Lipopolysaccharide-Induced Dynamic Lipid Membrane Reorganization: Tubules,Perforations, and Stacks

Lipopolysaccharide (LPS) is a unique lipoglycan, with two major physiological roles: 1), as a major structural component of the outer membrane of Gram-negative bacteria and 2), as a highly potent mammalian toxin when released from cells into solution (endotoxin). LPS is an amphiphile that spontaneously inserts into the outer leaflet of lipid bilayers to bury its hydrophobic lipidic domain, leaving the hydrophilic polysaccharide chain exposed to the exterior polar solvent. Divalent cations have long been known to neutralize and stabilize LPS in the outer membrane, whereas LPS in the presence of monovalent cations forms highly mobile negatively-charged aggregates. Yet, much of our understanding of LPS and its interactions with the cell membrane does not take into account its amphiphilic biochemistry and charge polarization. Herein, we report fluorescence microscopy and atomic force microscopy analysis of the interaction between LPS and fluid-phase supported lipid bilayer assemblies (sLBAs), as model membranes. Depending on cation availability, LPS induces three remarkably different effects on simple sLBAs. Net-negative LPS-Na⁺ leads to the formation of 100-μm-long flexible lipid tubules from surface-associated lipid vesicles and the destabilization of the sLBA resulting in micron-size hole formation. Neutral LPS-Ca²⁺ gives rise to 100-μm-wide single- or multilamellar planar sheets of lipid and LPS formed from surface-associated lipid vesicles. Our findings have important implications about the physical interactions between LPS and lipids and demonstrate that sLBAs can be useful platforms to study the interactions of amphiphilic virulence factors with cell membranes. Additionally, our study supports the general phenomenon that lipids with highly charged or bulky headgroups can promote highly curved membrane architectures due to electrostatic and/or steric repulsions.     

Atomistic Scale Effects of Lipopolysaccharide Modifications on Bacterial Outer Membrane Defenses

Lipopolysaccharides (LPS) are a main constituent of the outer membrane of Gram-negative bacteria. Salmonella enterica, like many other bacterial species, are able to chemically modify the structure of their LPS molecules through the PhoPQ pathway as a defense mechanism against the host immune response. These modifications make the outer membrane more resistant to antimicrobial peptides (AMPs), large lipophilic drugs, and cation depletion, and are crucial for survival within a host organism. It is believed that these LPS modifications prevent the penetration of large molecules and AMPs through a strengthening of lateral interactions between neighboring LPS molecules. Here, we performed a series of long-timescale molecular dynamics simulations to study how each of three key S. enterica lipid A modifications affect bilayer properties, with a focus on membrane structural characteristics, lateral interactions, and the divalent cation bridging network. Our results discern the unique impact each modification has on strengthening the bacterial outer membrane through effects such as increased hydrogen bonding and tighter lipid packing. Additionally, one of the modifications studied shifts Ca²⁺ from the lipid A region, replacing it as a major cross-linking agent between adjacent lipids and potentially making bacteria less susceptible to AMPs that competitively displace cations from the membrane surface. These results further improve our understanding of outer membrane chemical properties and help elucidate how outer membrane modification systems, such as PhoPQ in S. enterica, are able to alter bacterial virulence.     

Structural and thermodynamic analyses of the interaction between melittin and lipopolysaccharide

