring pentacyclic triterpenes (PTs) were subjected to comprehensive studies aimed at the analysis of their interactions with phospholipids found naturally in mitochondrial membrane. To reach this goal, the selected compounds--α-amyrin (AMalf), betulinic acid (BAc), and ursolic acid (Urs)--were incorporated into two-component and multicomponent Langmuir monolayers acting as a model of mitochondrial membrane. As the lipids characteristic for mitochondria, phosphatidylcholine (POPC), phosphatidylethanolamine (POPE), and cardiolipin (BHCL) were chosen. Our studies were motivated by the fact that, according to the literature, the anticancer activity of PTs is correlated with their ability to incorporate into mitochondrial membrane and modify its properties. The undertaken studies were based on the surface pressure (π)-molecular area (A) isotherm registration complemented with the thermodynamic analysis and BAM visualization. It was found that all three terpenes with the exception of high betulinic acid proportion (30 and 50%) interact beneficially with POPC in two-component monolayers, while incorporation of BAc and Urs into POPE film is energetically unfavorable. As far as the model mitochondrial membrane composed of POPC/POPE/BHCL is concerned, the largest destructive influence (high positive values of ΔG(Exc) and decrease of the model monolayer condensation) was found in the case of terpene acids, while the effect of α-amyrin was energetically favorable. We postulated that the origin of the observed findings is connected with the specific interactions between bolaamphlilic terpene acids and POPE, known from its propensity to form intermolecular hydrogen bonds.

Topics: Betulinic Acid; Cardiolipins; Hydrogen Bonding; Mitochondrial Membranes; Models, Molecular; Pentacyclic Triterpenes; Phosphatidylcholines; Phosphatidylethanolamines; Thermodynamics; Triterpenes; Ursolic Acid

The present paper was aimed at evaluating the effect of cholesterol (CHO) on the voltage-induced lipid pore formation in bilayer membranes through a global characterization of the temporal dynamics of the fluctuation pattern of ion currents. The bilayer model used was black lipid membranes (BLMs) of palmitoyloleoylphosphatidylethanolamine and palmitoyloleoylphosphatidylcholine (POPE:POPC) at a 7:3 molar ratio in the absence (BLM0) or in the presence of 30 (BLM30), 40 (BLM40) or 50(BLM50)mol% of cholesterol with respect to total phospholipids. Electrical current intensities (I) were measured in voltage (ΔV) clamped conditions at ΔV ranging between 0 and ±200mV. The autocorrelation parameter α derived from detrended fluctuation analysis (DFA) on temporal fluctuation patterns of electrical currents allowed discriminating between non-correlated (α=0.5, white noise) and long-range correlated (0.5<α<1) behaviors. The increase in |ΔV| as well as in cholesterol content increased the number of conductance states, the magnitude of conductance level, the capacitance of the bilayers and increased the tendency towards the development of long-range autocorrelated (fractal) processes (0.5<α<1) in lipid channel generation. Experiments were performed above the phase transition temperature of the lipid mixtures, but compositions used predicted a superlattice-like organization. This leads to the conclusion that structural defects other than phase coexistence may promote lipid channel formation under voltage clamped conditions. Furthermore, cholesterol controls the voltage threshold that allows the percolation of channel behavior where isolated channels become an interconnected network.

Topics: Animals; Cattle; Cell Membrane; Cerebral Cortex; Cholesterol; Electric Capacitance; Ion Transport; Lipid Bilayers; Phosphatidylcholines; Phosphatidylethanolamines; Temperature

ABCB10 is one of the three ATP-binding cassette (ABC) transporters found in the inner membrane of mitochondria. In mammals ABCB10 is essential for erythropoiesis, and for protection of mitochondria against oxidative stress. ABCB10 is therefore a potential therapeutic target for diseases in which increased mitochondrial reactive oxygen species production and oxidative stress play a major role. The crystal structure of apo-ABCB10 shows a classic exporter fold ABC transporter structure, in an open-inwards conformation, ready to bind the substrate or nucleotide from the inner mitochondrial matrix or membrane. Unexpectedly, however, ABCB10 adopts an open-inwards conformation when complexed with nonhydrolysable ATP analogs, in contrast to other transporter structures which adopt an open-outwards conformation in complex with ATP. The three complexes of ABCB10/ATP analogs reported here showed varying degrees of opening of the transport substrate binding site, indicating that in this conformation there is some flexibility between the two halves of the protein. These structures suggest that the observed plasticity, together with a portal between two helices in the transmembrane region of ABCB10, assist transport substrate entry into the substrate binding cavity. These structures indicate that ABC transporters may exist in an open-inwards conformation when nucleotide is bound. We discuss ways in which this observation can be aligned with the current views on mechanisms of ABC transporters.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Amino Acid Sequence; Animals; ATP-Binding Cassette Transporters; Binding Sites; Crystallography, X-Ray; Humans; Lipid Bilayers; Models, Molecular; Molecular Conformation; Molecular Dynamics Simulation; Molecular Sequence Data; Mutation; Nucleotides; Phosphatidylcholines; Phosphatidylethanolamines; Protein Binding; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Sf9 Cells

The rapid rise of antibiotic resistance in pathogens becomes a serious and growing threat to medicine and public health. Naturally occurring antimicrobial peptides (AMPs) are an important line of defense in the immune system against invading bacteria and microbial infection. In this work, we present a combined computational and experimental study of the biological activity and membrane interaction of the computationally designed Bac2A-based peptide library. We used the MARTINI coarse-grained molecular dynamics with adaptive biasing force method and the umbrella sampling technique to investigate the translocation of a total of 91 peptides with different amino acid substitutions through a mixed anionic POPE/POPG (3:1) bilayer and a neutral POPC bilayer, which mimic the bacterial inner membrane and the human red blood cell (hRBC) membrane, respectively. Potential of mean force (PMF, free energy profile) was obtained to measure the free energy barrier required to transfer the peptides from the bulk water phase to the water-membrane interface and to the bilayer interior. Different PMF profiles can indeed identify different membrane insertion scenarios by mapping out peptide-lipid energy landscapes, which are correlated with antimicrobial activity and hemolytic activity. Computationally designed peptides were further tested experimentally for their antimicrobial and hemolytic activities using bacteria growth inhibition assay and hemolysis assay. Comparison of PMF data with cell assay results reveals a good correlation of the peptides between predictive transmembrane activity and antimicrobial/hemolytic activity. Moreover, the most active mutants with the balanced substitutions of positively charged Arg and hydrophobic Trp residues at specific positions were discovered to achieve the improved antimicrobial activity while minimizing red blood cell lysis. Such substitutions provide more effective and cooperative interactions to distinguish the peptide interaction with different lipid bilayers. This work provides a useful computational tool to better understand the mechanism and energetics of membrane insertion of AMPs and to rationally design more effective AMPs.

Topics: Amino Acid Sequence; Amino Acid Substitution; Antimicrobial Cationic Peptides; Biological Transport; Cell Membrane; Diffusion; Hemolysis; Humans; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Microbial Sensitivity Tests; Molecular Dynamics Simulation; Molecular Mimicry; Molecular Sequence Data; Peptide Library; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Engineering; Pseudomonas aeruginosa; Thermodynamics

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 C2 domain of PKCε binds to negatively charged phospholipids but little is known so far about the docking orientation of this domain when it is bound. By using a FRET assay we have studied the binding of this domain to model membranes. We have also used ATR-Fourier transform infrared spectroscopy with polarized light (ATR-FTIR) to determine the docking mode by calculating the β-sandwich orientation when the domain is bound to different types of model membranes. The vesicle lipid compositions were: POPC/POPE/POPA (22:36:42) imitating the inner leaflet of a plasma membrane, POPC/POPA (50:50) in which POPE has been eliminated with respect to the former composition and POPC/POPE/CL (43:36:21) imitating the inner mitochondrial membrane. Results show that the β-sandwich of the PKCα-C2 domain is inclined at an angle α close to 45° to the membrane normal. Some differences were found with respect to the extent of binding as a function of phospholipid composition and small changes on secondary structure were only evident when the domain was bound to model membranes of POPC/POPA: in this case, the percentage of β-sheet of the C2 domain increases if compared with the secondary structure of the domain in the absence of vesicles. With respect to the β-sandwich orientation, when the domain is bound to POPC/POPE/CL membranes it forms an angle with the normal to the surface of the lipid bilayer (39°) smaller than that one observed when the domain interacts with vesicles of POPC/POPA (49°).

Topics: Adenosine; Calcium; Fluorescence Resonance Energy Transfer; Glycerophospholipids; Humans; Lipid Bilayers; Lipids; Mitochondrial Membranes; Models, Molecular; Models, Statistical; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Protein Binding; Protein Conformation; Protein Kinase C-epsilon; Protein Structure, Secondary; Protein Structure, Tertiary; Spectrophotometry, Infrared

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

We present an overview of the application of dual-focus fluorescence correlation spectroscopy (2f-FCS) for the measurement of diffusion coefficients within free-standing lipid membranes. The first part gives a detailed theoretical analysis of the expected performance of 2f-FCS, in particular about the sensitivity of the method with regard to precise focus position and to aberrations caused by refractive index mismatch or cover slide thickness deviation. After describing the experimental details of the 2f-FCS setup and the preparation of free-standing black lipid membranes (BLMs), we apply the method to study the diffusion of lipids within BLMs as a function of lipid composition and of ion valency and ionic strength of the surrounding buffer.

Topics: Diffusion; Ions; Lipid Bilayers; Lipids; Osmolar Concentration; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Spectrometry, Fluorescence

Nearly all molecular dynamics simulations of bacterial membranes simplify the lipid bilayer by composing it of only one or two lipids. Previous attempts of developing a model E. coli membrane have used only 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol) POPG lipids. However, an important constituent of bacterial membranes are lipids containing a cyclopropane ring within the acyl chain. We have developed a complex membrane that more accurately reflects the diverse population of lipids within E. coli cytoplasmic membranes, including lipids with a cyclic moiety. Differences between the deuterium order profile of cyclic lipids and monounsaturated lipids are observed. Furthermore, the inclusion of the cyclopropane ring decreases the surface density of the bilayer and produces a more rigid membrane as compared to POPE/POPG membranes. Additionally, the diverse acyl chain length creates a thinner bilayer which matches the hydrophobic thickness of E. coli transmembrane proteins better than the POPE/POPG bilayer. We believe that the complex lipid bilayer more accurately describes a bacterial membrane and suggest the use of it in molecular dynamic simulations rather than simple POPE/POPG membranes.

Topics: Cell Membrane; Cyclopropanes; Escherichia coli K12; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Models, Chemical; Phosphatidylcholines; Phosphatidylethanolamines

Human-β-defensins HBD-1-3 are important components of the innate immune system. Synthetic peptides Phd-1-3 with a single disulphide bond, spanning the cationic C-terminal region of HBD-1-3, have antimicrobial activity. The interaction of Phd-1-3 with model membranes was investigated using isothermal titration calorimetry (ITC) and steady-state fluorescence polarization to understand the biophysical basis for the mechanism of antimicrobial action. Calorimetric titration of POPE:POPG (7:3) vesicles with peptides at 25°C and 37°C showed complex profiles with two distinct regions of heat changes. The data indicate binding of Phd-1-3 at 37°C to both negative and zwitterionic lipid vesicles is exothermic with low enthalpy values (ΔH~-1.3 to -2.8kcal/mol) as compared to amphipathic helical antibacterial peptides. The adsorption of peptides to negatively charged lipid membranes is modulated by electrostatic interactions that are described by surface partition equilibrium model using Gouy-Chapman theory. However, this model could not explain the isotherms of peptide binding to zwitterionic lipid vesicles. Fluorescence polarization of TMA-DPH (1-[4-(trimethylammonio) phenyl]-6-phenyl-1,3,5-hexatriene) and DPH (1,6-diphenyl-1,3,5-hexatriene) located in the head group and acyl chain region respectively, indicates that the peptides interact with interfacial region of negatively charged membranes. Based on the results obtained, we conclude that adsorption of cationic peptides Phd-1-3 on lipid surface do not result in conformational change or pore formation. It is proposed that interaction of Phd-1-3 with the negatively charged lipid head group causes membrane destabilization, which in turn affects the efficient functioning of cytoplasmic membrane proteins in bacteria, resulting in cell death.

