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

We review the capabilities of Atomic Force Microscopy (AFM) in the study of phase transitions in Supported Lipid Bilayers (SLBs). AFM represents a powerful technique to cover the resolution range not available to fluorescence imaging techniques and where spectroscopic data suggest what the relevant lateral scale for domain formation might be. Phase transitions of lipid bilayers involve the formation of domains characterized by different heights with respect to the surrounding phase and are therefore easily identified by AFM in liquid solution once the bilayer is confined to a flat surface. Even if not endowed with high time resolution, AFM allows light to be shed on some aspects related to lipid phase transitions in the case of both a single lipid component and lipid mixtures containing sterols also. We discuss here the obtained results in light of the peculiarities of supported lipid bilayer model systems.

Topics: Diffusion; Hydrogen-Ion Concentration; Ions; Kinetics; Lipid Bilayers; Lipids; Materials Testing; Microscopy, Atomic Force; Phase Transition; Phosphatidylethanolamines; Phosphatidylglycerols; Phospholipids; Static Electricity; Temperature; Thermodynamics

Lipid membrane interfaces host reactions essential for the functioning of cells. The hydrogen-bonding environment at the membrane interface is particularly important for binding of proteins, drug molecules, and ions. We present here the implementation and applications of a depth-first search algorithm that analyzes dynamic lipid interaction networks. Lipid hydrogen-bond networks sampled transiently during simulations of lipid bilayers are clustered according to main types of topologies that characterize three-dimensional arrangements of lipids connected to each other via short water bridges. We characterize the dynamics of hydrogen-bonded lipid clusters in simulations of model POPE and POPE:POPG membranes that are often used for bacterial membrane proteins, in a model of the Escherichia coli membrane with six different lipid types, and in POPS membranes. We find that all lipids sample dynamic hydrogen-bonded networks with linear, star, or circular arrangements of the lipid headgroups, and larger networks with combinations of these three types of topologies. Overall, linear lipid-water bridges tend to be short. Water-mediated lipid clusters in all membranes with PE lipids tend to be somewhat small, with about four lipids in all membranes studied here. POPS membranes allow circular arrangements of three POPS lipids to be sampled frequently, and complex arrangements of linear, star, and circular paths may also be sampled. These findings suggest a molecular picture of the membrane interface whereby lipid molecules transiently connect in clusters with somewhat small spatial extension.

Topics: Algorithms; Escherichia coli; Hydrogen Bonding; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylethanolamines; Phosphatidylglycerols

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

Antibiotics (AB) resistance is a major threat to global health, thus the development of novel AB classes is urgently needed. Lantibiotics (i.e. nisin) are natural compounds that effectively control bacterial populations, yet their clinical potential is very limited. Nisin targets membrane-embedded cell wall precursor - lipid II - via capturing its pyrophosphate group (PPi), which is unlikely to evolve, and thus represents a promising pharmaceutical target. Understanding of exact molecular mechanism of initial stages of membrane-bound lipid II recognition by water-soluble nisin is indispensable. Here, using molecular simulations, we demonstrate that the structure of lipid II is determined to a large extent by the surrounding water-lipid milieu. In contrast to the bulk solvent, in the bilayer only two conformational states remain capable of nisin binding. In these states PPi manifests a unique arrangement of hydrogen bond acceptors on the bilayer surface. Such a "pyrophosphate pharmacophore" cannot be formed by phospholipids, which explains high selectivity of nisin/lipid II recognition. Similarly, the "recognition module" of nisin, being rather flexible in water, adopts the only stable conformation in the presence of PPi analogue (which mimics the lipid II molecule). We establish the "energy of the pyrophosphate pharmacophore" approach, which effectively distinguishes nisin conformations that can form a complex with PPi. Finally, we propose a molecular model of nisin recognition module/lipid II complex in the bacterial membrane. These results will be employed for further study of lipid II targeting by antimicrobial (poly)cyclic peptides and for design of novel AB prototypes.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Computational Chemistry; Dimethyl Sulfoxide; Diphosphates; Hydrogen Bonding; Lipid Bilayers; Membrane Lipids; Models, Chemical; Models, Molecular; Molecular Conformation; Nisin; Nuclear Magnetic Resonance, Biomolecular; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Binding; Protein Conformation; Solubility; Uridine Diphosphate N-Acetylmuramic Acid; Water

Magainin 2 and PGLa are cationic, amphipathic antimicrobial peptides which when added as equimolar mixture exhibit a pronounced synergism in both their antibacterial and pore-forming activities. Here we show for the first time that the peptides assemble into defined supramolecular structures along the membrane interface. The resulting mesophases are quantitatively described by state-of-the art fluorescence self-quenching and correlation spectroscopies. Notably, the synergistic behavior of magainin 2 and PGLa correlates with the formation of hetero-domains and an order-of-magnitude increased membrane affinity of both peptides. Enhanced membrane association of the peptide mixture is only observed in the presence of phophatidylethanolamines but not of phosphatidylcholines, lipids that dominate bacterial and eukaryotic membranes, respectively. Thereby the increased membrane-affinity of the peptide mixtures not only explains their synergistic antimicrobial activity, but at the same time provides a new concept to increase the therapeutic window of combinatorial drugs.

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Boron Compounds; Cell Membrane; Drug Combinations; Drug Synergism; Ethanolamines; Fluorescent Dyes; Lipid Bilayers; Magainins; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Binding; Skin; Spectrometry, Fluorescence; Xenopus laevis; Xenopus Proteins

Gad-1 and Gad-2 are helical, histidine-rich antimicrobial peptides (AMPs) from paralogous genes in cod.

Topics: Cardiolipins; Escherichia coli; Histidine; Lipid Bilayers; Phosphatidylethanolamines; Phosphatidylglycerols; Pore Forming Cytotoxic Proteins

Many plant-derived compounds possess antimicrobial, antioxidant and even anticancer activities. Therefore, they are considered as substances that can be used instead of synthetic compounds in various applications. In this work, the essential oil from hop cones was extracted and analyzed, and then its effects on model bacteria membranes were studied to verify whether the hop essential oils could be used as ecological pesticides. The experiments involved surface pressure-area measurements, penetration studies and Brewster angle microscopy (BAM) imaging of lipid monolayers as well as hydrodynamic diameter, zeta potential, steady-state fluorescence anisotropy and Cryo-Transmission Electron Microscopy (cryo-TEM) measurements of liposomes. Finally the bactericidal tests on plant pathogen bacteria Pseudomonas syringae pv. lachrymans PCM 1410 were performed. The obtained results showed that the components of the essential oils from hop cones incorporate into lipid monolayers and bilayers and alter their fluidity. However, the observed effect is determined by the system composition, its condensation and the oil concentration. Interestingly, at a given dose, the effect of the essential oil on membranes was found to stabilize. Moreover, BAM images proved that hop oil prevents the formation of a large fraction of a condensed phase at the interface. Both the studies on model membranes as well as the in vitro tests allow one to conclude that the hop essential oil could likely be considered as the candidate to be used in agriculture as a natural pesticide.

Topics: Anti-Bacterial Agents; Cardiolipins; Humulus; Lipid Bilayers; Membrane Fluidity; Microbial Sensitivity Tests; Oils, Volatile; Phosphatidylethanolamines; Phosphatidylglycerols; Pseudomonas syringae; Unilamellar Liposomes

Biobutanol production by fermentation is potentially a sustainable alternative to butanol production from fossil fuels. However, the toxicity of butanol to fermentative bacteria, resulting largely from cell membrane fluidization, limits production titers and is a major factor limiting the uptake of the technology. Here, studies were undertaken, in vitro and in silico, on the butanol effects on a representative bacterial (i.e. Escherichia coli) inner cell membrane. A critical butanol : lipid ratio for stability of 2 : 1 was observed, computationally, consistent with complete interdigitation. However, at this ratio the bilayer was ∼20% thicker than for full interdigitation. Furthermore, butanol intercalation induced acyl chain bending and increased disorder, measured as a 27% lateral diffusivity increase experimentally in a supported lipid bilayer. There was also a monophasic Tm reduction in butanol-treated large unilamellar vesicles. Both behaviours are inconsistent with an interdigitated gel. Butanol thus causes only partial interdigitation at physiological temperatures, due to butanol accumulating at the phospholipid headgroups. Acyl tail disordering (i.e. splaying and bending) fills the subsequent voids. Finally, butanol short-circuits the bilayer and creates a coupled system where interdigitated and splayed phospholipids coexist. These findings will inform the design of strategies targeting bilayer stability for increasing biobutanol production titers.