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is the very first site of interactions with the antimicrobial peptides. In this work, we have determined a solution conformation of melittin, a well-known membrane active amphiphilic peptide from honey bee venom, by transferred nuclear Overhauser effect (Tr-NOE) spectroscopy in its bound state with lipopolysaccharide. The LPS bound conformation of melittin is characterized by a helical structure restricted only to the C-terminus region (residues A15-R24) of the molecule. Saturation transfer difference (STD) NMR studies reveal that several C-terminal residues of melittin including Trp19 are in close proximity with LPS. Isothermal titration calorimetry (ITC) data demonstrates that melittin binding to LPS or lipid A is an endothermic process. The interaction between melittin and lipid A is further characterized by an equilibrium association constant (K_a) of 2.85 × 10⁶ M^− 1 and a stoichiometry of 0.80, melittin/lipid A. The estimated free energy of binding (ΔG⁰), − 8.8 kcal mol^− 1, obtained from ITC experiments correlates well with a partial helical structure of melittin in complex with LPS. Moreover, a synthetic peptide fragment, residues L13-Q26 or mel-C, derived from the C-terminus of melittin has been found to contain comparable outer membrane permeabilizing activity against Escherichia coli cells. Intrinsic tryptophan fluorescence experiments of melittin and mel-C demonstrate very similar emission maxima and quenching in presence of LPS micelles. The Red Edge Excitation Shift (REES) studies of tryptophan residue indicate that both peptides are located in very similar environment in complex with LPS. Collectively, these results suggest that a helical conformation of melittin, at its C-terminus, could be an important element in recognition of LPS in the outer membrane.     

Lipid Clustering Correlates with Membrane Curvature as Revealed by Molecular Simulations of Complex Lipid Bilayers

Cell membranes are complex multicomponent systems, which are highly heterogeneous in the lipid distribution and composition. To date, most molecular simulations have focussed on relatively simple lipid compositions, helping to inform our understanding of in vitro experimental studies. Here we describe on simulations of complex asymmetric plasma membrane model, which contains seven different lipids species including the glycolipid GM3 in the outer leaflet and the anionic lipid, phosphatidylinositol 4,5-bisphophate (PIP₂), in the inner leaflet. Plasma membrane models consisting of 1500 lipids and resembling the in vivo composition were constructed and simulations were run for 5 µs. In these simulations the most striking feature was the formation of nano-clusters of GM3 within the outer leaflet. In simulations of protein interactions within a plasma membrane model, GM3, PIP₂, and cholesterol all formed favorable interactions with the model α-helical protein. A larger scale simulation of a model plasma membrane containing 6000 lipid molecules revealed correlations between curvature of the bilayer surface and clustering of lipid molecules. In particular, the concave (when viewed from the extracellular side) regions of the bilayer surface were locally enriched in GM3. In summary, these simulations explore the nanoscale dynamics of model bilayers which mimic the in vivo lipid composition of mammalian plasma membranes, revealing emergent nanoscale membrane organization which may be coupled both to fluctuations in local membrane geometry and to interactions with proteins.     

Functional reconstitution of a membrane protein in a diacetylenic polymerizable lecithin

It is shown that polymerized diacetylenic lecithins may be used for the functional reconstitution of a membrane protein. Purple membrane patches isolated from Halobacterium halobium and liposomes of the polymerizable diacetylenic lecithin 1,2-bis(10,12 tricosadiynoyl)-sn-glycero-3-phosphocholine were sonicated together to form mixed vesicles highly enriched in the polymerizable lipid. A net inward proton flow on illumination as determined by the change of pH of the external medium demonstrated the stability of the vesicular form in this mixed lipid system as well as vectorial orientation of the bacteriorhodopsin in the bilayer. When bacteriorhodopsin was incorporated in non-polymerizable lipids, irradiation with ultraviolet light resulted in complete loss of function. In the diacetylenic lipids, the loss of function was slower than the increase in polymer concentration. This demonstrates the utility of the diacetylenic lecithin system for study of interactions between membrane proteins and polymerizable lipids, as well as its potential in the development of biosensors based on membrane proteins.     

The Lipopeptide Antibiotic Paenibacterin Binds to the Bacterial Outer Membrane and Exerts Bactericidal Activity through Cytoplasmic Membrane Damage