Topics: Adsorption; Bacteria; beta-Defensins; Calorimetry, Indirect; Dihydropyridines; Diphenylhexatriene; Fluorescence Polarization; Humans; Membranes, Artificial; Phosphatidylcholines; Phosphatidylethanolamines

Understanding the role of specific bilayer components in controlling the function of G-protein coupled receptors (GPCRs) will be a key factor in the development of novel pharmaceuticals. Cholesterol-dependence in particular has become an area of keen interest with respect to GPCR function; not least since the 2.6Å crystal structure of the β2 adrenergic receptor revealed a putative cholesterol binding motif conserved throughout class-A GPCRs. Furthermore, experimental evidence for cholesterol-dependent GPCR function has been demonstrated in a limited number of cases. This modulation of receptor function has been attributed to both direct interactions between cholesterol and receptor, and indirect effects caused by the influence of cholesterol on bilayer order and lateral pressure. Despite the widespread occurrence of cholesterol binding motifs, available experimental data on the functional involvement of cholesterol on GPCRs are currently limited to a small number of receptors. Here we investigate the role of cholesterol in the function of the neurotensin receptor 1 (NTS1) a class-A GPCR. Specifically we show how cholesterol, and the analogue cholesteryl hemisuccinate, influence activity, stability, and oligomerisation of both purified and reconstituted NTS1. The results caution against using such motifs as indicators of cholesterol-dependent GPCR activity.

Topics: Amino Acid Motifs; Biophysics; Cell Membrane; Cholesterol; Cholesterol Esters; Crystallography, X-Ray; Fluorescence Resonance Energy Transfer; Humans; Ligands; Lipid Bilayers; Models, Molecular; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Pressure; Protein Binding; Receptors, Adrenergic, beta-2; Receptors, Neurotensin; Time Factors

An important feature of antimicrobial peptides is their ability to distinguish pro- from eukaryotic membranes. In vitro experiments on the antimicrobial peptide NK-2 indicate that the discrimination between zwitterionic phosphatidylethanolamine lipids exposed by prokaryotes and phosphatidylcholine lipids exposed by eukaryotes plays an important role. The underlying mechanism is not understood. Here we present molecular dynamics simulations in conjunction with a coarse grained model and thermodynamic integration showing that NK-2 binds more strongly to palmitoyloleoylphosphatidylethanolamine (POPE) than to palmitoyloleoylphosphatidylcholine (POPC) bilayers. Finite size effects on the relative free energy have been corrected for with a method that may also be useful in future studies of the affinities of macromolecules for lipid membranes. Our results support the previous hypothesis that the stronger binding to PE compared to PC arises from a better accessibility of the phosphates of the lipids to the cationic peptide in a sense that a similar number of peptide-lipid salt bridges requires to break more favorable electrostatic headgroup-headgroup interactions for PC relative to PE. The transfer of NK-2 from POPC to POPE is found to lead to a decrease in electrostatic peptide-lipid but an increase in lipid-lipid and ion-lipid interactions, correlating with a dehydration of the lipids and the ions but an increased hydration of the peptide. The increase in affinity of NK-2 for POPE compared to POPC hence arises from a complex interplay of competing interactions. This work opens the perspective to study how the affinity of antimicrobial peptides changes with amino acid sequence and lipid composition.

Topics: Algorithms; Antimicrobial Cationic Peptides; Biophysics; Ions; Lipid Bilayers; Lipids; Models, Molecular; Models, Statistical; Molecular Conformation; Peptides; Phosphates; Phosphatidylcholines; Phosphatidylethanolamines; Protein Binding; Static Electricity; Thermodynamics

Accurate simulation of complex lipid bilayers has long been a goal in condensed phase molecular dynamics (MD). Structure and function of membrane-bound proteins are highly dependent on the lipid bilayer environment and are challenging to study through experimental methods. Within Amber, there has been limited focus on lipid simulations, although some success has been seen with the use of the General Amber Force Field (GAFF). However, to date there are no dedicated Amber lipid force fields. In this paper we describe a new charge derivation strategy for lipids consistent with the Amber RESP approach and a new atom and residue naming and type convention. In the first instance, we have combined this approach with GAFF parameters. The result is LIPID11, a flexible, modular framework for the simulation of lipids that is fully compatible with the existing Amber force fields. The charge derivation procedure, capping strategy, and nomenclature for LIPID11, along with preliminary simulation results and a discussion of the planned long-term parameter development are presented here. Our findings suggest that LIPID11 is a modular framework feasible for phospholipids and a flexible starting point for the development of a comprehensive, Amber-compatible lipid force field.

Topics: Cholesterol; Inositol; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids

For cationic antimicrobial peptides to become useful therapeutic agents, it is important to understand their mechanism of action. To obtain high resolution data, this involves studying the structure and membrane interaction of these peptides in tractable model bacterial membranes rather than directly utilizing more complex bacterial surfaces. A number of lipid mixtures have been used as bacterial mimetics, including a range of lipid headgroups, and different ratios of neutral to negatively charged headgroups. Here we examine how the structure and membrane interaction of aurein 2.2 and some of its variants depend on the choice of lipids, and how these models correlate with activity data in intact bacteria (MICs, membrane depolarization). Specifically, we investigated the structure and membrane interaction of aurein 2.2 and aurein 2.3 in 1:1 cardiolipin/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (CL/POPG) (mol/mol), as an alternative to 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)/POPG and a potential model for Gram positive bacteria such as S. aureus. The structure and membrane interaction of aurein 2.2, aurein 2.3, and five variants of aurein 2.2 were also investigated in 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)/POPG (mol/mol) lipids as a possible model for other Gram positive bacteria, such as Bacillus cereus. Solution circular dichroism (CD) results demonstrated that the aurein peptides adopted α-helical structure in all lipid membranes examined, but demonstrated a greater helical content in the presence of POPE/POPG membranes. Oriented CD and ³¹P NMR results showed that the aurein peptides had similar membrane insertion profiles and headgroup disordering effects on POPC/POPG and CL/POPG bilayers, but demonstrated reduced membrane insertion and decreased headgroup disordering on mixing with POPE/POPG bilayers at low peptide concentrations. Since the aurein peptides behaved very differently in POPE/POPG membrane, minimal inhibitory concentrations (MICs) of the aurein peptides in B. cereus strain C737 were determined. The MIC results indicated that all aurein peptides are significantly less active against B. cereus than against S. aureus and S. epidermidis. Overall, the data suggest that it is important to use a relevant model for bacterial membranes to gain insight into the mode of action of a given antimicrobial peptide in specific bacteria.

Topics: Anti-Infective Agents; Antimicrobial Cationic Peptides; Cell Membrane; Circular Dichroism; Gram-Positive Bacteria; Lipid Bilayers; Magnetic Resonance Spectroscopy; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols

We utilized epifluorescence microscopy to investigate the morphological changes in labeled lipid bilayers supported on quartz surfaces (SLBs) induced by the interaction of cationic antimicrobial peptides with the lipid membranes. The SLBs were prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and mixtures thereof as well as from Escherichia coli lipid extract. We succeeded in the preparation of POPG and POPG-rich SLBs without the necessity to use fusogenic agents such as calcium by using the Langmuir-Blodgett/Langmuir-Schaefer transfer method. The adsorption of the peptides to the SLBs was initially driven by electrostatic interactions with the PG headgroups and led to the formation of lipid protrusions bulging out from the lipid layer facing the bulk, originating particularly from domain boundaries and membrane defects. The shape, size, and frequency of the lipid protrusions are mainly controlled by the peptide macroscopic properties and the membrane composition. A restructuring of the lipid protrusions into other structures can also occur over time.

Topics: Antimicrobial Cationic Peptides; Calcium; Lipid Bilayers; Microscopy, Fluorescence; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Static Electricity

One approach to the growing health problem of antibiotic resistant bacteria is the development of antimicrobial peptides (AMPs) as alternative treatments. The mechanism by which these AMPs selectively attack the bacterial membrane is not well understood, but is believed to depend on differences in membrane lipid composition. N-acylation of the small amidated hexapeptide, RRWQWR-NH(2) (LfB6), derived from the 25 amino acid bovine lactoferricin (LfB25) can be an effective means to improve its antimicrobial properties. Here, we investigate the interactions of C6-LfB6, N-acylated with a 6 carbon fatty acid, with model lipid bilayers with two distinct compositions: 3:1 POPE:POPG (negatively charged) and POPC (zwitterionic). Results from solid-state (2)H and (31)P NMR experiments are compared with those from an ensemble of all-atom molecular dynamic simulations running in aggregate more than 8.6ms. (2)H NMR spectra reveal no change in the lipid acyl chain order when C6-LfB6 is bound to the negatively charged membrane and only a slight decrease in order when it is bound to the zwitterionic membrane. (31)P NMR spectra show no significant perturbation of the phosphate head groups of either lipid system in the presence of C6-LfB6. Molecular dynamic simulations show that for the negatively charged membrane, the peptide's arginines drive the initial association with the membrane, followed by attachment of the tryptophans at the membrane-water interface, and finally by the insertion of the C6 tails deep into the bilayer. In contrast, the C6 tail leads the association with the zwitterionic membrane, with the tryptophans and arginines associating with the membrane-water interface in roughly the same amount of time. We find similar patterns in the order parameters from our simulations. Moreover, we find in the simulations that the C6 tail can insert 1-2Å more deeply into the zwitterionic membrane and can exist in a wider range of angles than in the negatively charged membrane. We propose this is due to the larger area per lipid in the zwitterionic membrane, which provides more space for the C6 to insert and assume different orientations.

Topics: Acylation; Anisotropy; Anti-Infective Agents; Binding Sites; Escherichia coli; Hydrogen Bonding; Lactoferrin; Membrane Lipids; Membranes, Artificial; Microbial Sensitivity Tests; Molecular Conformation; Molecular Dynamics Simulation; Nuclear Magnetic Resonance, Biomolecular; Oligopeptides; Peptide Fragments; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Binding; Staphylococcus aureus; Structure-Activity Relationship

The identification of phosphocholine and phosphoethanolamine lipids by MALDI TOF/TOF, including characterisation of the headgroup and delineation of the acyl chain at each position of the glycerol backbone, has been explored using lipids representative of each type. The relative intensities of fragments involving the neutral loss of one or other of the acyl chains from ion adducts of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC) were compared. For POPC and POPE, a statistical preference for the loss of the chain from the sn-1 position was observed in the presence of lithium. For OPPC this selectivity was reversed for one of the fragments. In the absence of lithium, fragmentation was favoured at the sn-2 position for all lipids. In all cases, spectra obtained in the presence of lithium yielded more intense product ion peaks. Although Collision Induced Dissociation (CID) could be used for complete lipid characterisation, LIFT™ was found to be a better method due to the presence of a greater number of distinguishing product ion peaks and a better shot-to-shot reproducibility of peak intensities.

Topics: Ethanolamines; Lithium; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipid Ethers; Phosphorylcholine; Reproducibility of Results; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

We synthesize and characterize alkylthiohydroquinones (ATHs) in order to investigate their interactions with lipid model membranes, POPE and POPC. We observe the formation of structures with different morphologies, or curvature of the lipid bilayer, depending on pH and increasing temperature. We attribute their formation to changes in the balance charge/polarity induced by the ATHs. Mixtures of ATHs with POPE at pH 4 form two cubic phases, P4(3)32 and Im3m, that reach a maximum lattice size at 40 °C while under basic conditions these phases only expand upon heating from room temperature. The cubic phases coexist with lamellar or hexagonal phases and are associated with inhomogeneous distribution of the ATH molecules over the lipid matrix. The zwitterionic POPC does not form cubic phases but instead shows lamellar structures with no clear influence of the 2,6-BATH.