Topics: 1-Butanol; Cell Membrane; Escherichia coli; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylethanolamines; Phosphatidylglycerols; Transition Temperature; Unilamellar Liposomes

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

An understanding of the mechanism of action of antimicrobial peptides is fundamental to the development of new and more active antibiotics. In the present work, we use a wide range of techniques (SANS, SAXD, DSC, ITC, CD, and confocal and electron microscopy) in order to fully characterize the interaction of a cecropin A-melittin hybrid antimicrobial peptide, CA(1-7)M(2-9), of known antimicrobial activity, with a bacterial model membrane of POPE/POPG in an effort to unravel its mechanism of action. We found that CA(1-7)M(2-9) disrupts the vesicles, inducing membrane condensation and forming an onionlike structure of multilamellar stacks, held together by the intercalated peptides. SANS and SAXD revealed changes induced by the peptide in the lipid bilayer thickness and the bilayer stiffening in a tightly packed liquid-crystalline lamellar phase. The analysis of the observed abrupt changes in the repeat distance upon the phase transition to the gel state suggests the formation of an L

Topics: Amino Acid Sequence; Antimicrobial Cationic Peptides; Melitten; Phosphatidylethanolamines; Phosphatidylglycerols

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

Amphiphilic aminoglycoside derivatives are promising new antibacterials active against Gram-negative bacteria such as Pseudomonas aeruginosa, including colistin resistant strains. In this study, we demonstrated that addition of cardiolipin to the culture medium delayed growth of P. aeruginosa, favored asymmetrical growth and enhanced the efficiency of a new amphiphilic aminoglycoside derivative, the 3',6-dinonylneamine. By using membrane models mimicking P. aeruginosa plasma membrane composition (POPE:POPG:CL), we demonstrated the ability of 3'6-dinonylneamine to induce changes in the biophysical properties of membrane model lipid systems in a cardiolipin dependent manner. These changes include an increased membrane permeability associated with a reduced hydration and a decreased ability of membrane to mix and fuse as shown by monitoring calcein release, Generalized Polarization of Laurdan and fluorescence dequenching of octadecyl rhodamine B, respectively. Altogether, results shed light on how cardiolipin may be critical for improving antibacterial action of new amphiphilic aminoglycoside derivatives.

Topics: 2-Naphthylamine; Aminoglycosides; Anti-Bacterial Agents; Cardiolipins; Cell Membrane Permeability; Dose-Response Relationship, Drug; Fluoresceins; Laurates; Membrane Fusion; Phosphatidylethanolamines; Phosphatidylglycerols; Pseudomonas aeruginosa; Unilamellar Liposomes

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

Peptidoglycan (PG) biosynthesis and assembly are needed for bacterial cell wall formation. Lipid II is the precursor in the PG biosynthetic pathway and carries a nascent PG unit that is processed by glycosyltransferases. Despite its immense therapeutic value as a target of several classes of antibiotics, the conformational ensemble of lipid II in bacterial membranes and its interactions with membrane-anchored enzymes remain elusive. In this work, lipid II and its elongated forms (lipid VI and lipid XII) were modeled and simulated in bilayers of POPE (palmitoyl-oleoyl-phosphatidyl-ethanolamine) and POPG (palmitoyl-oleoyl-phosphatidyl-glycerol) that mimic the prototypical composition of Gram-negative cytoplasmic membranes. In addition, penicillin-binding protein 1b (PBP1b) from Escherichia coli was modeled and simulated in the presence of a nascent PG to investigate their interactions. Trajectory analysis reveals that as the glycan chain grows, the non-reducing end of the nascent PG displays much greater fluctuation along the membrane normal and minimally interacts with the membrane surface. In addition, dihedral angles within the pyrophosphate moiety are determined by the length of the PG moiety and its surrounding environment. When a nascent PG is bound to PBP1b, the stem peptide remains in close contact with PBP1b by structural rearrangement of the glycan chain. Most importantly, the number of nascent PG units required to reach the transpeptidase domain are determined to be 7 or 8. Our findings complement experimental results to further understand how the structure of nascent PG can dictate the assembly of the PG scaffold.

Topics: Cell Membrane; Cell Wall; Diphosphates; Escherichia coli; Penicillin-Binding Proteins; Peptidoglycan; Peptidyl Transferases; Phosphatidylethanolamines; Phosphatidylglycerols; Polysaccharides; Uridine Diphosphate N-Acetylmuramic Acid

Enzyme IIC (EIIC) is a membrane-embedded sugar transport protein that is part of the phosphoenolpyruvate-dependent phosphotransferases. Crystal structures of two members of the glucose EIIC superfamily, bcChbC in the inward-facing conformation and bcMalT in the outward-facing conformation, were previously solved. Comparing the two structures led us to the hypothesis that sugar translocation could be achieved by an elevator-type transport mechanism in which a transport domain binds to the substrate and, through rigid body motions, transports it across the membrane. To test this hypothesis and to obtain more accurate descriptions of alternate conformations of the two proteins, we first performed collective variable-based steered molecular dynamics (CVSMD) simulations starting with the two crystal structures embedded in model lipid bilayers, and steered their transport domain toward their own alternative conformation. Our simulations show that large rigid-body motions of the transport domain (55° in rotation and 8 Å in translation) lead to access of the substrate binding site to the alternate side of the membrane. H-bonding interactions between the sugar and the protein are intact, although the side chains of the binding-site residues were not restrained in the simulation. Pairs of residues in bcMalT that are far apart in the crystal structure become close to each other in the simulated model. Some of these pairs can be cross-linked by a mercury ion when mutated to cysteines, providing further support for the CVSMD-generated model. In addition, bcMalT binds to maltose with similar affinities before and after the cross-linking, suggesting that the binding site is preserved after the conformational change. In combination, these results support an elevator-type transport mechanism in EIIC.

Topics: Bacillus cereus; Binding Sites; Hydrogen Bonding; Lipid Bilayers; Maltose; Membrane Transport Proteins; Molecular Dynamics Simulation; Mutation; Phosphatidylethanolamines; Phosphatidylglycerols; Phosphoenolpyruvate Sugar Phosphotransferase System

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

Topics: Amino Acid Sequence; Antimicrobial Cationic Peptides; Bacteria; Cardiolipins; Cell Membrane; Cholesterol; Dimyristoylphosphatidylcholine; Fluorine; Humans; Isotopes; Lipid Bilayers; Magnetic Resonance Spectroscopy; Peptides; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Binding; Protein Conformation, alpha-Helical; Protein Folding; Proteolysis; Sweat

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

Understanding how membrane proteins interact with their environment is fundamental to the understanding of their structure, function and interactions. We have performed coarse-grained molecular dynamics simulations on a series of membrane proteins in a membrane environment to examine the perturbations of the lipids by the presence of protein. We analyze these perturbations in terms of elastic membrane deformations and local lipid protein interactions. However these two factors are insufficient to describe the variety of effects that we observe and the changes caused by membranes proteins to the structure and dynamics of their lipid environment. Other factors that change the conformation available to lipid molecules are evident and are able to modify lipid structure far from the protein surface, and thus mediate long-range interactions between membrane proteins. We suggest that these multiple modifications to lipid behavior are responsible, at the molecular level, for the lipophobic effect we have proposed to account for our observations of membrane protein organization.

Topics: Animals; Bacteria; Cell Membrane; Elasticity; Humans; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Membrane Proteins; Molecular Dynamics Simulation; Phosphatidylethanolamines; Phosphatidylglycerols; Spinacia oleracea

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

G protein gated inward rectifier K(+) (GIRK) channels open and thereby silence cellular electrical activity when inhibitory G protein coupled receptors (GPCRs) are stimulated. Here we describe an assay to measure neuronal GIRK2 activity as a function of membrane-anchored G protein concentration. Using this assay we show that four Gβγ subunits bind cooperatively to open GIRK2, and that intracellular Na(+) - which enters neurons during action potentials - further amplifies opening mostly by increasing Gβγ affinity. A Na(+) amplification function is characterized and used to estimate the concentration of Gβγ subunits that appear in the membrane of mouse dopamine neurons when GABAB receptors are stimulated. We conclude that GIRK2, through its dual responsiveness to Gβγ and Na(+), mediates a form of neuronal inhibition that is amplifiable in the setting of excess electrical activity.