Paenibacterin is a broad-spectrum lipopeptide antimicrobial agent produced by Paenibacillus thiaminolyticus OSY-SE. The compound consists of a cyclic 13-residue peptide and an N-terminal C₁₅ fatty acyl chain. The mechanism of action of paenibacterin against Escherichia coli and Staphylococcus aureus was investigated in this study. The cationic lipopeptide paenibacterin showed a strong affinity for the negatively charged lipopolysaccharides (LPS) from the outer membrane of Gram-negative bacteria. Addition of LPS (100 μg/ml) completely eliminated the antimicrobial activity of paenibacterin against E. coli. The electrostatic interaction between paenibacterin and LPS may have displaced the divalent cations on the LPS network and thus facilitated the uptake of antibiotic into Gram-negative cells. Paenibacterin also damaged the bacterial cytoplasmic membrane, as evidenced by the depolarization of membrane potential and leakage of intracellular potassium ions from cells of E. coli and S. aureus. Therefore, the bactericidal activity of paenibacterin is attributed to disruption of the outer membrane of Gram-negative bacteria and damage of the cytoplasmic membrane of both Gram-negative and Gram-positive bacteria. Despite the evidence of membrane damage, this study does not rule out additional bactericidal mechanisms potentially exerted by paenibacterin.     

Calcium Modulation of Ligand Affinity in the Cyclic GMP–gated Ion Channels of Cone Photoreceptors

To investigate modulation of the activation of cGMP-gated ion channels in cone photoreceptors, we measured currents in membrane patches detached from the outer segments of single cones isolated from striped bass retina. The sensitivity of these channels to activation by cGMP depends on the history of exposure to divalent cations of the membrane''s cytoplasmic surface. In patches maintained in 20 μM Ca⁺⁺ and 100 μM Mg⁺⁺ after excision, the current amplitude dependence on cGMP is well described by a Hill equation with average values of K _1/2, the concentration necessary to activate half the maximal current, of 86 μM and a cooperativity index, n, of 2.57. Exposing the patch to a solution free of divalent cations irreversibly increases the cGMP sensitivity; the average value of K _1/2 shifts to 58.8 μM and n shifts to 1.8. Changes in cGMP sensitivity do not affect other functional parameters of the ion channels, such as the interaction and permeation of mono- and divalent cations. Modulation of cGMP activation depends on the action of an endogenous factor that progressively dissociates from the channel as Ca⁺⁺ concentration is lowered below 1 μM. The activity of the endogenous modulator is not well mimicked by exogenously added calmodulin, although this protein competes with the endogenous modulator for a common binding site. Thus, the modulation of cGMP affinity in cones depends on the activity of an unidentified molecule that may not be calmodulin.     

Dynamics and Interactions of OmpF and LPS: Influence on Pore Accessibility and Ion Permeability

The asymmetric outer membrane of Gram-negative bacteria is formed of the inner leaflet with phospholipids and the outer leaflet with lipopolysaccharides (LPS). Outer membrane protein F (OmpF) is a trimeric porin responsible for the passive transport of small molecules across the outer membrane of Escherichia coli. Here, we report the impact of different levels of heterogeneity in LPS environments on the structure and dynamics of OmpF using all-atom molecular dynamics simulations. The simulations provide insight into the flexibility and dynamics of LPS components that are highly dependent on local environments, with lipid A being the most rigid and O-antigen being the most flexible. Increased flexibility of O-antigen polysaccharides is observed in heterogeneous LPS systems, where the adjacent O-antigen repeating units are weakly interacting and thus more dynamic, compared to homogeneous LPS systems in which LPS interacts strongly with each other with limited overall flexibility due to dense packing. The model systems were validated by comparing molecular-level details of interactions between OmpF surface residues and LPS core sugars with experimental data, establishing the importance of LPS core oligosaccharides in shielding OmpF surface epitopes recognized by monoclonal antibodies. There are LPS environmental influences on the movement of bulk ions (K⁺ and Cl⁻), but the ion selectivity of OmpF is mainly affected by bulk ion concentration.     