Topics: Hydrogen-Ion Concentration; Hydroquinones; Lipid Bilayers; Models, Molecular; Molecular Structure; Particle Size; Phosphatidylcholines; Phosphatidylethanolamines; Stereoisomerism; Surface Properties; Temperature

An oxidized form of cholesterol, atheronal, is a form found in vivo that has been associated with human pathologies. We have studied mixtures of this oxidized sterol with the phospholipids phosphatidylethanolamine and phosphatidylcholine. We used phospholipids either with palmitoyl and oleoyl acyl chains on the C1 and C2 carbon atoms of glycerol or with both acyl chains being palmitoleoyl. We also compared the effects of atheronal on the curvature properties of these lipids with the action of cholesterol. We studied the bilayer to hexagonal phase transition temperature of mixtures of these lipids using differential scanning calorimetry as well as the dimensions of the hexagonal phase cylinders using X-ray diffraction. Disordering of the lamellar phase was also qualitatively assessed by the loss of sharp diffraction peaks. Our results demonstrate that the modulation of membrane curvature in these systems depends not only on the nature of the sterol, but also on the acyl chain composition of the phospholipids used. In addition, some of the effects of atheronal could be ascribed to reaction of the aldehyde and ketone groups of this oxidized sterol with the amino group of phosphatidylethanolamine.

Topics: Calorimetry, Differential Scanning; Cholesterol; Phosphatidylcholines; Phosphatidylethanolamines; Scattering, Small Angle; X-Ray Diffraction

Phosphatidylinositol 4-phosphate (PtdIns(4)P) lipid is an essential component of eukaryotic membranes and a marker of the Golgi complex. Here, we developed metabolically stabilized (ms) analogs of PtdIns(4)P and the inositol 1,4-bisphosphate (IP(2)) head group derivative and demonstrated that these compounds can substitute the natural lipid fully retaining its physiological activities. The methylenephosphonate (MP) and phosphorothioate (PT) analogs of PtdIns(4)P and the aminohexyl (AH)-IP(2) probe are recognized by the PtdIns(4)P-specific PH domain of four phosphate adaptor protein 1 (FAPP1). Binding of FAPP1 to the PtdIns(4)P derivatives stimulates insertion of the PH domain into the lipid layers and induces tubulation of membranes. Both ms analogs and IP(2) probes could be invaluable for identifying protein effectors and characterizing PtdIns(4)P-dependent signaling cascades within the trans-Golgi network (TGN).

Topics: Adaptor Proteins, Signal Transducing; Binding Sites; Inositol Phosphates; Ligands; Lipid Bilayers; Magnetic Resonance Spectroscopy; Molecular Probes; Molecular Structure; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylinositol Phosphates; Phosphatidylinositols; Protein Conformation; Protein Structure, Tertiary; Recombinant Fusion Proteins; Signal Transduction

Pseudomonas aeruginosa, when cultured under the appropriate conditions, secretes rhamnolipids to the external medium. These glycolipids constitute one of the most interesting classes of biosurfactants so far. A dirhamnolipid fraction was isolated and purified from the crude biosurfactant, and its action on model and biological membranes was studied. Dirhamnolipid induced leakage of internal contents, as measured by the release of carboxyfluorescein, in phosphatidylcholine unilamellar vesicles, at concentrations below its CMC. Membrane solubilization was not observed within this concentration range. The presence of inverted cone-shaped lipids in the membrane, namely lysophosphatidylcholine, accelerated leakage, whereas cone-shaped lipids, like phosphatidylethanolamine, decreased leakage rate. Increasing concentrations of cholesterol protected the membrane against dirhamnolipid-induced leakage, which was totally abolished by the presence of 50 mol% of the sterol. Dirhamnolipid caused hemolysis of human erythrocytes through a lytic mechanism, as shown by the similar rates of K(+) and hemoglobin leakage, and by the absence of effect of osmotic protectants. Scanning electron microscopy showed that the addition of the biosurfactant changed the usual disc shape of erythrocytes into that of spheroechinocytes. The results are discussed within the frame of the biological actions of dirhamnolipid, and the possible future applications of this biosurfactant.

Topics: Cell Membrane; Cell Shape; Cholesterol; Erythrocytes; Fluoresceins; Glycolipids; Hemoglobins; Hemolysis; Humans; Kinetics; Lysophosphatidylcholines; Membranes, Artificial; Permeability; Phosphatidylcholines; Phosphatidylethanolamines; Polyethylene Glycols; Potassium; Pseudomonas aeruginosa; Unilamellar Liposomes

A significant modification to the additive all-atom CHARMM lipid force field (FF) is developed and applied to phospholipid bilayers with both choline and ethanolamine containing head groups and with both saturated and unsaturated aliphatic chains. Motivated by the current CHARMM lipid FF (C27 and C27r) systematically yielding values of the surface area per lipid that are smaller than experimental estimates and gel-like structures of bilayers well above the gel transition temperature, selected torsional, Lennard-Jones and partial atomic charge parameters were modified by targeting both quantum mechanical (QM) and experimental data. QM calculations ranging from high-level ab initio calculations on small molecules to semiempirical QM studies on a 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) bilayer in combination with experimental thermodynamic data were used as target data for parameter optimization. These changes were tested with simulations of pure bilayers at high hydration of the following six lipids: DPPC, 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1,2-dilauroyl-sn-phosphatidylcholine (DLPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-phosphatidylcholine (DOPC), and 1-palmitoyl-2-oleoyl-sn-phosphatidylethanolamine (POPE); simulations of a low hydration DOPC bilayer were also performed. Agreement with experimental surface area is on average within 2%, and the density profiles agree well with neutron and X-ray diffraction experiments. NMR deuterium order parameters (S(CD)) are well predicted with the new FF, including proper splitting of the S(CD) for the aliphatic carbon adjacent to the carbonyl for DPPC, POPE, and POPC bilayers. The area compressibility modulus and frequency dependence of (13)C NMR relaxation rates of DPPC and the water distribution of low hydration DOPC bilayers also agree well with experiment. Accordingly, the presented lipid FF, referred to as C36, allows for molecular dynamics simulations to be run in the tensionless ensemble (NPT), and is anticipated to be of utility for simulations of pure lipid systems as well as heterogeneous systems including membrane proteins.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Dimyristoylphosphatidylcholine; Lipid Bilayers; Lipids; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Quantum Theory; Thermodynamics; X-Ray Diffraction

Membrane fusion is essential to both cellular vesicle trafficking and infection by enveloped viruses. While the fusion protein assemblies that catalyze fusion are readily identifiable, the specific activities of the proteins involved and nature of the membrane changes they induce remain unknown. Here, we use many atomic-resolution simulations of vesicle fusion to examine the molecular mechanisms for fusion in detail. We employ committor analysis for these million-atom vesicle fusion simulations to identify a transition state for fusion stalk formation. In our simulations, this transition state occurs when the bulk properties of each lipid bilayer remain in a lamellar state but a few hydrophobic tails bulge into the hydrophilic interface layer and make contact to nucleate a stalk. Additional simulations of influenza fusion peptides in lipid bilayers show that the peptides promote similar local protrusion of lipid tails. Comparing these two sets of simulations, we obtain a common set of structural changes between the transition state for stalk formation and the local environment of peptides known to catalyze fusion. Our results thus suggest that the specific molecular properties of individual lipids are highly important to vesicle fusion and yield an explicit structural model that could help explain the mechanism of catalysis by fusion proteins.

Topics: Computer Simulation; Hemagglutinins, Viral; Hydrophobic and Hydrophilic Interactions; Membrane Fusion; Models, Molecular; Orthomyxoviridae; Phosphatidylcholines; Phosphatidylethanolamines; Transport Vesicles; Water

The FYVE domain associates with phosphatidylinositol 3-phosphate [PtdIns(3)P] in membranes of early endosomes and penetrates bilayers. Here, we detail principles of membrane anchoring and show that the FYVE domain insertion into PtdIns(3)P-enriched membranes and membrane-mimetics is substantially increased in acidic conditions. The EEA1 FYVE domain binds to POPC/POPE/PtdIns(3)P vesicles with a Kd of 49 nM at pH 6.0, however associates approximately 24 fold weaker at pH 8.0. The decrease in the affinity is primarily due to much faster dissociation of the protein from the bilayers in basic media. Lowering the pH enhances the interaction of the Hrs, RUFY1, Vps27p and WDFY1 FYVE domains with PtdIns(3)P-containing membranes in vitro and in vivo, indicating that pH-dependency is a general function of the FYVE finger family. The PtdIns(3)P binding and membrane insertion of the FYVE domain is modulated by the two adjacent His residues of the R(R/K)HHCRXCG signature motif. Mutation of either His residue abolishes the pH-sensitivity. Both protonation of the His residues and nonspecific electrostatic contacts stabilize the FYVE domain in the lipid-bound form, promoting its penetration and increasing the membrane residence time.

Topics: Binding Sites; Histidine; Humans; Hydrogen-Ion Concentration; Membrane Lipids; Models, Molecular; Mutation; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylinositol Phosphates; Protein Binding; Protein Interaction Domains and Motifs; Vesicular Transport Proteins

Using atomic-scale molecular dynamics simulations, we consider the intrinsic cell membrane potential that is found to originate from a subtle interplay between lipid transmembrane asymmetry and the asymmetric distribution of monovalent salt ions on the two sides of the cell membrane. It turns out that both the asymmetric distribution of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipids across a membrane and the asymmetric distribution of NaCl and KCl induce nonzero drops in the transmembrane potential. However, these potential drops are opposite in sign. As the PC leaflet faces a NaCl saline solution and the PE leaflet is exposed to KCl, the outcome is that the effects of asymmetric lipid and salt ion distributions essentially cancel one another almost completely. Overall, our study highlights the complex nature of the intrinsic potential of cell membranes under physiological conditions.

Topics: Cell Membrane; Glycerophospholipids; Membrane Potentials; Models, Molecular; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Potassium Chloride; Pressure; Sodium Chloride; Static Electricity; Temperature; Time Factors

We visualized nanometer-scale phospholipid particle fusion by scanning tunneling microscopy (STM) on an alkanethiol-modified gold substrate, induced by duramycin, a tetracyclic antibiotic peptide with 19 amino residues. Ultrasonic homogenization generated a suspension mainly consisting of minimal lipid particles (MLP) from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) in a phosphate buffer solution, confirmed by dynamic light scattering (DLS). In situ STM discerned individual MLP as particles (diameter approximately 8 nm) spread on Au(111), modified with alkanethiol, within the suspension. The MLP became fragile by the presence of duramycin, and the MLP were easily scratched by the scanning tip into multilayers along the surface. This process of particle fusion on the gold surface coincides with the aggregation of MLP in the suspension, observed by DLS. It was demonstrated that STM is capable of discerning and monitoring the nanometer-scale features of phospholipid particles altered by antibiotics with biochemical impact. STM might allow in situ, real-space, nanometer-scale observations of minute particles composed of phospholipids within the real cells with the highest magnification ratio.

Topics: Bacteriocins; Microscopy, Scanning Tunneling; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids

Multinanosecond molecular dynamics (MD) simulations have been employed to characterize the interaction of an integral membrane protein (IMP), the leucine transmitter from Aquifex aeolicus (Yamashita et al., Nature 2005, 437, 215-223), with hydrated lipid bilayer membranes in their physiologically relevant liquid crystalline phases. Analysis of the MD trajectories for dimyristoyl phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), and 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) focused on the contacts between aromatic and basic side chains of the IMP with the lipid head groups and water. Structural fluctuations of the IMP were investigated as well as the contact dynamics of neighboring lipids. In characterizing the IMP-membrane systems, the behaviors of the protein's cytoplasmic and periplasmic parts are considered separately. All three lipid membranes show a rather similar overall level of association with the IMP. However, for DMPC there is a better matching of the membrane core to the hydrophobic transmembrane portion of the IMP. The closed cytoplasmic end of the IMP exhibits a higher degree of association with lipids than the more open periplasmic end, an observation which correlates with the more compact structure and a slower dynamics of surrounding lipids.