Topics: Action Potentials; Animals; Biological Assay; Dopaminergic Neurons; G Protein-Coupled Inwardly-Rectifying Potassium Channels; Gene Expression Regulation; GTP-Binding Protein beta Subunits; GTP-Binding Proteins; Humans; Mice; Pars Compacta; Patch-Clamp Techniques; Phosphatidylethanolamines; Phosphatidylglycerols; Pichia; Primary Cell Culture; Protein Multimerization; Protein Subunits; Proteolipids; Receptors, GABA-B; Recombinant Proteins; Signal Transduction; Sodium

G protein gated inward rectifier potassium (GIRK) channels are gated by direct binding of G protein beta-gamma subunits (Gβγ), signaling lipids, and intracellular Na(+). In cardiac pacemaker cells, hetero-tetramer GIRK1/4 channels and homo-tetramer GIRK4 channels play a central role in parasympathetic slowing of heart rate. It is known that the Na(+) binding site of the GIRK1 subunit is defective, but the functional difference between GIRK1/4 hetero-tetramers and GIRK4 homo-tetramers remains unclear. Here, using purified proteins and the lipid bilayer system, we characterize Gβγ and Na(+) regulation of GIRK1/4 hetero-tetramers and GIRK4 homo-tetramers. We find in GIRK4 homo-tetramers that Na(+) binding increases Gβγ affinity and thereby increases the GIRK4 responsiveness to G protein stimulation. GIRK1/4 hetero-tetramers are not activated by Na(+), but rather are in a permanent state of high responsiveness to Gβγ, suggesting that the GIRK1 subunit functions like a GIRK4 subunit with Na(+) permanently bound.

Topics: Action Potentials; Animals; Cell Differentiation; G Protein-Coupled Inwardly-Rectifying Potassium Channels; Gene Expression Regulation; GTP-Binding Protein beta Subunits; GTP-Binding Protein gamma Subunits; HEK293 Cells; Humans; Mice; Mouse Embryonic Stem Cells; Myocardium; Myocytes, Cardiac; Oleic Acids; Patch-Clamp Techniques; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Multimerization; Protein Subunits; Proteolipids; Recombinant Proteins; Signal Transduction; Sodium; Transfection

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

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

Topics: Binding Sites; Calcium; Cations, Divalent; Cell Membrane; Chlorides; Escherichia coli; Molecular Dynamics Simulation; Phosphatidylethanolamines; Phosphatidylglycerols; Thermodynamics

The conjugation of nanoparticles with antimicrobial peptides (AMP) is emerging as a promising route to achieve superior antimicrobial activity. However, the nature of peptide-nanoparticle interactions in these systems remains unclear. This study describes a system consisting of a cysteine containing antimicrobial peptide conjugated with silver nanoparticles, in which the two components exhibit a dynamic interaction resulting in a significantly enhanced stability and biological activity compared to that of the individual components. This was investigated using NMR spectroscopy in conjunction with other biophysical techniques. Using fluorescence assisted cell sorting and membrane mimics we carried out a quantitative comparison of the activity of the AMP-nanoparticle system and the free peptide. Taken together, the study provides new insights into nanoparticle-AMP interactions at a molecular level and brings out the factors that will be useful for consideration while designing new conjugates with enhanced functionality.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Biomimetic Materials; Cell Line; Cell Membrane; Cell Survival; Cysteine; Drug Stability; Escherichia coli; Humans; Keratinocytes; Magnetic Resonance Spectroscopy; Metal Nanoparticles; Microbial Viability; Phosphatidylethanolamines; Phosphatidylglycerols; Silver; 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

Fluoroquinolones are antibiotics that have a large spectrum of action against bacteria, especially Gram-negative. A strategy to enhance their pharmacological behavior, and try to counteract bacterial resistance, is their coordination to divalent metal ions and 1,10-phenanthroline. These stable complexes modify fluoroquinolones potency and specificity, possibly due to their alternative translocation through the bacterial membranes. In this work, we determined the interaction of ciprofloxacin and its copper(II) ternary complex with unilamellar liposomes of DMPC, POPE/POPG (0.75:0.25), POPE/POPG/cardiolipin (0.67:0.23:0.10), and E. coli total extract, using time-resolved and steady-state fluorescence spectroscopy. The association constants determined show that the interaction of both compounds depends on membrane lipids composition and is always higher for the metalloantibiotic, a trend already observed for other fluoroquinolone metalloantibiotics. Nevertheless, the interaction of ciprofloxacin metalloantibiotic is, normally, higher, which reflects the fluoroquinolone species that are present in solution at physiological pH. In overall, the results obtained suggest that ciprofloxacin and its metalloantibiotic have different translocation pathways, proposing that the diffusion of the metalloantibiotic is a hydrophobic mechanism and suggesting that this new metalloantibiotic may be a good choice to replace the pure ciprofloxacin and bypass, at least, one of the mechanisms of the bacterial resistance to fluoroquinolones.

Topics: Anti-Bacterial Agents; Cardiolipins; Ciprofloxacin; Dimyristoylphosphatidylcholine; Escherichia coli; Fluoroquinolones; Gram-Negative Bacteria; Hydrophobic and Hydrophilic Interactions; Phosphatidylethanolamines; Phosphatidylglycerols; Spectrometry, Fluorescence; Unilamellar Liposomes

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

Antimicrobial lipopeptides (AMLPs) are antimicrobial drug candidates that preferentially target microbial membranes. One class of AMLPs, composed of cationic tetrapeptides attached to an acyl chain, have minimal inhibitory concentrations in the micromolar range against a range of bacteria and fungi. Previously, we used coarse-grained molecular dynamics simulations and free energy methods to study the thermodynamics of their interaction with membranes in their monomeric state. Here, we extended the study to the biologically relevant micellar state, using, to our knowledge, a novel reaction coordinate based on hydrophobic contacts. Using umbrella sampling along this reaction coordinate, we identified the critical transition states when micelles insert into membranes. The results indicate that the binding of these AMLP micelles to membranes is thermodynamically favorable, but in contrast to the monomeric case, there are significant free energy barriers. The height of these free energy barriers depends on the membrane composition, suggesting that the AMLPs' ability to selectively target bacterial membranes may be as much kinetic as thermodynamic. This mechanism highlights the importance of considering oligomeric state in solution as criterion when optimizing peptides or lipopeptides as antibiotic leads.

Topics: Anti-Infective Agents; Hydrophobic and Hydrophilic Interactions; Kinetics; Lipid Bilayers; Lipopeptides; Membrane Fusion; Micelles; Molecular Dynamics Simulation; Phosphatidylethanolamines; Phosphatidylglycerols; Thermodynamics

Electron microscopy and atomic force microscopy images of cholesterol-dependent cytolysins and related proteins that form large pores in lipid membranes have revealed the presence of incomplete rings, or arcs. Some evidence indicates that these arcs are inserted into the membrane and induce membrane leakage, but other experiments seem to refute that. Could such pores, only partially lined by protein, be kinetically and thermodynamically stable? How would the lipids be structured in such a pore? Using the antimicrobial peptide protegrin-1 as a model, we test the stability of pores only partially lined by peptide using all-atom molecular dynamics simulations in POPC and POPE/POPG membranes. The data show that, whereas pure lipid pores close rapidly, pores partially lined by protegrin arcs are stable for at least 300 ns. Estimates of the thermodynamic stability of these arcs using line tension data and implicit solvent calculations show that these arcs can be marginally stable in both zwitterionic and anionic membranes. Arcs provide an explanation for the observed ion selectivity in protegrin electrophysiology experiments and could possibly be involved in other membrane permeabilization processes where lipids are thought to participate, such as those induced by antimicrobial peptides and colicins, as well as the Bax apoptotic pore.

Topics: Amino Acid Sequence; Antimicrobial Cationic Peptides; Lipid Bilayers; Molecular Dynamics Simulation; Molecular Sequence Data; Phosphatidylethanolamines; Phosphatidylglycerols

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

The emergence of antibiotic resistant pathogens is one of the major medical concerns of the 21st century, prompting renewed interest in the development of novel antimicrobial compounds. Here we use microsecond-scale all-atom molecular dynamics simulations to characterize the structure, dynamics, and membrane-binding mechanism of a synthetic antimicrobial lipopeptide, C16-KGGK. Our simulations suggest that these lipopeptides prefer to aggregate in solution and alter the intrinsic order of the lipid bilayer upon binding. From these results and previous coarse-grained simulations, we have developed a simple model for the binding and insertion process for these lipopeptides.