S-Adenosyl methionine synthetase 1 limits fat storage in Caenorhabditis elegans

Cytosolic lipid droplets are versatile, evolutionarily conserved organelles that are important for the storage and utilization of lipids in almost all cell types. To obtain insight into the physiological importance of lipid droplet size, we isolated and characterized a new S-adenosyl methionine synthetase 1 (SAMS-1)-deficient Caenorhabditis elegans mutant, which have enlarged lipid droplets throughout its life cycle. We found that the sams-1 mutant showed a markedly reduced body size and progeny number; impaired synthesis of phosphatidylcholine, a major membrane phospholipid; and elevated expression of key lipogenic genes, such as dgat-2, resulting in the accumulation of triacylglyceride in fewer, but larger, lipid droplets. The sams-1 mutant store more than 50 % (wild type: 10 %) of its intestinal fat in large lipid droplets, ≥10 μm³ in size. In response to starvation, SAMS-1 deficiency causes reduced depletion of a subset of lipid droplets located in the anterior intestine. Given the importance of liberation of fatty acids from lipid droplets, we propose that the physiological function of SAMS-1, a highly conserved enzyme involved in one-carbon metabolism, is the limitation of fat storage to ensure proper growth and reproduction.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0386-6) contains supplementary material, which is available to authorized users.     

The Influence of Natural Lipid Asymmetry upon the Conformation of a Membrane-inserted Protein (Perfringolysin O)

Eukaryotic membrane proteins generally reside in membrane bilayers that have lipid asymmetry. However, in vitro studies of the impact of lipids upon membrane proteins are generally carried out in model membrane vesicles that lack lipid asymmetry. Our recently developed method to prepare lipid vesicles with asymmetry similar to that in plasma membranes and with controlled amounts of cholesterol was used to investigate the influence of lipid composition and lipid asymmetry upon the conformational behavior of the pore-forming, cholesterol-dependent cytolysin perfringolysin O (PFO). PFO conformational behavior in asymmetric vesicles was found to be distinct both from that in symmetric vesicles with the same lipid composition as the asymmetric vesicles and from that in vesicles containing either only the inner leaflet lipids from the asymmetric vesicles or only the outer leaflet lipids from the asymmetric vesicles. The presence of phosphatidylcholine in the outer leaflet increased the cholesterol concentration required to induce PFO binding, whereas phosphatidylethanolamine and phosphatidylserine in the inner leaflet of asymmetric vesicles stabilized the formation of a novel deeply inserted conformation that does not form pores, even though it contains transmembrane segments. This conformation may represent an important intermediate stage in PFO pore formation. These studies show that lipid asymmetry can strongly influence the behavior of membrane-inserted proteins.     

The Antimicrobial Mechanism of Action of Epsilon-Poly-l-Lysine

Epsilon-poly-l-lysine (ε-PL) is a natural antimicrobial cationic peptide which is generally regarded as safe (GRAS) as a food preservative. Although its antimicrobial activity is well documented, its mechanism of action is only vaguely described. The aim of this study was to clarify ε-PL''s mechanism of action using Escherichia coli and Listeria innocua as model organisms. We examined ε-PL''s effect on cell morphology and membrane integrity and used an array of E. coli deletion mutants to study how specific outer membrane components affected the action of ε-PL. We furthermore studied its interaction with lipid bilayers using membrane models. In vitro cell studies indicated that divalent cations and the heptose I and II phosphate groups in the lipopolysaccharide layer of E. coli are critical for ε-PL''s binding efficiency. ε-PL removed the lipopolysaccharide layer and affected cell morphology of E. coli, while L. innocua underwent minor morphological changes. Propidium iodide staining showed that ε-PL permeabilized the cytoplasmic membrane in both species, indicating the membrane as the site of attack. We compared the interaction with neutral or negatively charged membrane systems and showed that the interaction with ε-PL relied on negative charges on the membrane. Suspended membrane vesicles were disrupted by ε-PL, and a detergent-like disruption of E. coli membrane was confirmed by atomic force microscopy imaging of supported lipid bilayers. We hypothesize that ε-PL destabilizes membranes in a carpet-like mechanism by interacting with negatively charged phospholipid head groups, which displace divalent cations and enforce a negative curvature folding on membranes that leads to formation of vesicles/micelles.     

A biochemical study of the reconstitution of d-lactate dehydrogenase-deficient membrane vesicles using fluorine-labeled components

Fluorine-19 labeled compounds have been incorporated into lipids and proteins of Escherichia coli. ¹⁹F-Labeled membrane vesicles, prepared by growing a fatty acid auxotroph of a d-lactate dehydrogenase-deficient strain on 8,8-difluoromyristic acid, can be reconstituted for oxidase and transport activities by binding exogenous d-lactate dehydrogenase. ¹⁹F-Labeled d-lactate dehydrogenases prepared by addition of fluorotryptophans to a tryptophan-requiring strain are able to reconstitute d-lactate dehydrogenase-deficient membrane vesicles. Thus, lipid and protein can be labeled independently and used to investigate protein-lipid interactions in membranes.     