Topics: Bacteria; Dimyristoylphosphatidylcholine; Lipid Bilayers; Membrane Proteins; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines

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

Topics: Electrochemistry; Hydrogen Bonding; Lipid Bilayers; Membrane Potentials; Membranes, Artificial; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids

In this study, we examined the adsorption of cytochrome c (cyt c) on monolayers and liposomes formed from (i) pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), or cardiolipin (CL) and on (ii) the more thermodynamically stable binary mixtures of POPE/CL (0.8:0.2 mol/mol) and POPC/CL (0.6:0.4 mol/mol). Constant surface pressure experiments showed that the maximum and minimum interactions occurred in the pure CL (anionic phospholipid) and the pure POPE (zwitterion) monolayers, respectively. Observation by atomic force microscopy (AFM) of the images of Langmuir-Blodgett (LB) films extracted at 30 mN m-1 suggests that the different interactions of cyt c with POPE/CL and the POPC/CL monolayers could be due to lateral phase separation occurring in the POPE/CL mixture. The competition between 8-anilino-1-naphthalene sulfonate (ANS) and cyt c for the same binding sites in liposomes that have identical nominal compositions with respect to those of the monolayers was used to obtain binding parameters. In agreement with the monolayer experiments, the most binding was observed in POPE/CL liposomes. All of our observations strongly support the existence of selective adsorption of cyt c on CL, which is modulated differently by different neutral phospholipids (POPE and POPC).

Topics: Adsorption; Cardiolipins; Cytochromes c; Lipid Bilayers; Liposomes; Microscopy, Atomic Force; Phosphatidylcholines; Phosphatidylethanolamines; Pressure

Disulfide-bonded beta-hairpin structures are common among antimicrobial peptides. Disulfide bonds are known to be important for antimicrobial activity, but the underlying structural reason is not well understood. We have investigated the membrane-bound structure of a disulfide-deleted analogue of the antimicrobial peptide protegrin-1, in which the four Cys residues were replaced by Ala. The secondary structure, dynamics, and topology of this Ala-PG1 peptide in the membrane were determined by using magic-angle-spinning NMR spectroscopy. Conformation-dependent (13)C isotropic chemical shifts of multiple (13)C-labeled residues were obtained from 1D cross-polarization and direct-polarization spectra, and from 2D J-coupling-mediated (13)C-(13)C correlation spectra. Most labeled residues exhibited two conformations: a random coil and a beta-sheet structure. The dual-conformation property was present in both anionic lipid bilayers, which mimic the bacterial membrane, and zwitterionic cholesterol-containing bilayers, which mimic the eukaryotic cell membrane. The mobility of the peptide was measured by using a 2D C-H dipolar-shift correlation experiment. The random-coil fraction was highly mobile whereas the beta-sheet component was rigid. (1)H spin diffusion from the lipid chains to the peptide indicates that the beta-sheet component was well inserted into the anionic membrane, but surface bound in the cholesterol-containing neutral membrane. Thus, the removal of disulfide bonds changed some PG-1 molecules to highly mobile random coils that were poorly associated with the lipid membrane, but other molecules retained a beta-sheet conformation and had a similar membrane-binding topology to the parent peptide. Thus, the reduced antimicrobial activity of Ala-PG1 was largely due to the reduced number of insertion-competent beta-sheet molecules, rather than uniformly weakened activity of identically structured peptides.

Topics: Alanine; Amino Acid Sequence; Anti-Infective Agents; Antimicrobial Cationic Peptides; Carbon Radioisotopes; Cell Membrane; Cholesterol; Cysteine; Disulfides; Lipid Bilayers; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Conformation; Proteins

This paper compares six phospholipidic monolayers at the water/chloroform interface by performing dilational rheological measurements with a drop tensiometer apparatus. The chosen lipids differ both in their headgroup structure and fatty acyl chain saturation or symmetry. The study concentrated on monolayers formed with DPPC, DPPE, DOPC, DOPE, POPC and POPE. Using a generalized Maxwell rheological model, transposed at the interface, the intimate intermolecular interactions between amphiphilic molecules are studied on and off the monolayer plane. The equilibrium and nonequilibrium phenomena are analyzed and, respectively, correlated with monolayer cohesion and with monolayer/sub-surface interactions. The purpose of this work is to gain further insights into the influences (as slight as they are) of the weak changes in phospholipid structure and on the behavior of the monolayers. The results, widely described, provide further details on nuances existing between very similar molecules, and likewise, on the synergies created between the different effects.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Biochemistry; Chloroform; Fatty Acids; Membrane Lipids; Models, Chemical; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Rheology; Surface Properties

From equilibrium molecular dynamics simulations we have determined single-channel water permeabilities for Escherichia coli aquaporin Z (AqpZ) and aquaglyceroporin GlpF with the channels embedded in lipid bilayers. GlpF's osmotic water permeability constant pf exceeds by 2-3 times that of AqpZ and the diffusive permeability constant (pd) of GlpF is found to exceed that of AqpZ 2-9-fold. Achieving complete water selectivity in AqpZ consequently implies lower transport rates overall relative to the less selective, wider channel of GlpF. For AqpZ, the ratio pf/pd congruent with 12 is close to the average number of water molecules in the channel lumen, whereas for GlpF, pf/pd congruent with 4. This implies that single-file structure of the luminal water is more pronounced for AqpZ, the narrower channel of the two. Electrostatics profiles across the pore lumens reveal that AqpZ significantly reinforces water-channel interactions, and weaker water-water interactions in turn suppress water-water correlations relative to GlpF. Consequently, suppressed water-water correlations across the narrow selectivity filter become a key structural determinant for water permeation causing luminal water to permeate slower across AqpZ.

Topics: Aquaporins; Biophysics; Carbohydrate Metabolism; Computer Simulation; Crystallography, X-Ray; Databases, Protein; Diffusion; Escherichia coli; Escherichia coli Proteins; Histidine; Kinetics; Lipid Bilayers; Membrane Proteins; Models, Molecular; Models, Statistical; Models, Theoretical; Osmosis; Permeability; Phosphatidylcholines; Phosphatidylethanolamines; Protons; Static Electricity; Time Factors; Water

In this work, using atomic force microscopy (AFM), we have studied the influence of the temperature on the properties of the surface planar bilayers (SPBs) formed with: (i) the total lipid extract of Escherichia coli; (ii) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPC) (1:1, mol/mol); and, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol-amine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol). According to the height profile analysis we performed, the height of the SPBs of DMPC:POPC were temperature dependent. Separated domains were observed in the SPBs of the POPE:POPG mixture and the E. coli lipid extract. The implication of those domains for the correct insertion of membrane proteins into proteoliposomes is discussed.

Topics: Dimyristoylphosphatidylcholine; Escherichia coli; Lipid Bilayers; Liposomes; Membrane Proteins; Microscopy, Atomic Force; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Surface Properties; Thermodynamics

Cardiolipin (CL) is a phospholipid found in the energy-transducing membranes of bacteria and mitochondria and it is thought to be involved in relevant biological processes as apoptosis. In this work, the mixing properties of CL and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) at the air-water interface, have been examined using the thermodynamic framework analysis of compression isotherms. Accordingly, the values of the Gibbs energy of mixing, the more stable monolayers assayed were: POPC:CL (0.6:0.4, mol:mol) and POPE:CL (0.8:0.2, mol:mol). The results reflect that attractive forces are the greatest contributors to the total interaction in these compositions. Supported planar bilayers (SPBs) with such compositions were examined using atomic force microscopy (AFM) at different temperatures. With the POPC:CL mixture, rounded and featureless SPBs were obtained at 4 degrees C and 24 degrees C. In contrast, the extension of the POPE:CL mixture revealed the existence of different lipid domains at 24 degrees C and 37 degrees C. Three lipid domains coexisted which can be distinguished by measuring the step height difference between the uncovered mica and the bilayer. While the low and intermediate domains were temperature dependent, the high domain was composition dependent. When cytochrome c (cyt c) was injected into the fluid cell, the protein showed a preferential adsorption onto the high domain of the POPC:CL. These results suggest that the high domain is mainly formed by CL.

Topics: Cardiolipins; In Vitro Techniques; Intracellular Membranes; Lipid Bilayers; Membrane Lipids; Microscopy, Atomic Force; Mitochondria; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Thermodynamics

The relationship between the molecular organization of lipid headgroups and the activity of surface-acting enzyme was examined using a bacterial cholesterol oxidase (COD) as a model. The initial rate of cholesterol oxidation by COD in fluid state 1-palmitoyl-2-oleoyl-phosphatidylethanolamine/1-palmitoyl-2-oleoyl-phosphatidylcholine/cholesterol (POPE/POPC/CHOL) bilayers was measured as a function of POPE-to-phospholipid mole ratio (X(PE)) and cholesterol-to-lipid mole ratio (X(CHOL)) at 37 degrees C. At X(PE) = 0, the COD activity changed abruptly at X(CHOL) approximately 0.40, whereas major activity peaks were detected at X(PE) approximately 0.18, 0.32, 0.50, 0.64, and 0.73 when X(CHOL) was fixed to 0.33 or 0.40. At a fixed X(CHOL) of 0.50, the COD activity increased progressively with PE content and exhibited small peaks or kinks at X(PE) approximately 0.40, 0.50, 0.58, 0.69, and 0.81. When X(PE) and X(CHOL) were systematically varied within a narrow 2-D lipid composition window, an onset of COD activity at X(CHOL) approximately 0.40 and the elimination of the activity peak at X(PE) approximately 0.64 for X(CHOL) >0.40 were clearly observed. Except for X(PE) approximately 0.40 and 0.58, the observed critical PE mole ratios agree closely (+/-0.03) with those predicted by a headgroup superlattice model (Virtanen, J.A., et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4964-4969; Cannon, B., et al. (2006) J. Phys. Chem. B 110, 6339-6350), which proposes that lipids with headgroups of different sizes tend to adopt regular, superlattice-like distributions at discrete and predictable compositions in fluid lipid bilayers. Our results indicate that headgroup superlattice domains exist in lipid bilayers and that they may play a crucial role in modulating the activity of enzymes acting on the cell membrane surface.

Topics: Cholesterol; Cholesterol Oxidase; Computer Simulation; Lipid Bilayers; Models, Theoretical; Oxidation-Reduction; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids

Activation of phospholipase Cbeta (PLCbeta) by G-proteins results in increased intracellular Ca(2+) and activation of protein kinase C. We have previously found that activated PLCbeta-Gbetagamma complex can be rapidly deactivated by Galpha(GDP) subunits without dissociation, which led to the suggestion that Galpha(GDP) binds to PLCbeta-Gbeta gamma and perturbs the activating interaction without significantly affecting the PLCbeta-Gbeta gamma binding energy. Here, we have used high pressure fluorescence spectroscopy to determine the volume change associated with this interaction. Since PLCbeta and G-protein subunits associate on membrane surfaces, we worked under conditions where the membrane surface properties are not expected to change. We also determined the pressure range in which the proteins remain membrane bound: PLCbeta binding was stable throughout the 1-2000 bars range, Gbeta gamma binding was stable only at high membrane concentrations, whereas Galpha(s)(GDP) dissociated from membranes above 1 kbar. High pressure dissociated PLCbeta-Gbeta gamma with a DeltaV = 34 +/- 5 ml/mol. This same volume change is obtained for a peptide derived from Gbeta which also activates PLCbeta. In the presence of Galpha(s)(GDP), the volume change associated with PLCbeta-Gbeta gamma interaction is reduced to 25 +/- 1 ml/mol. These results suggest that activation of PLCbeta by Gbeta gamma is conferred by a small (i.e., 3-15 ml/mol) volume element.