Topics: Algorithms; Anti-Infective Agents; Lipid Bilayers; Lipopeptides; Membrane Lipids; Models, Molecular; Molecular Dynamics Simulation; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Binding; Time Factors

Domain formation in bacteria-mimetic membranes due to cationic peptide binding was recently proposed based on calorimetric data. We now use (2)H solid-state NMR to critically examine the presence and absence of domains in bacterial membranes containing zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) lipids. Chain-perdeuterated POPE and POPG are used in single-component membranes, binary POPE/POPG (3:1) membranes, and membranes containing one of four cationic peptides: two antimicrobial peptides (AMPs) of the β-hairpin family of protegrin-1 (PG-1), and two cell-penetrating peptides (CPPs), HIV TAT and penetratin. (2)H quadrupolar couplings were measured to determine the motional amplitudes of POPE and POPG acyl chains as a function of temperature. Homogeneously mixed POPE/POPG membranes should give the same quadrupolar couplings for the two lipids, whereas the presence of membrane domains enriched in one of the two lipids should cause distinct (2)H quadrupolar couplings that reflect different chain disorder. At physiological temperature (308 K), we observed no or only small coupling differences between POPE and POPG in the presence of any of the cationic peptides. However, around ambient temperature (293 K), at which gel- and liquid-crystalline phases coexist in the peptide-free POPE/POPG membrane, the peptides caused distinct quadrupolar couplings for the two lipids, indicating domain formation. The broad-spectrum antimicrobial peptide PG-1 ordered ∼40% of the POPE lipids while disordering POPG. The Gram-negative selective PG-1 mutant, IB549, caused even larger differences in the POPE and POPG disorder: ∼80% of POPE partitioned into the ordered phase, whereas all of the POPG remained in the disordered phase. In comparison, TAT rigidified POPE and POPG similarly in the binary membrane at ambient temperature, indicating that TAT does not cause dynamic heterogeneity but interacts with the membrane with a different mechanism. Penetratin maintained the POPE order but disordered POPG, suggesting moderate domain separation. These results provide insight into the extent of domain formation in bacterial membranes and the possible peptide structural requirements for this phenomenon.

Topics: Amino Acid Sequence; Antimicrobial Cationic Peptides; Bacteria; Carrier Proteins; Cell Membrane; Cell-Penetrating Peptides; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Phosphatidylethanolamines; Phosphatidylglycerols; tat Gene Products, Human Immunodeficiency Virus; Temperature

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

Many observations of the role of the membrane in the function and organization of transmembrane (TM) proteins have been explained in terms of hydrophobic mismatch between the membrane and the inserted protein. For a quantitative investigation of this mechanism in the lipid-protein interactions of functionally relevant conformations adopted by a multi-TM segment protein, the bacterial leucine transporter (LeuT), we employed a novel method, Continuum-Molecular Dynamics (CTMD), that quantifies the energetics of hydrophobic mismatch by combining the elastic continuum theory of membrane deformations with an atomistic level description of the radially asymmetric membrane-protein interface from MD simulations. LeuT has been serving as a model for structure-function studies of the mammalian neurotransmitter:sodium symporters (NSSs), such as the dopamine and serotonin transporters, which are the subject of intense research in the field of neurotransmission. The membrane models in which LeuT was embedded for these studies were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid, or 3:1 mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) lipids. The results show that deformation of the host membrane alone is not sufficient to alleviate the hydrophobic mismatch at specific residues of LeuT. The calculations reveal significant membrane thinning and water penetration due to the specific local polar environment produced by the charged K288 of TM7 in LeuT, that is membrane-facing deep inside the hydrophobic milieu of the membrane. This significant perturbation is shown to result in unfavorable polar-hydrophobic interactions at neighboring hydrophobic residues in TM1a and TM7. We show that all the effects attributed to the K288 residue (membrane thinning, water penetration, and the unfavorable polar-hydrophobic interactions at TM1a and TM7), are abolished in calculations with the K288A mutant. The involvement of hydrophobic mismatch is somewhat different in the functionally distinct conformations (outward-open, occluded, inward-open) of LeuT, and the differences are shown to connect to structural elements (e.g., TM1a) known to play key roles in transport. This finding suggests a mechanistic hypothesis for the enhanced transport activity observed for the K288A mutant, suggesting that the unfavorable hydrophobic-hydrophilic interactions hinder the motion of TM1a in the fun

Topics: Cell Membrane; Hydrophobic and Hydrophilic Interactions; Molecular Dynamics Simulation; Mutation; Phosphatidylethanolamines; Phosphatidylglycerols; Plasma Membrane Neurotransmitter Transport Proteins; Protein Conformation

RCK domains control activity of a variety of K(+) channels and transporters through binding of cytoplasmic ligands. To gain insight toward mechanisms of RCK domain activation, we solved the structure of the RCK domain from the Ca(2+)-gated K(+) channel, MthK, bound with Ba(2+), at 3.1 Å resolution. The Ba(2+)-bound RCK domain was assembled as an octameric gating ring, as observed in structures of the full-length MthK channel, and shows Ba(2+) bound at several positions. One of the Ba(2+) sites, termed C1, overlaps with a known Ca(2+)-activation site, determined by residues D184 and E210. Functionally, Ba(2+) can activate reconstituted MthK channels as observed in electrophysiological recordings, whereas Mg(2+) (up to 100 mM) was ineffective. Ba(2+) activation was abolished by the mutation D184N, suggesting that Ba(2+) activates primarily through the C1 site. Our results suggest a working hypothesis for a sequence of ligand-dependent conformational changes that may underlie RCK domain activation and channel gating.

Topics: Amino Acid Motifs; Archaeal Proteins; Barium; Binding Sites; Calcium; Coordination Complexes; Crystallography, X-Ray; Hydrogen-Ion Concentration; Ion Channel Gating; Lipid Bilayers; Membrane Potentials; Methanobacteriaceae; Models, Molecular; Phosphatidylethanolamines; Phosphatidylglycerols; Potassium Channels, Calcium-Activated; Protein Structure, Quaternary; Protein Structure, Tertiary

In this work we have investigated the selectivity of lactose permease (LacY) of Escherichia coli (E. coli) for its surrounding phospholipids when reconstituted in binary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-Palmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) with 1-palmitoyl-2-oleoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (POPG). Förster resonance energy transfer (FRET) measurements have been performed to investigate the selectivity between a single tryptophan mutant of LacY used as donor (D), and two analogues of POPE and POPG labeled with pyrene in the acyl chains (Pyr-PE and Pyr-PG) used as acceptors. As a difference from previous works, now the donor has been single-W151/C154G/D68C LacY. It has been reported that the replacement of the aspartic acid in position 68 by cysteine inhibits active transport in LacY. The objectives of this work were to elucidate the phospholipid composition of the annular region of this mutant and to determine whether the mutation performed, D68C, induced changes in the protein-lipid selectivity. FRET efficiencies for Pyr-PE were always higher than for Pyr-PG. The values of the probability of each site in the annular ring being occupied by a label (μ) were similar at the studied temperatures (24 °C and 37 °C), suggesting that the lipid environment is not significantly affected when increasing the temperature. By comparing the results with those obtained for single-W151/C154G LacY, we observe that the mutation in the 68 residue indeed changes the selectivity of the protein for the phospholipids. This might be probably due to a change in the conformational dynamics of LacY.

Topics: Escherichia coli; Escherichia coli Proteins; Fluorescence Resonance Energy Transfer; Models, Molecular; Monosaccharide Transport Proteins; Phosphatidylethanolamines; Phosphatidylglycerols; Point Mutation; Symporters

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

Four synthesized biocidal guanidine hydrochloride polymers with different alkyl chain length, including polyhexamethylene guanidine hydrochloride and its three new analogs, were used to investigate their interactions with phospholipids vesicles mimicking bacterial membrane. Characterization was conducted by using fluorescence dye leakage, isothermal titration calorimetry, and differential scanning calorimetry. The results showed that the gradually lengthened alkyl chain of the polymer increased the biocidal activity, accompanied with the increased dye leakage rate and the increased binding constant and energy change value of polymer-membrane interaction. The polymer-membrane interaction induced the change of pretransition and main phase transition (decreased temperature and increased width) of phospholipids vesicles, suggesting the conformational change in the phospholipids headgroups and disordering in the hydrophobic regions of lipid membranes. The above information revealed that the membrane disruption actions of guanidine hydrochloride polymers are the results of the polymer's strong binding to the phospholipids membrane and the subsequent perturbations of the polar headgroups and hydrophobic core region of the phospholipids membrane. The alkyl chain structure significantly affects the binding constant and energy change value of the polymer-membrane interactions and the perturbation extent of the phospholipids membrane, which lead to the different biocidal activity of the polymer analogs. This work provides important information about the membrane disruption action mechanism of biocidal guanidine hydrochloride polymers.