In Intact Cone Photoreceptors,a Ca2+-dependent,Diffusible Factor Modulates the cGMP-gated Ion Channels Differently than in Rods

We investigated the modulation of cGMP-gated ion channels in single cone photoreceptors isolated from a fish retina. A new method allowed us to record currents from an intact outer segment while controlling its cytoplasmic composition by superfusion of the electropermeabilized inner segment. The sensitivity of the channels to agonists in the intact outer segment differs from that measured in membrane patches detached from the same cell. This sensitivity, measured as the ligand concentration necessary to activate half-maximal currents, K _1/2, also increases as Ca²⁺ concentration decreases. In electropermeabilized cones, K _1/2 for cGMP is 335.5 ± 64.4 μM in the presence of 20 μM Ca²⁺, and 84.3 ± 12.6 μM in its absence. For 8Br-cGMP, K _1/2 is 72.7 ± 11.3 μM in the presence of 20 μM Ca²⁺ and 15.3 ± 4.5 μM in its absence. The Ca²⁺-dependent change in agonist sensitivity is larger in extent than that measured in rods. In electropermeabilized tiger salamander rods, K _1/2 for 8Br-cGMP is 17.9 ± 3.8 μM in the presence of 20 μM Ca²⁺ and 7.2 ± 1.2 μM in its absence. The Ca²⁺-dependent modulation is reversible in intact cone outer segments, but is progressively lost in the absence of divalent cations, suggesting that it is mediated by a diffusible factor. Comparison of data in intact cells and detached membrane fragments from cones indicates that this factor is not calmodulin. At 40 μM 8Br-cGMP, the Ca²⁺-dependent change in sensitivity in cones is half-maximal at K _Ca = 286 ± 66 nM Ca²⁺. In rods, by contrast, K _Ca is ∼50 nM Ca²⁺. The difference in magnitude and Ca²⁺ dependence of channel modulation between photoreceptor types suggests that this modulation may play a more significant role in the regulation of photocurrent gain in cones than in rods.     

Annular Anionic Lipids Stabilize the Integrin αIIbβ3 Transmembrane Complex

Cationic membrane-proximal amino acids determine the topology of membrane proteins by interacting with anionic lipids that are restricted to the intracellular membrane leaflet. This mechanism implies that anionic lipids interfere with electrostatic interactions of membrane proteins. The integrin αIIbβ3 transmembrane (TM) complex is stabilized by a membrane-proximal αIIb(Arg⁹⁹⁵)-β3(Asp⁷²³) interaction; here, we examine the influence of anionic lipids on this complex. Anionic lipids compete for αIIb(Arg⁹⁹⁵) contacts with β3(Asp⁷²³) but paradoxically do not diminish the contribution of αIIb(Arg⁹⁹⁵)-β3(Asp⁷²³) to TM complex stability. Overall, anionic lipids in annular positions stabilize the αIIbβ3 TM complex by up to 0.50 ± 0.02 kcal/mol relative to zwitterionic lipids in a headgroup structure-dependent manner. Comparatively, integrin receptor activation requires TM complex destabilization of 1.5 ± 0.2 kcal/mol, revealing a sizeable influence of lipid composition on TM complex stability. We implicate changes in lipid headgroup accessibility to small molecules (physical membrane characteristics) and specific but dynamic protein-lipid contacts in this TM helix-helix stabilization. Thus, anionic lipids in ubiquitous annular positions can benefit the stability of membrane proteins while leaving membrane-proximal electrostatic interactions intact.     