Topics: Animals; Baculoviridae; Biophysics; Buffers; Calcium; Cell Line; Dose-Response Relationship, Drug; Fluorescence Resonance Energy Transfer; GTP-Binding Protein beta Subunits; GTP-Binding Protein gamma Subunits; Insecta; Isoenzymes; Lipid Bilayers; Lipids; Macromolecular Substances; Membranes; Models, Biological; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipase C beta; Pressure; Protein Binding; Protein Kinase C; Protein Structure, Tertiary; Proteins; Signal Transduction; Spectrometry, Fluorescence; Thermodynamics; Type C Phospholipases

Integral membrane proteins are characterized by having a preference for aromatic residues, e.g., tryptophan (W), at the interface between the lipid bilayer core and the aqueous phase. The reason for this is not clear, but it seems that the preference is related to a complex interplay between steric and electrostatic forces. The flat rigid paddle-like structure of tryptophan, associated with a quadrupolar moment (aromaticity) arising from the pi-electron cloud of the indole, interacts primarily with moieties in the lipid headgroup region hardly penetrating into the bilayer core. We have studied the interaction between the nitrogen moiety of lipid molecule headgroups and the pi-electron distribution of gramicidin (gA) tryptophan residues (W9, W11, W13, and W15) using molecular dynamics (MD) simulations of gA embedded in two hydrated lipid bilayers composed of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoylphosphatidyl-choline (POPC), respectively. We use a force field model for tryptophan in which polarizability is only implicit, but we believe that classical molecular dynamics force fields are sufficient to capture the most prominent features of the cation-pi interaction. Our criteria for cation-pi interactions are based on distance and angular requirements, and the results from our model suggest that cation-pi interactions are relevant for W(PE)1), W(PE)13, W(PE)15, and, to some extent, W(PC)11 and W(PC)13. In our model, W9 does not seem to engage in cation-pi interactions with lipids, neither in POPE nor POPC. The criteria for the cation-pi effect are satisfied more often in POPE than in POPC, whereas the H-bonding ability between the indole donor and the carbonyl acceptor is similar in POPE and POPC. This suggests an increased affinity for lipids with ethanolamine headgroups to transmembrane proteins enriched in interfacial tryptophans.

Topics: Amino Acids; Cations; Computer Simulation; Lipid Bilayers; Membrane Fluidity; Membrane Proteins; Models, Chemical; Models, Molecular; Phosphatidylcholines; Phosphatidylethanolamines; Protein Conformation; Tryptophan; Water

Sphingosine 1-phosphate is a bioactive sphingolipid that regulates cell growth and suppresses programmed cell death. The biosynthesis of sphingosine 1-phosphate is catalyzed by sphingosine kinase (SK) but the mechanism by which the subcellular localization and activity of SK is regulated in response to various stimuli is not fully understood. To elucidate the origin and structural determinant of the specific subcellular localization of SK, we performed biophysical and cell studies of human SK1 (hSK1) and selected mutants. In vitro measurements showed that hSK1 selectively bound phosphatidylserine over other anionic phospholipids and strongly preferred the plasma membrane-mimicking membrane to other cellular membrane mimetics. Mutational analysis indicates that conserved Thr54 and Asn89 in the putative membrane-binding surface are essential for lipid selectivity and membrane targeting both in vitro and in the cell. Also, phosphorylation of Ser225 enhances the membrane affinity and plasma membrane selectivity of hSK1, presumably by modulating the interaction of Thr54 and Asn89 with the membrane. Collectively, these studies suggest that the specific plasma membrane localization and activation of SK1 is mediated largely by specific lipid-protein interactions.

Topics: Amino Acid Sequence; Asparagine; Cell Line; Cell Membrane; DNA Mutational Analysis; Green Fluorescent Proteins; Humans; Kinetics; Lipids; Lysophospholipids; Mass Spectrometry; Microscopy, Confocal; Microscopy, Fluorescence; Models, Biological; Molecular Sequence Data; Mutation; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipids; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Pressure; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Serine; Sphingosine; Surface Plasmon Resonance; Threonine; Time Factors; Transfection

Molecular dynamics computer simulations of pentachlorophenol (PCP) in palmitoyl-oleoyl-phosphatidylethanolamine and palmitoyl-oleoyl-phosphatidylcholine lipid bilayers were carried out to investigate the distribution of PCP and the effects of PCP on the phospholipid bilayer structure. Starting from two extreme starting structures, including PCP molecules outside the lipid bilayer, the PCP distribution converges in simulations of up to 50 ns. PCP preferentially occupies the region between the carbonyl groups and the double bonds in the acyl chains of the lipid molecules in the bilayer. In the presence of PCP, the lipid chain order increases somewhat in both chains, and the average tilt angle of the lipid chains decreases. The increase in the lipid chain order in the presence of PCP was more pronounced in the palmitoyl-oleoyl-phosphatidylcholine bilayer compared to the palmitoyl-oleoyl-phosphatidylethanolamine bilayer. The number of trans conformations of lipid chain dihedrals does not change significantly. PCP aligns parallel to the alkyl chains of the lipid to optimize the packing in the dense ordered chain region of the bilayer. The hydroxyl group of PCP forms hydrogen bonds with both water and lipid oxygen atoms in the water/lipid interface region.

Topics: Computer Simulation; Diffusion; Lipid Bilayers; Macromolecular Substances; Membrane Fluidity; Membranes, Artificial; Models, Chemical; Models, Molecular; Molecular Conformation; Motion; Pentachlorophenol; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Surface Properties; Water

Previous studies have shown that human calcitonin (hCT) and its C-terminal fragment hCT(9-32) translocate in nasal epithelium. Moreover, hCT(9-32) was used as a carrier to internalize efficiently the green fluorescent protein, drugs, and plasmid DNA. To understand the mechanism of the membrane crossing process, we determined structural parameters of the carrier peptide hCT(9-32) in a membrane environment using solid-state NMR. For that purpose, we synthesized a multiply labeled hCT(9-32) peptide comprising four positions with fully (15)N- and (13)C-labeled amino acids. Multilamellar vesicle samples containing varying mixing ratios of hCT(9-32) and phospholipids found in the plasma membrane of nasal epithelium were prepared. The typical axially symmetric powder patterns of (31)P NMR spectra confirmed the presence of lamellar bilayers in our samples. The chemical shift anisotropy of the (31)P NMR spectra of the samples in the presence of hCT(9-32) is slightly reduced, revealing weak interaction of the peptide with the lipid headgroups. The peptide does not penetrate the lipid membrane as indicated by very similar (2)H NMR order parameters of the phospholipid fatty acid chains in the absence and presence of the carrier peptide. This membrane topology was confirmed by measurements of paramagnetic enhancement of relaxation rates. The conformation of hCT(9-32) was investigated by cross polarization magic angle spinning NMR methods. All peptide signals were resolved and fully assigned in two-dimensional proton-driven (13)C spin diffusion experiments. The isotropic chemical shifts of (13)CO, (13)Calpha, and (13)Cbeta provide information about the secondary structure of the carrier peptide. The conformation of hCT(9-32) was further corroborated by quantitative phi torsion angle measurements. Two monomeric structural models are consistent with the data: (i) a linear backbone conformation of hCT(9-32) and (ii) an antiparallel beta-sheet structure. These structures are maintained over a wide range of peptide:lipid mixing ratios. No direct indications for fibril formation of hCT(9-32) were found. Dipolar coupling measurements indicate rather high amplitudes of motion of the peptide.

Topics: Amino Acid Sequence; Calcitonin; Carbon Isotopes; Carrier Proteins; Humans; Membrane Lipids; Models, Molecular; Molecular Sequence Data; Nasal Mucosa; Nuclear Magnetic Resonance, Biomolecular; Peptide Fragments; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Phosphorus Isotopes; Protein Conformation; Protein Structure, Secondary; Thermodynamics

Site-directed spin labeling was used to determine the membrane orientation and insertion of the C2A domain from synaptotagmin I. A series of single cysteine mutants of the C2A domain of synaptotagmin I was prepared and labeled with a sulfhydryl specific spin label. Upon Ca2+ or membrane binding, the EPR line shapes of these mutants reveal dramatic decreases in label mobility within the Ca2+-binding loops. This loss in mobility is likely due in part to a reduction in local backbone fluctuations within the loop regions. Power saturation was then used to determine the position of each spin-labeled site along the bilayer normal, and these EPR distance constraints were used along with the high-resolution solution structure of C2A to generate a model for the orientation and position of the domain at the membrane interface. This model places the polypeptide backbone of both the first and third Ca2+-binding loops in contact with the membrane interface, with several labeled side chains lying within the bilayer interior. All three Ca2+-binding sites lie near a plane defined by the lipid phosphates. This model indicates that there is some desolvation of this domain upon binding and that hydrophobic as well as electrostatic interactions contribute to the binding of C2A. When compared to the C2 domain from cPLA2 (Frazier et al. (2002) Biochemistry 41, 6282), a similar orientation for the beta-sandwich region is found; however, the cPLA2 C2 domain is translocated 5-7 A deeper into the membrane hydrocarbon. This difference in depth is consistent with previous biophysical data and with the difference that long-range electrostatic interactions and desolvation are expected to make to the binding of these two C2 domains.

Topics: Binding Sites; Calcium; Calcium-Binding Proteins; Cysteine; Electron Spin Resonance Spectroscopy; Lipid Bilayers; Membrane Glycoproteins; Mutagenesis, Site-Directed; Nerve Tissue Proteins; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Spin Labels; Synaptotagmin I; Synaptotagmins; Thermodynamics

By means of the quartz crystal microbalance (QCM) technique, we investigated the interaction of porcine heterotetrametric annexin A2t with solid supported lipid membranes. Dissociation and rate constants of annexin A2t binding to various lipid mixtures were determined as a function of Ca2+ concentrations in solution. In contrast to what has been observed for annexin A1, the binding affinity and kinetics of annexin A2t binding are not influenced by cholesterol. In the experimental setup chosen, the annexin A2t binding is strictly Ca2+-dependent and only affected by the amount of phosphatidylserine (PS) in the membrane and the Ca2+ concentration in solution. By Ca2+-titration experiments at constant annexin A2t concentration, we investigated the reversibility of annexin A2t adsorption and desorption. Surprisingly, Ca2+-titration curves display a significant hysteresis. Protein desorption curves starting from annexin A2t bound to the membrane at 1 mM CaCl2 exhibit high cooperativity with half-maximum Ca2+ concentrations in the submicromolar range. However, protein adsorption curves starting from an EGTA-containing solution with soluble annexin A2t always show two inflection points upon addition of Ca2+ ions. These two inflection points may be indicative of two protein populations differently bound to the solid-supported membrane. The ratio of these two annexin A2t populations depends on the amount of PS molecules and cholesterol in the membrane as well as on the Ca2+ concentration. We propose a model discussing the results obtained in terms of two binding sites differing in their affinity due to lipid rearrangement.

Topics: Adsorption; Animals; Annexin A2; Brain; Cattle; Cholesterol; Crystallization; Lipid Bilayers; Liposomes; Membrane Lipids; Oscillometry; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipids; Protein Binding; Quartz; Swine

In this work, we present the first characterization of the cell lysing mechanism of MSI-78, an antimicrobial peptide. MSI-78 is an amphipathic alpha-helical peptide designed by Genaera Corporation as a synthetic analog to peptides from the magainin family. (31)P-NMR of mechanically aligned samples and differential scanning calorimetry (DSC) were used to study peptide-containing lipid bilayers. DSC showed that MSI-78 increased the fluid lamellar to inverted hexagonal phase transition temperature of 1,2-dipalmitoleoyl-phosphatidylethanolamine indicating the peptide induces positive curvature strain in lipid bilayers. (31)P-NMR of lipid bilayers composed of MSI-78 and 1-palmitoyl-2-oleoyl-phosphatidylethanolamine demonstrated that the peptide inhibited the fluid lamellar to inverted hexagonal phase transition of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine, supporting the DSC results, and the peptide did not induce the formation of nonlamellar phases, even at very high peptide concentrations (15 mol %). (31)P-NMR of samples containing 1-palmitoyl-2-oleoyl-phosphatidylcholine and MSI-78 revealed that MSI-78 induces significant changes in the bilayer structure, particularly at high peptide concentrations. At lower concentrations (1-5%), the peptide altered the morphology of the bilayer in a way consistent with the formation of a toroidal pore. Higher concentrations of peptide (10-15%) led to the formation of a mixture of normal hexagonal phase and lamellar phase lipids. This work shows that MSI-78 induces significant changes in lipid bilayers via positive curvature strain and presents a model consistent with both the observed spectral changes and previously published work.