Topics: Anti-Infective Agents; Biophysical Phenomena; Calorimetry; Candida albicans; Escherichia coli; Guanidine; Guanidines; Lipid Bilayers; Microbial Sensitivity Tests; Models, Chemical; Molecular Structure; Phosphatidylethanolamines; Phosphatidylglycerols; Phospholipids; Polymers; Pseudomonas aeruginosa; Spectrometry, Mass, Electrospray Ionization; Spectroscopy, Fourier Transform Infrared; Staphylococcus aureus

Molecular dynamics (MD) simulations are used to study the pathway for the insertion of the cationic antimicrobial peptide protegrin-1 (PG1) into mixed anionic lipid bilayers composed of palmitoyl-oleoyl-phosphatidylglycerol (POPG) and palmitoyl-oleoyl-phosphatidylethanolamine (POPE) in a 1:3 ratio (POPG/POPE). We calculate the potential of mean force (PMF) during the transfer of the peptide from the bulk aqueous phase to the transmembrane (TM) configuration using the adaptive biasing force (ABF) method. We find that the PMF has two energy minima separated by an energy barrier. One minimum corresponds to the fully transmembrane inserted state, with a free energy of -20.1 kcal/mol. The second PMF minimum, which corresponds to adsorption to the membrane surface, has a value of -2.5 kcal/mol. The PMF also shows the existence of a free energy barrier of +6.3 kcal/mol for the insertion process. Using the Kramers theory Langevin equation and the Grote-Hynes theory generalized Langevin equation, we calculated the transmission coefficient for PG1 diffusion through the potential barrier. We focus on the use of the PMF and the time correlation function of the fluctuation of the instantaneous force to calculate the rate constants for insertion/deinsertion of PG1 from the mixed POPG/POPE membrane. The influence of the activation free energy barrier on the dynamics of the insertion and permeation of peptides through the membrane are discussed.

Topics: Antimicrobial Cationic Peptides; Lipid Bilayers; Molecular Dynamics Simulation; Permeability; Phosphatidylethanolamines; Phosphatidylglycerols; Thermodynamics

Biochemical and structural work has revealed the importance of phospholipids in biogenesis, folding and functional modulation of membrane proteins. Therefore, the nature of protein-phospholipid interaction is critical to understand such processes. Here, we have studied the interaction of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) mixtures with the lactose permease (LacY), the sugar/H(+) symporter from Escherichia coli and a well characterized membrane transport protein. FRET measurements between single-W151/C154G LacY reconstituted in a lipid mixture composed of POPE and POPG at different molar ratios and pyrene-labeled PE or PG revealed a different phospholipid distribution between the annular region of LacY and the bulk lipid phase. Results also showed that both PE and PG can be part of the annular region, being PE the predominant when the PE:PG molar ratio mimics the membrane of E. coli. Furthermore, changes in the thermotropic behavior of phospholipids located in this annular region confirm that the interaction between LacY and PE is stronger than that of LacY and PG. Since PE is a proton donor, the results obtained here are discussed in the context of the transport mechanism of LacY.

Topics: Escherichia coli; Escherichia coli Proteins; Fluorescence Resonance Energy Transfer; Monosaccharide Transport Proteins; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Structure, Tertiary; Symporters

We report the insertion of a transmembrane protein, lactose permease (LacY) from Escherichia coli (E. coli), in supported lipid bilayers (SLBs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), in biomimetic molar proportions. We provide evidence of the preferential insertion of LacY in the fluid domains. Analysis of the self-assembled protein arrangements showed that LacY: (i) is inserted as a monomer within fluid domains of SLBs of POPE:POPG (3:1, mol/mol), (ii) has a diameter of approx. 7.8nm; and (iii) keeps an area of phospholipids surrounding the protein that is compatible with shells of phospholipids.

Topics: Escherichia coli; Lipid Bilayers; Membrane Transport Proteins; Microscopy, Atomic Force; Phosphatidylethanolamines; Phosphatidylglycerols; Phospholipids

The free energies of adsorption of the monomer or dimer of the cationic beta-hairpin antimicrobial peptide protegrin-1 (PG1) in a specific binding orientation on a lipid bilayer are determined using molecular dynamics (MD) simulations and Poisson-Boltzmann calculations. The bilayer is composed of anionic palmitoyl-oleoyl-phosphatidylglycerol (POPG) and palmitoyl-oleoyl-phosphatidylethanolamine (POPE) with ratio 1:3 (POPG/POPE). PG1 is believed to kill bacteria by binding on their membranes. There, it forms pores that lyse the bacteria. Herein we focus on the thermodynamics of binding. In particular, we explore the role of counterion release from the lipid bilayer upon adsorption of either the monomeric or the dimeric form of PG1. Twenty-two 4-ns-long MD trajectories of equilibrated systems are generated to determine the free energy profiles for the monomer and dimer as a function of the distance between the peptide(s) and the membrane surface. The MD simulations are conducted at 11 different separations from the membrane for each of the two systems, one with PG1, the second with a PG1 dimer of only a specific orientation of the monomer and dimer without taking into account the change of entropy for the peptide. To calculate the potential of mean force for each peptide/membrane system, a variant of constrained MD and thermodynamic integration is used. We observed that PG1 dimer binds more favorably to the POPG/POPE membrane. A simple method for relating the free energy profile to the PG1-membrane binding constant is employed to predict a free energy of adsorption of -2.4 +/- 0.8 kcal/mol. A corresponding PG1-dimer-membrane binding constant is calculated as -3.5 +/- 1.1 kcal/mol. Free energy profiles from MD simulation were extensively analyzed and compared with results of Poisson-Boltzmann theory. We find the peptide-membrane attraction to be dominated by the entropy increase due to the release of counterions in a POPG/POPE lipid bilayer.

Topics: Adsorption; Antimicrobial Cationic Peptides; Dimerization; Lipid Bilayers; Models, Chemical; Molecular Dynamics Simulation; Phosphatidylethanolamines; Phosphatidylglycerols; Thermodynamics

Phosphatidylethanolamine (PE) and phosphatidylgycerol (PG) are the main components of the inner membrane of Escherichia coli. Mixtures of PE and PG mimicking the proportions found in E. coli have been extensively used to reconstitute transmembrane proteins as lactose permease (LacY) in proteoliposomes because in this environment the protein shows maximal activity. Hence, the study of the physicochemical properties of this phospholipid matrix becomes of potential interest. In previous studies, we used atomic force microscopy (AFM) and force spectroscopy (FS) to study the topographic and nanomechanical properties of supported lipid bilayers (SLBs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and of POPE and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol). The study reported here was extended for completeness to asymmetric SLBs obtained by the Langmuir-Blodgett (LB) method. Thus, we prepared SLBs with the proximal leaflet extracted at 30 mN x m(-1) and the distal leaflet extracted at 25 mN x m(-1). We prepared SLBs with both leaflets with same composition (POPG/POPG), and also with the proximal leaflet of POPE and the distal leaflet of POPG or POPE:POPG (3:1, mol/mol). The topography of the SLBs acquired in liquid was compared with the topography of the monolayers acquired in air. Breakthrough (F(y)) and adhesion forces (F(adh)) of SLBs were extracted from force curves. The values obtained are discussed in terms of the possible involvement of the nanomechanical properties of the SLBs in membrane protein insertion. The results provide means for the observation that insertion of LacY in POPE:POPG (3:1, mol/mol) occurs preferentially in the fluid phase, which is the phase with the lower F(y) and the higher F(adh).