The interplay between lipids and dopamine on α-synuclein oligomerization and membrane binding

The deposition of α-syn (α-synuclein) as amyloid fibrils and the selective loss of DA (dopamine) containing neurons in the substantia nigra are two key features of PD (Parkinson''s disease). α-syn is a natively unfolded protein and adopts an α-helical conformation upon binding to lipid membrane. Oligomeric species of α-syn have been proposed to be the pathogenic species associated with PD because they can bind lipid membranes and disrupt membrane integrity. DA is readily oxidized to generate reactive intermediates and ROS (reactive oxygen species) and in the presence of DA, α-syn form of SDS-resistant soluble oligomers. It is postulated that the formation of the α-syn:DA oligomers involves the cross-linking of DA-melanin with α-syn, via covalent linkage, hydrogen and hydrophobic interactions. We investigate the effect of lipids on DA-induced α-syn oligomerization and studied the ability of α-syn:DA oligomers to interact with lipids vesicles. Our results show that the interaction of α-syn with lipids inhibits the formation of DA-induced α-syn oligomers. Moreover, the α-syn:DA oligomer cannot interact with lipid vesicles or cause membrane permeability. Thus, the formation of α-syn:DA oligomers may alter the actions of α-syn which require membrane association, leading to disruption of its normal cellular function.     

Inhibition of the phosphoinositide 3-kinase pathway decreases innate resistance to lipopolysaccharide toxicity in TLR4 deficient mice

Background

Upon lipopolysaccharide (LPS) stimulation, activation of both the Toll-like receptor 4 (TLR4) and phosphoinositide 3-kinase (PI3K) pathways serves to balance proinflammatory and anti-inflammatory responses. Although the antagonist to TLR4 represents an emerging promising target for the treatment of sepsis; however, the role of the PI3K pathway under TLR4-null conditions is not well understood. This goal of this study was to investigate the effect of inhibition of PI3K on innate resistance to LPS toxicity in a murine model.

Results

The overall survival of the cohorts receiving intraperitoneal injections of 100, 500, or 1000 μg LPS from Escherichia coli serotype 026:B6 after 7 d was 100%, 10%, and 10%, respectively. In contrast, no mortality was noted after 500-μg LPS injection in Tlr4^-/- mice. When the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 was injected (1 mg/25 g body weight) 1 h prior to the administration of LPS, the overall survival of the Tlr4^-/- mice was 30%. In the Tlr4^-/- mice, the LPS injection induced no NF-κB activation but an increased Akt phosphorylation in the lung and liver, when compared to that of the C57BL/6 mice. Injection of 500 μg LPS led to a significant induction in O₂^- detected by electron paramagnetic resonance (EPR) spin trapping spectroscopy in the lung and liver at 3 and 6 h in C57BL/6 but not Tlr4^-/- mice. Addition of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 only significantly increased the O₂^- level in the lung and liver of the Tlr4^-/- mice but not in the C57BL/6 mice following 500-μg LPS injection. In addition, the serum IL-1β and IL-2 levels were more elevated in C57BL/6 mice than in Tlr4^-/- mice. Notably, IL-1β and IL-2 were significantly increased in Tlr4^-/- mice but not in the C57BL/6 mice when the PI3K pathway was inhibited by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 prior to LPS injection.

Conclusions

In this study, we demonstrate that innate resistance to LPS toxicity in Tlr4^-/- mice is impaired by inhibition of the PI3K pathway, with a corresponding increase in mortality and production of tissue O₂^- and inflammatory cytokines.     

Pardaxin Permeabilizes Vesicles More Efficiently by Pore Formation than by Disruption

Pardaxin is a 33-amino-acid neurotoxin from the Red Sea Moses sole Pardachirus marmoratus, whose mode of action shows remarkable sensitivity to lipid chain length and charge, although the effect of pH is unclear. Here we combine optical spectroscopy and dye release experiments with laser scanning confocal microscopy and natural abundance ¹³C solid-state nuclear magnetic resonance to provide a more complete picture of how pardaxin interacts with lipids. The kinetics and efficiency of release of entrapped calcein is highly sensitive to pH. In vesicles containing zwitterionic lipids (PC), release occurs most rapidly at low pH, whereas in vesicles containing 20% anionic lipid (PG), release occurs most rapidly at high pH. Pardaxin forms stable or transient pores in PC vesicles that allow release of contents without loss of vesicle integrity, whereas the inclusion of PG promotes total vesicle collapse. In agreement with this, solid-state nuclear magnetic resonance reveals that pardaxin takes up a trans-membrane orientation in 14-O-PC/6-O-PC bicelles, whereas the inclusion of 14-0-PG restricts it to contacts with lipid headgroups, promoting membrane lysis. Pore formation in zwitterionic vesicles is more efficient than lysis of anionic vesicles, suggesting that electrostatic interactions may trap pardaxin in several suboptimal interconverting conformations on the membrane surface.     