Topics: Antimicrobial Cationic Peptides; Lipid Bilayers; Macromolecular Substances; Membrane Fluidity; Molecular Conformation; Motion; Permeability; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Porosity; Protein Precursors; Stress, Mechanical; Xenopus Proteins

The sarcoplasmic reticulum channel (ryanodine receptor) from cardiac myocytes was reconstituted into planar lipid bilayers consisting of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) in varying ratios. The channel activity parameters, i.e., open probability and average open time and its resolved short and long components, were determined as a function of POPE mole fraction (X(PE)) at 22.4 degrees C. Interestingly, all of these parameters exhibited a narrow and pronounced peak at X(PE) approximately 0.80. Differential scanning calorimetric measurements on POPE/POPC liposomes with increasing X(PE) indicated that the lipid bilayer enters a composition-driven transition from the liquid-crystalline state to the gel state at 22.4 degrees C when X(PE) approaches 0.80. Thus, the peaking of the reconstituted channel activity at X(PE) approximately 0.80 in the planar bilayer could result from the appearance of gel/liquid-crystalline domain boundaries at this POPE content. Lipid packing at domain boundaries is known to be looser as compared to the homogenous gel or liquid-crystalline state. We propose that the attractive potential of packing defects at lipid domain boundaries and entropic excluded-volume effects could result in the direct interactions of the transmembrane region of the channel protein with the lipid-packing defects at the lipid/protein interface, which could thus provide a favorable environment for the open state of the protein. The present findings indicate that the activity of the sarcoplasmic reticulum calcium channel could be modulated by lipid domain formation upon slight changes in membrane lipid composition in vivo.

Topics: Calcium Channels; Electric Capacitance; Ion Channel Gating; Lipid Bilayers; Membrane Fluidity; Membrane Microdomains; Membrane Potentials; Membranes, Artificial; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Protein Conformation; Ryanodine Receptor Calcium Release Channel; Structure-Activity Relationship

We demonstrate ordered orientation of the hydration water at the surface of phospholipid bilayers by use of coherent anti-Stokes Raman scattering (CARS) microscopy, a highly sensitive vibrational imaging method recently developed. We investigated negatively charged POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine) and neutral POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) multilamellar onions dispersed in deuterated dodecane. The imaging contrast based on the CARS signal from the H2O stretching vibration shows a clear dependence on the excitation field polarization. Our results provide direct experimental evidence that water molecules close to the phospholipid bilayer surface are ordered with the symmetry axis along the direction normal to the bilayer. Moreover, the amount of ordered water molecules depends on the lipid polar group. The spectral profile for the inter-lamellar water shows that the water molecules bound to the bilayer surface are less hydrogen-bonded and exhibit a higher vibrational frequency than bulk water.

Topics: In Vitro Techniques; Lipid Bilayers; Microscopy; Models, Molecular; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Spectrum Analysis, Raman; Water

Previous experimental work has shown that the functional properties of the mechanosensitive channel of large conductance (MscL) are affected by variations in lipid composition. Here, we utilize molecular dynamics simulations of Mycobacterium tuberculosis MscL to investigate such lipid composition effects on a molecular level. In particular, two sets of simulations were performed. In the first, trajectories using lipids with different headgroups (phosphatidylcholine and phosphatidylethanolamine) were compared. Protein-lipid interactions were clearly altered by the headgroup changes, leading to conformational differences in the C-terminal region of M. tuberculosis MscL. In the second set of simulations, lipid tails were gradually shortened, thinning the membrane over a molecular dynamics trajectory. These simulations showed evidence of hydrophobic matching between MscL and the lipid membrane, as previously proposed. For all simulations, protein-lipid interaction energies in the second transmembrane region were correlated to mutagenic data, emphasizing the importance of lipid interactions for proper MscL function.

Topics: Biophysical Phenomena; Biophysics; Computer Simulation; Crystallography, X-Ray; Escherichia coli; Escherichia coli Proteins; Ion Channels; Lipid Bilayers; Lipids; Models, Biological; Models, Molecular; Molecular Conformation; Mutagenesis; Mycobacterium tuberculosis; Phosphatidylcholines; Phosphatidylethanolamines; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Proteins; Time Factors

To understand the molecular basis and to prevent diseases such as Alzheimer's disease (AD), the targets of the triggering agent have to be elucidated. beta-Amyloid peptide (Abeta) is the major component of extracellular senile plaques characteristic of AD. For a very long time, the aggregated form of the Abeta was supposed to be responsible for the neurodegeneration that occurs in AD. Recently, the attention has been diverted to the monomeric or oligomeric forms of Abeta and their interaction with cellular targets. In our investigation, the physiological and medically important insertion of externally applied Abeta monomers into the bilayer of lipid vesicles is demonstrated. Abeta(25-35) has been localized in the region of the lipid alkyl chain, and it has a severe disordering effect on the lamellar order of the lipid bilayer. Both of these results are of biomedical relevance.

Topics: Amino Acid Sequence; Amyloid beta-Peptides; Deuterium; Fourier Analysis; Leucine; Lipid Bilayers; Models, Molecular; Neutron Diffraction; Peptide Fragments; Phosphatidylcholines; Phosphatidylethanolamines; Scattering, Radiation

Pardaxin is a membrane-lysing peptide originally isolated from the fish Pardachirus marmoratus. The effect of the carboxy-amide of pardaxin (P1a) on bilayers of varying composition was studied using (15)N and (31)P solid-state NMR of mechanically aligned samples and differential scanning calorimetry (DSC). (15)N NMR spectroscopy of [(15)N-Leu(19)]P1a found that the orientation of the peptide's C-terminal helix depends on membrane composition. It is located on the surface of lipid bilayers composed of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and is inserted in lipid bilayers composed of 1,2-dimyristoyl-phosphatidylcholine (DMPC). The former suggests a carpet mechanism for bilayer disruption whereas the latter is consistent with a barrel-stave mechanism. The (31)P chemical shift NMR spectra showed that the peptide significantly disrupts lipid bilayers composed solely of zwitterionic lipids, particularly bilayers composed of POPC, in agreement with a carpet mechanism. P1a caused the formation of an isotropic phase in 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) lipid bilayers. This, combined with DSC data that found P1a reduced the fluid lamellar-to-inverted hexagonal phase transition temperature at very low concentrations (1:50,000), is interpreted as the formation of a cubic phase and not micellization of the membrane. Experiments exploring the effect of P1a on lipid bilayers composed of 4:1 POPC:cholesterol, 4:1 POPE:cholesterol, 3:1 POPC:1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), and 3:1 POPE:POPG were also conducted, and the presence of anionic lipids or cholesterol was found to reduce the peptide's ability to disrupt bilayers. Considered together, these data demonstrate that the mechanism of P1a is dependent on membrane composition.

Topics: Animals; Calorimetry, Differential Scanning; Cell Membrane; Cholesterol; Fish Venoms; Fishes; Lipid Bilayers; Magnetic Resonance Spectroscopy; Membranes; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Temperature

The C2 domain is a ubiquitous Ca(2+)-binding motif that triggers the membrane docking of many key signaling proteins during intracellular Ca(2+) signals. Site-directed spin labeling was carried out on the C2 domain of cytosolic phospholipase A(2) in order to determine the depth of penetration and orientation of the domain at the membrane interface. Membrane depth parameters, Phi, were obtained by EPR spectroscopy for a series of selectively spin-labeled C2 domain cysteine mutants, and for spin-labeled lipids and spin-labeled bacteriorhodopsin cysteine mutants. Values of Phi were combined with several other constraints, including the solution NMR structure, to generate a model for the position of the C2 domain at the membrane interface. This modeling yielded an empirical expression for Phi, which for the first time defines its behavior from the bulk aqueous phase to the center of the lipid bilayer. In this model, the backbones of both the first and third Ca(2+)-binding loops are inserted approximately 10 A into the bilayer, with residues inserted as deep as 15 A. The backbone of the second Ca(2+)-binding loop is positioned near the lipid phosphate, and the two beta-sheets of the C2 domain are oriented so that the individual strands make angles of 30-45 degrees with respect to the bilayer surface. Upon membrane docking, spin labels in the Ca(2+)-binding loops exhibit decreases in local motion, suggesting either changes in tertiary contacts due to protein conformational changes and/or interactions with lipid.

Topics: Calcium-Binding Proteins; Cell Membrane; Cytosol; Electron Spin Resonance Spectroscopy; Lipid Bilayers; Models, Chemical; Models, Molecular; Mutagenesis, Site-Directed; Peptide Fragments; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipases A; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Spin Labels; Thermodynamics

The co-release of ATP with norepinephrine from sympathetic nerve terminals in the heart may augment adrenergic stimulation of cardiac Ca(2+) channel activity. To test for a possible direct effect of extracellular ATP on L-type Ca(2+) channels, single channels were reconstituted from porcine sarcolemma into planar lipid bilayers so that intracellular signaling pathways could be controlled. Extracellular ATP (2-100 microM) increased the open probability of the reconstituted channels, with a maximal increase of approximately 2.6-fold and an EC(50) of 3.9 microM. The increase in open probability was due to an increase in channel availability and a decrease in channel inactivation rate. Other nucleotides displayed a rank order of effectiveness of ATP > alpha,beta-methylene-ATP > 2-methylthio-ATP > UTP > adenosine 5'-O-(3-thiotriphosphate) >> ADP; adenosine had no effect. Several antagonists of P2 receptors had no impact on the ATP-dependent increase in open probability, indicating that receptor activation was not required. These results suggest that extracellular ATP and other nucleotides can stimulate the activity of cardiac L-type Ca(2+) channels via a direct interaction with the channels.

Topics: Adenosine; Adenosine Triphosphate; Animals; Calcium Channels, L-Type; Extracellular Space; Heart; Heart Ventricles; Ion Channel Gating; Lipid Bilayers; Patch-Clamp Techniques; Phosphatidylcholines; Phosphatidylethanolamines; Sarcolemma; Swine; Uridine; Uridine Triphosphate

Although its function is unknown, alpha-synuclein is widely distributed in neural tissue and is the major component in the pathological aggregates found in patients with Parkinson's disease, Alzheimer's disease, Down's syndrome, and multiple system atrophy. In this report, we have quantified the binding alpha-synucleins to lipid membranes. In contrast to previous studies, we find, using real time equilibrium fluorescence methods, that alpha-synuclein binds strongly to large, unilamellar vesicles with either anionic or zwitterionic headgroups. Membrane binding is also strong for beta-synuclein, phosphorylated alpha-synuclein, and a synuclein mutant that is associated with familial Parkinson's disease. In solution at less than 400 nM, synuclein has a tendency to undergo concentration-dependent oligomerization as determined by changes in intrinsic fluorescence and fluorescence resonance energy transfer. Above this concentration, the protein begins to aggregate into structures visible by light scattering. Although membrane binding does not affect the secondary structure of alpha-synuclein, it greatly inhibits the ability of this protein to self-associate. Taken together, our results indicate that pathological conditions may be associated with a disruption in synuclein-membrane interactions.