Topics: Lipid Bilayers; Microscopy, Atomic Force; Phosphatidylethanolamines; Phosphatidylglycerols

Oritavancin, a lipoglycopeptide with marked bactericidal activity against vancomycin-resistant Staphylococcus aureus and enterococci, induces calcein release from CL:POPE and POPG:POPE liposomes, an effect enhanced by an increase in POPG:POPE ratio, and decreased when replacing POPG by DPPG (Domenech et al., Biochim Biophys Acta 2009; 1788:1832-40). Using vesicles prepared from lipids extracted from S. aureus, we showed that oritavancin induces holes, erosion of the edges, and decrease of the thickness of the supported lipid bilayers (atomic force microscopy; AFM). Oritavancin also induced an increase of membrane permeability (calcein release) on a time- and dose-dependent manner. These effects were probably related to the ability of the drug to bind to lipid bilayers as shown by 8-anilino-1- naphthalene sulfonic acid (ANS) assay. Interaction of oritavancin with phospholipids at the level of their glycerol backbone and hydrophobic domain was studied by monitoring changes of Laurdan excitation generalized polarization (GP(ex)) and 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy upon temperature increase. Oritavancin increased GP(ex) values and the transition temperature, indicating a more ordered structure at the level of the glycerol backbone. Oritavancin slightly decreased DPH fluorescence depolarization intensities, suggesting an increase in fluidity at the level of acyl chains. Together, our data confirm the interaction of oritavancin with lipids and the potential role of a rigidifying effect at the level of glycerol backbone for membrane permeabilization. This work shows how AFM and biophysical methods may help in characterizing drug-membrane interactions, and sheds further light on the mode of action of oritavancin.

Topics: Anilino Naphthalenesulfonates; Anti-Bacterial Agents; Diphenylhexatriene; Fluorescence Polarization; Glycopeptides; Lipid Bilayers; Lipids; Lipoglycopeptides; Microscopy, Atomic Force; Permeability; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Binding; Staphylococcus aureus; Time Factors; Unilamellar Liposomes

We show that the activity of an ion channel is correlated with the phase state of the lipid bilayer hosting the channel. By measuring unitary conductance, dwell times, and open probability of the K(+) channel KcsA as a function of temperature in lipid bilayers composed of POPE and POPG in different relative proportions, we obtain that all those properties show a trend inversion when the bilayer is in the transition region between the liquid-disordered and the solid-ordered phase. These data suggest that the physical properties of the lipid bilayer influence ion channel activity likely via a fine-tuning of its conformations. In a more general interpretative framework, we suggest that other parameters such as pH, ionic strength, and the action of amphiphilic drugs can affect the physical behavior of the lipid bilayer in a fashion similar to temperature changes resulting in functional changes of transmembrane proteins.

Topics: Bacterial Proteins; Electric Conductivity; Ion Channel Gating; Lipid Bilayers; Phase Transition; Phosphatidylethanolamines; Phosphatidylglycerols; Potassium Channels; Streptomyces lividans; Temperature; Time Factors

Voltage-dependent K(+) (Kv) channels form the basis of the excitability of nerves and muscles. KvAP is a well-characterized archeal Kv channel that has been widely used to investigate many aspects of Kv channel biochemistry, biophysics, and structure. In this study, a minimal kinetic gating model for KvAP function in two different phospholipid decane bilayers is developed. In most aspects, KvAP gating is similar to the well-studied eukaryotic Shaker Kv channel: conformational changes occur within four voltage sensors, followed by pore opening. Unlike the Shaker Kv channel, KvAP possesses an inactivated state that is accessible from the pre-open state of the channel. Changing the lipid composition of the membrane influences multiple gating transitions in the model, but, most dramatically, the rate of recovery from inactivation. Inhibition by the voltage sensor toxin VSTx1 is most easily explained if VSTx1 binds only to the depolarized conformation of the voltage sensor. By delaying the voltage sensor's return to the hyperpolarized conformation, VSTx1 favors the inactivated state of KvAP.

Topics: Alkanes; Archaea; Ion Channel Gating; Kinetics; Lipid Bilayers; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Potassium Channels, Voltage-Gated; Shaker Superfamily of Potassium Channels; Spider Venoms

Supported lipid bilayers composed of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) were assembled by the vesicle fusion technique on mica and studied by temperature-controlled atomic force microscopy. The role of different physical parameters on the main phase transition was elucidated. Both mixed (POPE/POPG 3:1) and pure POPE bilayers were studied. By increasing the ionic strength of the solution and the incubation temperature, a shift from a decoupled phase transition of the two leaflets, to a coupled transition, with domains in register, was obtained. The observed behavior points to a modulation of the substrate/bilayer and interleaflet coupling induced by the environment and preparation conditions of supported lipid bilayers. The results are discussed in view of the role of different interactions in the system. The influence of the substrate on the lipid bilayers, in terms of interleaflet coupling, can also help us in understanding the possible effect that submembrane elements like the cytoskeleton might have on the structure and dynamics of biomembranes.

Topics: Computer Simulation; Lipid Bilayers; Membrane Fluidity; Models, Chemical; Models, Molecular; Molecular Conformation; Phosphatidylethanolamines; Phosphatidylglycerols

We report an atomic force microscopy study on the lateral spatial redistribution of an integral membrane protein reconstituted in supported lipid bilayers (SLBs) subjected to a thermally induced phase transition. KcsA proteins were reconstituted in proteoliposomes of POPE/POPG (3:1, mol/mol), and SLBs, including the proteins, were then obtained by the vesicle fusion technique on mica. By decreasing the temperature, the lipid bilayer passed from a liquid disordered (l(d)) phase in which the proteins are homogeneously distributed to a coexistence of solid ordered (s(o)) and l(d) domains with the proteins preferentially distributed in the l(d) domains. The inhomogeneous distribution eventually led to protein clustering. The obtained results are discussed in light of the role that the lipid/protein interaction can have in determining the function of integral membrane proteins.

Topics: Aluminum Silicates; Bacterial Proteins; Lipid Bilayers; Microscopy, Atomic Force; Phase Transition; Phosphatidylethanolamines; Phosphatidylglycerols; Potassium Channels; Recombinant Proteins; Spectroscopy, Fourier Transform Infrared; Temperature

We conducted over 150 ns of simulation of a protegrin-1 octamer pore in a lipid bilayer composed of palmitoyloleoyl-phosphatidylethanolamine (POPE) and palmitoyloleoyl-phosphatidylglycerol (POPG) lipids mimicking the inner membrane of a bacterial cell. The simulations improve on a model of a pore proposed from recent NMR experiments and provide a coherent understanding of the molecular mechanism of antimicrobial activity. Although lipids tilt somewhat toward the peptides, the simulated protegrin-1 pore more closely follows the barrel-stave model than the toroidal-pore model. The movement of ions is investigated through the pore. The pore selectively allows negatively charged chloride ions to pass through at an average rate of one ion every two nanoseconds. Only two events are observed of sodium ions crossing through the pore. The potential of mean force is calculated for the water and both ion types. It is determined that the chloride ions move through the pore with ease, similarly to the water molecules with the exception of a zone of restricted movement midway through the pore. In bacteria, ions moving through the pore will compromise the integrity of the transmembrane potential. Without the transmembrane potential as a countermeasure, water will readily flow inside the higher osmolality cytoplasm. We determine that the diffusivity of water through a single PG-1 pore is sufficient to cause fast cell death by osmotic lysis.

Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Candida albicans; Chlorides; Computer Simulation; Escherichia coli; Ion Transport; Lipid Bilayers; Listeria monocytogenes; Magnetic Resonance Spectroscopy; Microbial Sensitivity Tests; Models, Biological; Models, Molecular; Osmolar Concentration; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Conformation; Proteins; Sodium; Time Factors; Water

The site-specific motion of Arg residues in a membrane-bound disulfide-linked antimicrobial peptide, protegrin-1 (PG-1), was investigated by using magic-angle-spinning solid-state NMR spectroscopy to better understand the membrane insertion and lipid interaction of this cationic membrane-disruptive peptide. The C-H and N-H dipolar couplings and 13C chemical shift anisotropies were measured in the anionic POPE/POPG membrane, and were found to be reduced from the rigid-limit values by varying extents; this indicates the presence of segmental motion. An Arg residue at the beta-turn region of the peptide showed much weaker spin interactions, which indicates larger amplitudes of motion than an Arg residue in the beta-strand region of the peptide. This is consistent with the exposure of the beta turn to the membrane surface and the immersion of the beta strand in the hydrophobic middle of the membrane, and supports the previously proposed oligomerization of the peptide into beta barrels in the anionic membrane. The 13C T2 and 1H T(1rho) relaxation times indicate that the beta-turn backbone undergoes large-amplitude intermediate-timescale motion in the fluid phase of the membrane; this causes significant line broadening and loss of spectral intensity. This study illustrates the strong correlation between the dynamics and structure of membrane proteins, and the capability of solid-state NMR spectroscopy to provide detailed information on site-specific dynamics in complex membrane-protein assemblies.