Remodeling of Lipid Vesicles into Cylindrical Micelles by α-Synuclein in an Extended α-Helical Conformation

α-Synuclein (αS) is a protein with multiple conformations and interactions. Natively unfolded in solution, αS accumulates as amyloid in neurological tissue in Parkinson disease and interacts with membranes under both physiological and pathological conditions. Here, we used cryoelectron microscopy in conjunction with electron paramagnetic resonance (EPR) and other techniques to characterize the ability of αS to remodel vesicles. At molar ratios of 1:5 to 1:40 for protein/lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol), large spherical vesicles are converted into cylindrical micelles ∼50 Å in diameter. Other lipids of the same charge (negative) exhibit generally similar behavior, although bilayer tubes of 150–500 Å in width are also produced, depending on the lipid acyl chains. At higher protein/lipid ratios, discoid particles, 70–100 Å across, are formed. EPR data show that, on cylindrical micelles, αS adopts an extended amphipathic α-helical conformation, with its long axis aligned with the tube axis. The observed geometrical relationship between αS and the micelle suggests that the wedging of its long α-helix into the outer leaflet of a membrane may cause curvature and an anisotropic partition of lipids, leading to tube formation.     

The interaction of spectrin · actin and synthetic phospholipids

Using differential scanning calorimetry and freeze fracture electron microscopy interactions were studied between lipids and a spectrin · action complex isolated from human erythrocyte membranes. With dispersions of $\text{1,2-}\text{dimyristoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$, $\text{1,2-}\text{dimyristoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphoglycerol}$ and mixtures of these two compounds, which for experimental reasons were chosen as the lipid counterpart, such an interaction could clearly be deduced from changes in the temperature and the enthalpy of the phase transition. Furthermore it was demonstrated that the interaction with this membrane protein protects the bilayer against the action of Ca²⁺ and Mg²⁺ and prevents fusion of lipid vesicles which easily occurs in some of the systems when divalent ions were added to the pure lipid vesicles.     

Progressive structuring of a branched antimicrobial Peptide on the path to the inner membrane target

In recent years, interest has grown in the antimicrobial properties of certain natural and non-natural peptides. The strategy of inserting a covalent branch point in a peptide can improve its antimicrobial properties while retaining host biocompatibility. However, little is known regarding possible structural transitions as the peptide moves on the access path to the presumed target, the inner membrane. Establishing the nature of the interactions with the complex bacterial outer and inner membranes is important for effective peptide design. Structure-activity relationships of an amphiphilic, branched antimicrobial peptide (B2088) are examined using environment-sensitive fluorescent probes, electron microscopy, molecular dynamics simulations, and high resolution NMR in solution and in condensed states. The peptide is reconstituted in bacterial outer membrane lipopolysaccharide extract as well as in a variety of lipid media mimicking the inner membrane of Gram-negative pathogens. Progressive structure accretion is observed for the peptide in water, LPS, and lipid environments. Despite inducing rapid aggregation of bacteria-derived lipopolysaccharides, the peptide remains highly mobile in the aggregated lattice. At the inner membranes, the peptide undergoes further structural compaction mediated by interactions with negatively charged lipids, probably causing redistribution of membrane lipids, which in turn results in increased membrane permeability and bacterial lysis. These findings suggest that peptides possessing both enhanced mobility in the bacterial outer membrane and spatial structure facilitating its interactions with the membrane-water interface may provide excellent structural motifs to develop new antimicrobials that can overcome antibiotic-resistant Gram-negative pathogens.