Topics: alpha-Synuclein; beta-Synuclein; Blotting, Western; Cell Membrane; Circular Dichroism; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Humans; Hydrogen-Ion Concentration; Lipids; Nerve Tissue Proteins; Neurodegenerative Diseases; Phosphatidylcholines; Phosphatidylethanolamines; Phosphorylation; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Silver Staining; Spectrometry, Fluorescence; Synucleins

The activity of membrane-associated protein kinase C (PKC) is tightly controlled by the physical properties of the membrane lipid bilayer, in particular, curvature stress, which is induced by bilayer-destabilizing lipid components. An important example of this is the weakened lipid headgroup interactions induced by phosphatidylethanolamine (PE) and cholesterol. In this work our previous observation with a mixed isoform PKC showing a biphasic dependence of activity as a function of membrane curvature stress [Slater et al. (1994) J. Biol. Chem. 269, 4866-4871] was here extended to individual isoforms. The Ca(2+)-dependent PKCalpha, PKCbeta, and PKCgamma, along with Ca(2+)-independent PKCdelta, but not PKCepsilon or PKCzeta, displayed a biphasic activity as a function of membrane PE content. The fluorescence anisotropy of N-(5-dimethylaminonaphthalene-1-sulfonyl)dioleoylphosphatidylserine (dansyl-PS), which probes the lipid environment of PKC, also followed a biphasic profile as a function of PE content for full-length PKCalpha, PKCbetaIotaIota, and PKCgamma as did the isolated C1 domain of PKCalpha. In addition, the rotational correlation time of both PKCalpha and PKCdelta C1-domain-associated sapintoxin D, a fluorescent phorbol ester, was also a biphasic function of membrane lipid PE content. These results indicate that the C1 domain acts as a sensor of the bilayer surface properties and that its conformational response to these effects may directly underlie the resultant effects on enzyme activity.

Topics: Animals; Brain; Fluorescence Polarization; Isoenzymes; Kinetics; Lipid Bilayers; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Protein Kinase C; Protein Kinase C beta; Protein Kinase C-alpha; Protein Kinase C-delta; Rats; Recombinant Proteins

The association of two species to form a bound complex, e.g., the binding of a ligand to a protein or the adsorption of a peptide on a lipid membrane, involves an entropy loss, reflecting the conversion of free translational and rotational degrees of freedom into bound motions. Previous theoretical estimates of the standard entropy change in bimolecular binding processes, DeltaS(o), have been derived from the root-mean-square fluctuations in protein crystals, suggesting DeltaS(o) approximately -50 e.u., i.e., TDeltaS degrees approximately -25 kT = -15 kcal/mol. In this work we focus on adsorption, rather than binding processes. We first present a simple statistical-thermodynamic scheme for calculating the adsorption entropy, including its resolution into translational and rotational contributions, using the known distance-orientation dependent binding (adsorption) potential. We then utilize this scheme to calculate the free energy of interaction and entropy of pentalysine adsorption onto a lipid membrane, obtaining TDeltaS(o) approximately -1.7 kT approximately -1.3 kcal/mol. Most of this entropy change is due to the conversion of one free translation into a bound motion, the rest arising from the confinement of two rotational degrees of freedom. The smaller entropy loss in adsorption compared to binding processes arises partly because a smaller number of degrees of freedom become restricted, but mainly due to the fact that the binding potential is much "softer."

Topics: Adsorption; Entropy; Ligands; Lipid Bilayers; Oligopeptides; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Proteins

We investigated the effects of clinically relevant ethanol concentrations (5-20 mM) on the single-channel kinetics of bovine aortic smooth muscle maxi-K channels reconstituted in lipid bilayers (1:1 palmitoyl-oleoyl-phosphatidylethanolamine: palmitoyl-oleoyl-phosphatidylcholine). Ethanol at 10 and 20 mM decreased the channel open probability (P(o)) by 75 +/- 20.3% mainly by increasing the mean closed time (+82 to +960%, n = 7). In some instances, ethanol also decreased the mean open time (-40.8 +/- 22. 5%). The P(o)-voltage relation in the presence of 20 mM ethanol exhibited a rightward shift in the midpoint of voltage activation (DeltaV(1/2) congruent with 17 mV), a slightly steeper relationship (change in slope factor, Deltak, congruent with -2.5 mV), and a decreased maximum P(o) (from approximately 0.82 to approximately 0. 47). Interestingly, channels inhibited by ethanol at low Ca(2+) concentrations (2.5 microM) were very resistant to ethanol in the presence of increased Ca(2+) (>/= 20 microM). Alcohol consumption in clinically relevant amounts may alter the contribution of maxi-K channels to the regulation of arterial tone.

Topics: Animals; Calcium; Cattle; Central Nervous System Depressants; Dose-Response Relationship, Drug; Ethanol; In Vitro Techniques; Ion Channel Gating; Ion Transport; Large-Conductance Calcium-Activated Potassium Channels; Lipid Bilayers; Membrane Potentials; Muscle, Smooth, Vascular; Patch-Clamp Techniques; Permeability; Phosphatidylcholines; Phosphatidylethanolamines; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Calcium-Activated

PMP1 is a 38-residue plasma membrane protein of the yeast Saccharomyces cerevisiae that regulates the activity of the H(+)-ATPase. The cytoplasmic domain conformation results in a specific interfacial distribution of five basic side chains, thought to strongly interact with anionic phospholipids. We have used the PMP1 18-38 fragment to carry out a deuterium nuclear magnetic resonance ((2)H-NMR) study for investigating the interactions between the PMP1 cytoplasmic domain and phosphatidylserines. For this purpose, mixed bilayers of 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphoserine (POPS) were used as model membranes (POPC/POPS 5:1, m/m). Spectra of headgroup- and chain-deuterated POPC and POPS phospholipids, POPC-d4, POPC-d31, POPS-d3, and POPS-d31, were recorded at different temperatures and for various concentrations of the PMP1 fragment. Data obtained from POPS deuterons revealed the formation of specific peptide-POPS complexes giving rise to a slow exchange between free and bound PS lipids, scarcely observed in solid-state NMR studies of lipid-peptide/protein interactions. The stoichiometry of the complex (8 POPS per peptide) was determined and its significance is discussed. The data obtained with headgroup-deuterated POPC were rationalized with a model that integrates the electrostatic perturbation induced by the cationic peptide on the negatively charged membrane interface, and a "spacer" effect due to the intercalation of POPS/PMP1f complexes between choline headgroups.

Topics: Amino Acid Sequence; Biophysical Phenomena; Biophysics; Deuterium; Fungal Proteins; Lipid Bilayers; Macromolecular Substances; Magnetic Resonance Spectroscopy; Membrane Proteins; Models, Chemical; Molecular Sequence Data; Nerve Tissue Proteins; Peptide Fragments; Phosphatidylcholines; Phosphatidylethanolamines; Protein Binding; Proteolipids; Proton-Translocating ATPases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Static Electricity

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

alpha-Synuclein is a highly conserved presynaptic protein of unknown function. A mutation in the protein has been causally linked to Parkinson's disease in humans, and the normal protein is an abundant component of the intraneuronal inclusions (Lewy bodies) characteristic of the disease. alpha-Synuclein is also the precursor to an intrinsic component of extracellular plaques in Alzheimer's disease. The alpha-synuclein sequence is largely composed of degenerate 11-residue repeats reminiscent of the amphipathic alpha-helical domains of the exchangeable apolipoproteins. We hypothesized that alpha-synuclein should associate with phospholipid bilayers and that this lipid association should stabilize an alpha-helical secondary structure in the protein. We report that alpha-synuclein binds to small unilamellar phospholipid vesicles containing acidic phospholipids, but not to vesicles with a net neutral charge. We further show that the protein associates preferentially with vesicles of smaller diameter (20-25 nm) as opposed to larger (approximately 125 nm) vesicles. Lipid binding is accompanied by an increase in alpha-helicity from 3% to approximately 80%. These observations are consistent with a role in vesicle function at the presynaptic terminal.

Topics: alpha-Synuclein; Amino Acid Sequence; Animals; Binding Sites; Canaries; Circular Dichroism; Humans; Liposomes; Molecular Sequence Data; Nerve Tissue Proteins; Osmolar Concentration; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylinositols; Protein Structure, Secondary; Sequence Alignment; Sequence Homology, Amino Acid; Structure-Activity Relationship; Synucleins

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

The phase behavior of mixtures of 1-palmitoyl-2-oleoyl-sn-glycerol (1,2-POG) with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) was studied by using DSC, small-angle X-ray diffraction and 31P-NMR. The results have been used to construct phase diagrams for both type of mixtures, in the 0-45 degreesC range. It is concluded that 1, 2-POG form complexes in the gel phases with both POPC and POPS. In the case of POPC, two complexes are postulated, the first one at a 1, 2-POG/POPC molar ratio of 40:60, and the second one at 70:30, defining three different regions in the phase diagram. Two eutectic points are proposed to occur: one at a very low 1,2-POG concentration and the other at a 1,2-POG concentration slightly lower than 70%. In the case of the 1,2-POG/POPS mixtures, the pattern was similar, but the first complex was seen to happen at a higher concentration, about 50 mol% of 1,2-POG, whereas the second was found at 80 mol% of 1,2-POG. This indicated a bigger presence of 1,2-POG in the complexes with POPS than with POPC. In the first region of the phase diagram, i.e. at concentrations of 1,2-POG lower than that required for the formation of the first complex, and at temperatures above the phase transition, lamellar phases were seen in all the cases. In region 2 of the phase diagram, i.e. at concentrations where the first and the second complexes coexist, a mixture of lamellar and non-lamellar phases was observed. Finally, at high concentrations of 1,2-POG, non-lamellar phases were detected as predominant, these phases being of an isotropic nature, according to 31P-NMR. An important conclusion of this study is that, using unsaturated lipids, similar to those found in biological membranes, it has been shown that diacylglycerols are found separated in domains, and that this process starts at very low concentrations of diacylglycerols. The formation of separated domains enriched in diacylglycerol is biologically relevant as it will allow them to have important effects on the membrane structure besides the fact that their concentration in the biomembrane is relatively low.

Topics: Calorimetry, Differential Scanning; Diglycerides; Magnetic Resonance Spectroscopy; Phosphatidylcholines; Phosphatidylethanolamines; X-Ray Diffraction

Dye leakage experiments were undertaken to investigate the membrane disruption properties of cholesterol oxidase. Inspection of the X-ray crystal structures of cholesterol oxidase suggested that an active-site "lid" opens in order to bind substrate [Li, J., Vrielink, A., Brick, P., & Blow, D. M. (1993) Biochemistry 32, 11507-11515]. We tested whether the interaction of the putative active-site lid with the membrane was sufficiently disruptive of the membrane structure to cause leakage or lysis of the cell membrane. Vesicles (100 nm) composed of egg phosphatidylcholine, 2-palmitoyl-3-oleoyl-1-sn-phosphatidylethanolamine, and 2-palmitoyl-3-oleoyl-1-sn-phosphatidylcholine were used in this study to mimic biomembranes. To separate the effects of membrane binding from conversion of cholesterol to cholest-4-en-3-one, the active-site mutant E361Q was utilized. In the reaction catalyzed by E361Q, isomerization of the cholest-5-en-3-one intermediate is suppressed and cholest-5-en-3-one is the major product isolated. Furthermore, E361Q produces cholest-5-en-3-one 20-fold more slowly than wild type produces cholest-4-en-3-one from cholesterol. Wild-type and E361Q cholesterol oxidases bind to vesicles with an apparent K(D) of approximately 25 microM, as measured by quenching of intrinsic tryptophan fluorescence, irrespective of headgroup size and cholesterol content. Membrane disruption was measured by leakage of the encapsulated marker carboxyfluorescein. Leakage was observed with cholesterol-containing vesicles and wild-type enzyme only; the rate of leakage was dependent on the rate of cholest-4-en-3-one production. E361Q did not induce membrane disruption, regardless of vesicle type tested. Thus, binding of cholesterol oxidase to the membrane and partitioning of cholesterol into the active site does not sufficiently perturb the bilayer to cause leakage of vesicle contents. Formation of the product cholest-4-en-3-one, however, does increase membrane permeability. Expansion of the lipid bilayer upon conversion of cholesterol to cholest-4-en-3-one is the likely cause of this increased permeability.