Topics: Antimicrobial Cationic Peptides; Arginine; Cations; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Magnetic Resonance Spectroscopy; Movement; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Structure, Secondary; Temperature; Time Factors

The insertion of charged amino acid residues into the hydrophobic part of lipid bilayers is energetically unfavorable yet found in many cationic membrane peptides and protein domains. To understand the mechanism of this translocation, we measured the (13)C-(31)P distances for an Arg-rich beta-hairpin antimicrobial peptide, PG-1, in the lipid membrane using solid-state NMR. Four residues, including two Arg's, scattered through the peptide were chosen for the distance measurements. Surprisingly, all residues show short distances to the lipid (31)P: 4.0-6.5 A in anionic POPE/POPG membranes and 6.5-8.0 A in zwitterionic POPC membranes. The shortest distance of 4.0 A, found for a guanidinium Czeta at the beta-turn, suggests N-H...O-P hydrogen bond formation. Torsion angle measurements of the two Arg's quantitatively confirm that the peptide adopts a beta-hairpin conformation in the lipid bilayer, and gel-phase 1H spin diffusion from water to the peptide indicates that PG-1 remains transmembrane in the gel phase of the membrane. For this transmembrane beta-hairpin peptide to have short (13)C-(31)P distances for multiple residues in the molecule, some phosphate groups must be embedded in the hydrophobic part of the membrane, with the local (31)P plane parallel to the beta-strand. This provides direct evidence for toroidal pores, where some lipid molecules change their orientation to merge the two monolayers. We propose that the driving force for this toroidal pore formation is guanidinium-phosphate complexation, where the cationic Arg residues drag the anionic phosphate groups along as they insert into the hydrophobic part of the membrane. This phosphate-mediated translocation of guanidinium ions may underlie the activity of other Arg-rich antimocrobial peptides and may be common among cationic membrane proteins.

Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Arginine; Guanidine; Hydrogen Bonding; Lipid Bilayers; Magnetic Resonance Spectroscopy; Membrane Proteins; Models, Molecular; Organophosphates; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Conformation; Proteins

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

Molecular dynamics (MD) simulations have been used to unmask details of specific interactions of anionic phospholipids with intersubunit binding sites on the surface of the bacterial potassium channel KcsA. Crystallographic data on a diacyl glycerol fragment at this site were used to model phosphatidylethanolamine (PE), or phosphatidylglycerol (PG), or phosphatidic acid (PA) at the intersubunit binding sites. Each of these models of a KcsA-lipid complex was embedded in phosphatidyl choline bilayer and explored in a 20 ns MD simulation. H-bond analysis revealed that in terms of lipid-protein interactions PA > PG >> PE and revealed how anionic lipids (PG and PA) bind to a site provided by two key arginine residues (R(64) and R(89)) at the interface between adjacent subunits. A 27 ns simulation was performed in which KcsA (without any lipids initially modeled at the R(64)/R(89) sites) was embedded in a PE/PG bilayer. There was a progressive specific increase over the course of the simulation in the number of H-bonds of PG with KcsA. Furthermore, two specific PG binding events at R(64)/R(89) sites were observed. The phosphate oxygen atoms of bound PG formed H-bonds to the guanidinium group of R(89), whereas the terminal glycerol H-bonded to R(64). Overall, this study suggests that simulations can help identify and characterize sites for specific lipid interactions on a membrane protein surface.

Topics: Anions; Bacterial Proteins; Binding Sites; Cell Membrane; Computer Simulation; Crystallography, X-Ray; Databases, Protein; Escherichia coli Proteins; Hydrogen Bonding; Ions; Lipid Bilayers; Lipids; Models, Biological; Models, Molecular; Molecular Conformation; Mutation; Oxygen; Phosphates; Phosphatidic Acids; Phosphatidylethanolamines; Phosphatidylglycerols; Phospholipids; Potassium Channels; Potassium Channels, Voltage-Gated; Pressure; Protein Folding; Streptomyces lividans; Temperature; Time Factors

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

Phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) are the main lipid components of the inner bacterial membrane. A computer model for such a membrane was built of palmitoyloleoyl PE (POPE) and palmitoyloleoyl PG (POPG) in the proportion 3:1, and sodium ions (Na+) to neutralize the net negative charge on each POPG (POPE-POPG bilayer). The bilayer was simulated for 25 ns. A final 10-ns trajectory fragment was used for analyses. In the bilayer interfacial region, POPEs and POPGs interact readily with one another via intermolecular hydrogen (H) bonds and water bridges. POPE is the main H-bond donor in either PEPE or PEPG H-bonds; PGPG H-bonds are rarely formed. Almost all POPEs are H-bonded and/or water bridged to either POPE or POPG but PE-PG links are favored. In effect, the atom packing in the near-the-interface regions of the bilayer core is tight. Na+ does not bind readily to lipids, and interlipid links via Na+ are not numerous. Although POPG and POPE comprise one bilayer, their bilayer properties differ. The average surface area per POPG is larger and the average vertical location of the POPG phosphate group is lower than those of POPE. Also, the alkyl chains of POPG are more ordered and less densely packed than the POPE chains. The main conclusion of this study is that in the PE-PG bilayer PE interacts more strongly with PG than with PE. This is a likely molecular-level event behind a regulating mechanism developed by the bacteria to control its membrane permeability and stability consisting in changes of the relative PG/PE concentration in the membrane.

Topics: Cell Membrane; Computer Simulation; Escherichia coli; Hydrogen Bonding; Lipid Bilayers; Membrane Fluidity; Models, Biological; Models, Chemical; Models, Molecular; Molecular Conformation; Motion; Permeability; Phosphatidylethanolamines; Phosphatidylglycerols; Surface Properties; Water

We have determined the mixing properties and lamellar organization of bacterial membrane mimetics composed of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and -phosphatidylglycerol (POPG) at various molar ratios applying differential scanning calorimetry, small and wide-angle X-ray scattering, as well as optical phase contrast microscopy. Combining the experimental thermodynamic data with a simulation of the liquidus and solidus lines, we were able to construct a phase diagram. Using this approach, we find that the lipids mix in all phases non-ideally in the thermodynamic sense. As expected, pure POPE assembles into multilamellar and pure POPG into unilamellar vesicles, respectively, which are stable within the studied temperature range. In contrast, mixtures of the two components form oligolamellar vesicles consisting of about three to five bilayers. The layers within these oligolamellar liposomes are positionally correlated within the gel phase, but become uncorrelated within the fluid phase exhibiting freely fluctuating bilayers, while the vesicles as a whole remain intact and do not break up into unilamellar forms. X-ray, as well as DSC data, respectively, reveal a miscibility gap due to a lateral phase segregation at POPG concentrations above about 70 mol%, similar to previously reported data on mixtures composed of disaturated PEs and PGs. Hence, the existence of a region of immiscibility is a general feature of PE/PG mixtures and the mixing properties are dominated by PE/PG headgroup interactions, but are largely independent of the composition of the hydrocarbon chains. This is in accordance with a recent theoretical prediction.

Topics: Bacteria; Biophysical Phenomena; Biophysics; Calorimetry, Differential Scanning; Cell Membrane; Escherichia coli; Hydrocarbons; Lipids; Liposomes; Microscopy, Confocal; Microscopy, Phase-Contrast; Models, Chemical; Phosphatidylethanolamines; Phosphatidylglycerols; Scattering, Radiation; Staphylococcus aureus; Temperature; Thermodynamics; X-Ray Diffraction; X-Rays

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

We have observed a discontinuous unbinding transition of lipid bilayer stacks composed of phosphatidylethanolamine and phosphatidylglycerol using x-ray diffraction. The unbinding is reversible and coincides with the main (L(beta)-->L(alpha)) transition of the lipid mixture. Interbilayer interaction potentials deduced from the diffraction data reveal that the bilayers in the L(beta) phase are only weakly bound. The unbinding transition appears to be driven by an abrupt increase in steric repulsion resulting from increased thermal undulations of the bilayers upon entering the fluid L(alpha) phase.