Topics: Animals; Binding Sites; Cholesterol Oxidase; Coleoptera; Lipid Bilayers; Mutation; Permeability; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylethanolamines; Protein Binding

Recently, evidence for cholesterol and phosphatidylcholine (PC) molecules to adapt superlattice arrangements in fluid lipid bilayers has been presented. Whether superlattice arrangements exist in other biologically relevant lipid membranes, such as phosphatidylethanolamine (PE)/PC, is still speculative. In this study, we have examined the physical properties of fluid 1-palmitoyl-2-oleoyl-PC (POPC) and 1-palmitoyl-2-oleoyl-PE (POPE) binary mixtures as a function of the POPE mole fraction (X(PE)) using fluorescence and Fourier transform infrared spectroscopy. At 30 degrees C, i.e., above the Tm of POPE and POPC, deviations, or dips, as well as local data scattering in the excimer-to-monomer fluorescence intensity ratio of intramolecular excimer forming dipyrenylphosphatidylcholine probe in POPE/POPC mixtures were detected at X(PE) approximately 0.04, 0.11, 0.16, 0.26, 0.33, 0.51, 0.66, 0.75, 0.82, 0.91, and 0.94. The above critical values of X(PE) coincide (within +/-0.03) with the critical mole fractions X(HX,PE) or X(R,PE) predicted by a headgroup superlattice model, which assumes that the lipid headgroups form hexagonal or rectangular superlattice, respectively, in the bilayer. Other spectroscopic data, generalized polarization of Laurdan and infrared carbonyl and phosphate stretching frequency, were also collected. Similar agreements between some of the observed critical values of X(PE) from these data and the X(HX,PE) or X(R,PE) values were also found. However, all techniques yielded critical values of X(PE) (e.g., 0.42 and 0.58) that cannot be explained by the present headgroup superlattice model. The effective cross-sectional area of the PE headgroup is smaller than that of the acyl chains. Hence, the relief of "packing frustration" of PE in the presence of PC (larger headgroup than PE) may be one of the major mechanisms in driving the PE and PC components to superlattice-like lateral distributions in the bilayer. We propose that headgroup superlattices may play a significant role in the regulation of membrane lipid compositions in cells.

Topics: Biophysical Phenomena; Biophysics; In Vitro Techniques; Lipid Bilayers; Macromolecular Substances; Models, Molecular; Phosphatidylcholines; Phosphatidylethanolamines; Spectrometry, Fluorescence; Spectroscopy, Fourier Transform Infrared

Biphalin, (Tyr-D-Ala-Gly-Phe-NH)2, is a highly potent dimeric analog of enkephalin. Its analgesic efficacy is due in part to its ability to permeate the blood-brain barrier. To aid in understanding the mechanism of the transmembrane movement we determined and analyzed the permeability and partition coefficients of biphalin and a series of analogues where F, Cl, I, NO2, or NH2 were placed in the para position of the aromatic rings of Phe4,4'. Liposomes composed of neutral phospholipids and cholesterol were used as the model membrane. The overall good correlation between permeability and water-membrane partition coefficients suggests that the movement of biphalins across the model membrane is controlled by diffusion and depends on the water-membrane partition coefficient. To explain the observed correlation between permeability and the electron withdrawing/donating character of the substituents in the phenylalanine ring, we examined various folding patterns of Leu-enkephalin, an endogenous pentapeptide that exhibits affinities toward the same classes of opioid receptors (delta and mu). The observed permeabilities and partition coefficients of biphalin and analogues, as well as the tyrosine side chain accessibility, are consistent with the presence of the type of folding where the tyrosine and phenylalanine side chains are in a close contact. We propose that the aromatic ring interaction can promote the peptide permeability by stabilizing a more compact structure of biphalin that would minimize the number of hydrogen bonds with water and therefore enhances partitioning into the model membrane.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Amino Acid Sequence; Analgesics; Blood-Brain Barrier; Calorimetry, Differential Scanning; Dimerization; Enkephalin, Leucine; Enkephalins; Hydrogen-Ion Concentration; Kinetics; Liposomes; Models, Biological; Permeability; Phosphatidylcholines; Phosphatidylethanolamines; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet

The 1-palmitoyl-2-oleoyl-phosphatidylethanolamine: 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE:POPC) system has been investigated by measuring, in the inverted hexagonal (HII) phase, the intercylinder spacings (using x-ray diffraction) and orientational order of the acyl chains (using 2H nuclear magnetic resonance). The presence of 20 wt% dodecane leads to the formation of a HII phase for the composition range from 0 to 39 mol% of POPC in POPE, as ascertained by x-ray diffraction and 2H nuclear magnetic resonance. The addition of the alkane induces a small decrease in chain order, consistent with less stretched chains. An increase in temperature or in POPE proportion leads to a reduction in the intercylinder spacing, primarily due to a decrease in the water core radius. A temperature increase also leads to a reduction in the orientational order of the lipid acyl chains, whereas the POPE proportion has little effect on chain order. A correlation is proposed to relate the radius of curvature of the cylinders in the inverted hexagonal phase to the chain order of the lipids adopting the HII phase. A simple geometrical model is proposed, taking into account the area occupied by the polar headgroup at the interface and the orientational order of the acyl chains reflecting the contribution of the apolar core. From these parameters, intercylinder spacings are calculated that agree well with the values determined experimentally by x-ray diffraction, for the variations of both temperature and POPE:POPC proportion. This model suggests that temperature increases the curvature of lipid layers, mainly by increasing the area subtended by the hydrophobic core through chain conformation disorder, whereas POPC content affects primarily the headgroup interface contribution. The frustration of lipid layer curvature is also shown to be reflected in the acyl chain order measured in the L alpha phase, in the absence of dodecane; for a given temperature, increased order is observed when the curling tendencies of the lipid plane are more pronounced.

Topics: Biophysical Phenomena; Biophysics; Deuterium; In Vitro Techniques; Magnetic Resonance Spectroscopy; Membrane Lipids; Membranes, Artificial; Molecular Structure; Phosphatidylcholines; Phosphatidylethanolamines; Temperature; Thermodynamics

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

The hypothesis that protein kinase C (PKC) activity is sensitive to phospholipid head group interactions was tested using lipid bilayers of defined composition with PKC purified from rat brain. The head group interactions were modulated by varying phosphatidylcholine cis-unsaturation, vesicle curvature, and by the addition of phosphatidylethanolamine and cholesterol. With unilamellar vesicles (including 20 mol% brain phosphatidylserine), increased phosphatidylcholine unsaturation potentiated basal and phorbol ester stimulated PKC activity. By contrast, in the presence of phosphatidylethanolamine, the activity decreased with increasing phosphatidylcholine unsaturation. Weakening phospholipid head group interactions spaces the head group region and increases interstitial water, and this effect was assessed from its effect on the fluorescence intensity of the phospholipid-labeled fluorophore 1-palmitoyl-2-N-(4-nitrobenzo-2-oxa-1,3-diazole)aminohexanoylphosphat idylcholin e (C6-NBD-PC). When the PKC activities with vesicles of varying phosphatidylcholine unsaturation, with and without phosphatidylethanolamine, were plotted as a function of the fluorescence intensity of C6-NBD-PC-labeled vesicles, a biphasic profile was obtained, which had an optimum value of intensity, relating to head group spacing, that corresponded to a maximal enzyme activity. A similar biphasic curve was also found when PKC activities were plotted as a function of published bilayer intrinsic curvature x-ray diffraction data, a parameter closely related to head group spacing. By contrast, no simple relationship was evident between PKC activity and 1,6-diphenyl-1,3,5-hexatriene anisotropy, taken as a measure of lipid order or fluidity. Therefore, increasing the level of phosphatidylcholine unsaturation, phosphatidylethanolamine, or cholesterol either potentiates or attenuates PKC activity, dependent on whether the initial condition is above or below its optimum.

Topics: Animals; Brain; Fluorescent Dyes; Kinetics; Lipid Bilayers; Membrane Fluidity; Micelles; Myelin Basic Protein; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipids; Phosphorylation; Protein Kinase C; Rats; Spectrometry, Fluorescence; Structure-Activity Relationship; X-Ray Diffraction

X-ray diffraction studies on oriented multilayers of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) in the lamellar gel (L beta) and inverted hexagonal (HII) phases at various temperatures (5-50 degrees C) and relative humidities (0-100%) are reported. One-dimensional electron density profiles of the L beta phase bilayers were constructed to a resolution of better than 4 A using direct methods to solve for the phase problem. In addition, the electron density profiles were fitted favorably using a model in which the atomic groups were assumed to be Gaussian distributed [Wiener, M. C., & White, S. H. (1992) Biophys. J. 61, 434-447]. The X-ray data clearly demonstrate that, at 100% relative humidity (RH), POPE samples exist in two distinct L beta phases, differing primarily in the amount of water between the lamellae. As the hexagonal phase transition temperature is approached, 100% RH POPE samples partially dehydrate, releasing approximately 5 water molecules per phospholipid and experiencing on average a 3-A decrease in repeat spacing. The lower temperature hydrated L beta phase POPE electron density distribution resembles that obtained from the L beta phase 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayers and is unlike the partially dehydrated POPE bilayers.

Topics: Desiccation; Humidity; Lipid Bilayers; Mathematics; Models, Theoretical; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; X-Ray Diffraction

The reduction in spectral splitting, or motional narrowing, of the deuterium spectra of D2O/phospholipid mixtures near the main chain melting phase transition was studied for palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and equimolar mixtures of the two at 10% hydration. For POPC the splitting was about 1700 Hz in both the fluid and gel phases, dropping to zero near the phase transition (as reported previously). For POPE the splitting remained approximately constant above the phase transition. Below the phase transition the spectrum showed a single broad line whose linewidth varied between 100 Hz and 800 Hz. This was interpreted as being due to small domains of water within a weakly hydrated crystal. POPC:POPE (1:1) samples exhibited motional narrowing behaviour similar to that for POPC except that the splitting above the phase transition was approximately twice that below the transition. The relatively broad temperature range (approximately 20 K) of the transition is explained using a simple physical model involving lipid fluctuations near the phase transition.

Topics: Deuterium; Deuterium Oxide; Magnetic Resonance Spectroscopy; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Thermodynamics; Water

A voltage-dependent cationic channel of large conductance is observed in phospholipid bilayers formed by the tip-dip method from proteoliposomes derived from mitochondrial membranes. It is blocked by peptide M, a 13 residue peptide having the properties of a mitochondrial signal sequence. To verify the reliability of the experimental approach, mitochondrial membranes from bovine adrenal cortex or porin-deficient mutant yeast were either fused to planar bilayers or incorporated in giant liposomes which were studied by patch clamp. Cationic channels were found with both techniques. They had the same conductance levels and voltage-dependence as those which have been described using the tip-dip method. Moreover, they were similarly blocked by peptide M. The voltage-dependence of block duration was analyzed in planar bilayer and tip-dip records. Results strengthen the idea that peptide M might cross the channel. Other mitochondrial channels were observed in planar bilayers and patch clamp of giant liposomes. Because they were never detected in tip-dip records, they are likely to be inactivated at the surface monolayer used to form the bilayer in this type of experiment.

Topics: Adrenal Cortex; Animals; Cattle; Electric Conductivity; Ion Channels; Lipid Bilayers; Liposomes; Membrane Potentials; Mitochondria; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Proteolipids; Saccharomyces cerevisiae; Trypsin

Motivated by the results of Neyton and Miller (1988. J. Gen. Physiol. 92:549-586), suggesting that the Ca(2+)-activated K+ channel has four high affinity ion binding sites, we propose a physically attractive variant of the single-vacancy conduction mechanism for this channel. Simple analytical expressions for conductance, current, flux ratio exponent, and reversal potential under bi-ionic conditions are found. A set of conductance data are analyzed to determine a realistic range of parameter values. Using these, we find qualitative agreement with a variety of experimental results previously reported in the literature. The exquisite selectivity of the Ca(2+)-activated K+ channel may be explained as a consequence of the concerted motion of the "stack" in the proposed mechanism.

Topics: Animals; Calcium; Electric Conductivity; Kinetics; Lipid Bilayers; Mathematics; Membrane Potentials; Models, Biological; Muscles; Phosphatidylcholines; Phosphatidylethanolamines; Potassium Channels; Rats

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