Topics: Lipid Bilayers; Liposomes; Models, Chemical; Phosphatidylethanolamines; Phosphatidylglycerols; X-Ray Diffraction

A lipid transfer protein that facilitates the transfer of glycolipids between donor and acceptor membranes has been investigated using a fluorescence resonance energy transfer assay. The glycolipid transfer protein (23-24 kDa, pI 9.0) catalyzes the high specificity transfer of lipids that have sugars beta-linked to either a ceramide or a diacylglycerol backbone, such as simple glycolipids and gangliosides, but not the transfer of phospholipids, cholesterol, or cholesterol esters. In this study, we examined the effect of different charged lipids on the rate of transfer of anthrylvinyl-labeled galactosylceramide (1 mol %) from a donor to acceptor vesicle population at neutral pH. Compared to neutral donor vesicle membranes, introduction of negatively charged lipid at 5 or 10 mol % into the donor vesicles significantly decreased the transfer rate. Introduction of the same amount of negative charge into the acceptor vesicle membrane did not impede the transfer rate as effectively. Also, positive charge in the donor vesicle membrane was not as effective at slowing the transfer rate as was negative charge in the donor vesicle. Increasing the ionic strength of the buffer with NaCl significantly reversed the charge effects. At neutral pH, the transfer protein (pI congruent with 9.0) is expected to be positively charged, which may promote association with the negatively charged donor membrane. Based on these and other experiments, we conclude that the transfer process follows first-order kinetics and that the off-rate of the transfer protein from the donor vesicle surface is the rate-limiting step in the transfer process.

Topics: Animals; Biological Transport; Carrier Proteins; Cattle; Energy Transfer; Fluorescent Dyes; Galactosylceramides; Glycosphingolipids; Kinetics; Lipid Bilayers; Phosphatidic Acids; Phosphatidylethanolamines; Phosphatidylglycerols; Phospholipids; Spectrometry, Fluorescence; Static Electricity; Surface Properties

The lamellar/nonlamellar phase preferences of lipid model membranes composed of mixtures of several cationic lipids with various zwitterionic and anionic phospholipids were examined by a combination of differential scanning calorimetry and (31)P NMR spectroscopy. All of the cationic lipids utilized in this study form only lamellar phases in isolation. Mixtures of these cationic lipids with zwitterionic strongly lamellar phase-preferring lipids such as phosphatidylcholine form only the lamellar liquid-crystalline phase even at high temperatures, as expected. Moreover, mixtures of these cationic lipids with strongly nonlamellar phase-preferring zwitterionic lipids such as phosphatidylethanolamine exhibit a markedly reduced propensity to form inverted nonlamellar phases, again as expected. However, when mixed with anionic lipids such as phosphatidylserine, phosphatidylglycerol, cardiolipin, or phosphatidic acid, a marked enhancement of nonlamellar phase-forming propensity occurs, despite the fact both components of the mixture are nominally lamellar phase-preferring. An examination of the lamellar/nonlamellar phase transition temperatures and the nature of the nonlamellar phases formed, as a function of temperature and of the composition of the mixture, indicates that the propensity to form inverted nonlamellar phases is maximal in mixtures where the mean surface charge of the membrane surface approaches neutrality and decreases markedly with increases in the density of positive or negative charge at the membrane surface. Moreover, the onset temperatures of the reversed hexagonal phase rise more steeply than do those of the inverted cubic phase as the ratio of cationic and anionic lipids is varied, suggesting that the formation of inverted hexagonal phases is more sensitive to this surface charge effect. These results indicate that surface charge per se is a significant and effective modulator of the lamellar/nonlamellar phase preferences of membrane lipids and that charged group interactions at membrane surfaces may have a major role in regulating this particular membrane property.

Topics: Anions; Calorimetry, Differential Scanning; Cations; Fatty Acids, Monounsaturated; Fluorescent Dyes; Glycerophospholipids; Lipid Bilayers; Magnetic Resonance Spectroscopy; Phosphatidic Acids; Phosphatidylethanolamines; Phosphatidylglycerols; Quaternary Ammonium Compounds; Structure-Activity Relationship; Surface Properties

The main steps in the construction of a computer model for a bacterial membrane are described. The membrane has been built of 72 lipid molecules, 54 of which being 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylethanolamine (POPE) and 18--1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidyl-rac-glycerol (POPG) molecules (thus in the proportion of 3:1). The membrane was hydrated with 1955 water molecules (approximately 27 water molecules per lipid). To neutralise the electronic charge (-e) on each POPG molecule, 18 sodium ions (Na+) were added to the membrane close to the POPG phosphate groups. The atomic charges on the POPE and POPG headgroups were obtained from ab initio quantum mechanical restrained electrostatic potential fitting (RESP) (Bayly et al., 1993, J. Phys. Chem. 97, 10269) using the GAMESS program at the 6-31G* level (Schmidt et al., 1993, J. Comput. Chem. 14, 1347). The model constructed in this way provided an initial structure for subsequent molecular dynamics simulation studies intended to elucidate the atomic level interactions responsible for the structure and dynamics of the bacterial membrane.

Topics: Bacteria; Computer Simulation; Membrane Lipids; Models, Molecular; Molecular Conformation; Phosphatidylethanolamines; Phosphatidylglycerols; Static Electricity; Thermodynamics; Water

PGLa, a 21-residue member of the magainin family of antibiotic peptides, is shown to be helical between residues 6 and 21 when associated with detergent micelles by multidimensional solution nuclear magnetic resonance (NMR) spectroscopy. Solid-state NMR experiments on specifically 15N-labeled peptides in oriented phospholipid bilayer samples show that the helix axis is parallel to the plane of the bilayers. 15N solid-state NMR powder pattern line shapes obtained on unoriented samples demonstrate that the amino-terminal residues are highly mobile and that the fluctuations of backbone sites decrease from Ala6 toward the carboxy terminus. The powder pattern observed for 15N-labeled Ala20 is essentially that expected for a rigid site. These findings are similar to those for the 23-residue magainin2 peptide in membrane environments.

Topics: Amino Acid Sequence; Antimicrobial Cationic Peptides; Lipid Bilayers; Micelles; Molecular Sequence Data; Nitrogen; Nuclear Magnetic Resonance, Biomolecular; Peptides; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Conformation; Protein Structure, Secondary; Solutions

Magainin 2, an antimicrobial peptide from the Xenopus skin, kills bacteria by permeabilizing the cell membranes. We have proposed that the peptide preferentially interacts with acidic phospholipids to form a peptide-lipid supramolecular complex pore, which allows mutually coupled transbilayer traffic of ions, lipids, and peptides, thus simultaneously dissipating transmembrane potential and lipid asymmetry [Matsuzaki, K., Murase, O., Fujii, N., and Miyajima, K. (1996) Biochemistry 35, 11361-11368]. In this paper, we examined the effect of membrane curvature strain on pore formation. Magainin effectively forms the pore only in phosphatidylglycerol bilayers at low peptide-to-lipid ratios, well below 1/100. In contrast, the permeabilization of phosphatidylserine, phosphatidic acid, or cardiolipin bilayers occurred at much higher peptide-to-lipid ratios (1/50 to 1/10) with some morphological change of the vesicles. The latter three classes of phospholipids are known to form hexagonal II structures under conditions of reduced interlipid electrostatic repulsions. Incorporation of phosphatidylethanolamine also inhibited the magainin-induced pore formation in the inhibitory order of dioleoylphosphatidylethanolamine > dielaidoylphosphatidylethanolamine. Addition of a small amount of palmitoyllysophosphatidylcholine enhanced the peptide-induced permeabilization of phosphatidylglycerol bilayers. Magainin greatly raised the bilayer to hexagonal II phase transition temperature of dipalmitoleoylphosphatidylethanolamine. These results suggest that the peptide imposes positive curvature strain, facilitating the formation of a torus-type pore, and that the presence of negative curvature-inducing lipids inhibits pore formation.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Circular Dichroism; Lipid Bilayers; Lysophosphatidylcholines; Magainins; Molecular Sequence Data; Peptides; Permeability; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Binding; Xenopus laevis; Xenopus Proteins

The membrane protein porin and a synthetic polypeptide of 21 hydrophobic residues were inserted into detergent micelles or lipid membranes, and the fluorescence of their single tryptophan residue was measured in the time-resolved and polarized mode. In all cases, the tryptophan fluorescence exhibits a long-lifetime component of about 20 ns. This long-lifetime component was exploited to detect slow orientational motions in the range of tens of nanoseconds via the anisotropy decay. For this purpose, the analysis of the anisotropy has to be extended to account for different orientations of the dipoles of the short- and long-lifetime components. This is demonstrated for porin and the polypeptide solubilized in micelles, in which the longest relaxation time reflects the rotational diffusion of the micelle. When the polypeptide is inserted into lipid membranes, it forms a membrane-spanning alpha-helix, and the slowest relaxation process is interpreted as reflecting orientational fluctuations of the helix.

Topics: Diffusion; Dimyristoylphosphatidylcholine; Fluorescence Polarization; Liposomes; Micelles; Models, Theoretical; Peptides; Phosphatidylethanolamines; Phosphatidylglycerols; Porins; Protein Conformation; Rhodobacter capsulatus; Rotation; Tryptophan