Anionic Lipid Monolayers Research Articles

Lipid membrane templated misfolding and self-assembly of intrinsically disordered tau protein

The aggregation of the intrinsically disordered tau protein into highly ordered β-sheet-rich fibrils is implicated in the pathogenesis of a range of neurodegenerative disorders. The mechanism of tau fibrillogenesis remains unresolved, particularly early events that trigger the misfolding and assembly of the otherwise soluble and stable tau. We investigated the role the lipid membrane plays in modulating the aggregation of three tau variants, the largest isoform hTau40, the truncated construct K18, and a hyperphosphorylation-mimicking mutant hTau40/3Epi. Despite being charged and soluble, the tau proteins were also highly surface active and favorably interacted with anionic lipid monolayers at the air/water interface. Membrane binding of tau also led to the formation of a macroscopic, gelatinous layer at the air/water interface, possibly related to tau phase separation. At the molecular level, tau assembled into oligomers composed of ~ 40 proteins misfolded in a β-sheet conformation at the membrane surface, as detected by in situ synchrotron grazing-incidence X-ray diffraction. Concomitantly, membrane morphology and lipid packing became disrupted. Our findings support a general tau aggregation mechanism wherein tau’s inherent surface activity and favorable interactions with anionic lipids drive tau-membrane association, inducing misfolding and self-assembly of the disordered tau into β-sheet-rich oligomers that subsequently seed fibrillation and deposition into diseased tissues.

X-Ray Reflectivity and Diffraction Studies of Doxorubicin Binding to Model Lipid Membranes

The effect of anticancer antibiotic doxorubicin on structural organization of anionic lipid monolayers has been studied. X-ray reflectivity and grazing incidence diffraction techniques were applied to monitor the changes in 2D structure and electron density distribution of Langmuir monolayer composed of negatively charged dipalmitoylphosphatidylglycerol (DPPG) and dioleoylphosphatidylserine (DOPS). For comparison, monolayer of zwitterionic dipalmitoylphosphatidylethanolamine (DPPE) also was investigated. The presented experimental results suggest that doxorubicin interaction with anionic lipid monolayers (DPPG and DOPS) proceeds preferentially via electrostatic attraction—positively charged amino groups of doxorubicin bind to negatively charged head groups of phospholipid molecules. Based on the obtained data, the penetration of doxorubicin into the hydrophobic part of anionic lipid monolayers does not occur. X-ray measurements on DPPE monolayer indicated that doxorubicin did not cause any significant alterations of molecular packing in condensed monolayer of zwitterionic DPPE molecules.

Fibrillar and Nonfibrillar Amyloid Beta Structures Drive Two Modes of Membrane-Mediated Toxicity.

In Alzheimer's disease, the amyloid-beta peptide (Aβ) is implicated in neuronal toxicity via interactions with the cell membrane. Monomeric Aβ (Aβm) is intrinsically disordered, but it can adopt a range of aggregated conformations with varying toxicities from short fibrillar oligomers (FO), to globular nonfibrillar oligomers (NFO), and full-length amyloid fibrils. NFO is considered to be the most toxic, followed by fibrils, and finally Aβm. To elucidate molecular-level membrane interactions that contribute to their different toxicities, we used liquid surface X-ray scattering and Langmuir trough insertion assays to compare Aβm, FO, and NFO surface activities and interactions with anionic DMPG lipid monolayers at the air/water interface. All Aβ species were highly surface active and rapidly adopted β-sheet rich structures upon adsorption to the air/water interface. Likewise, all Aβ species had affinity for the anionic membrane. Aβm rapidly converted to β-sheet rich assemblies upon binding the membrane, and these aggregated structures of Aβm and FO disrupted hexagonally packed lipid domains and resulted in membrane thinning and instability. In contrast, NFO perturbed membrane structure by extracting lipids from the air/water interface and causing macroscale membrane deformations. Altogether, our results support two models for membrane-mediated Aβ toxicity: fibril-induced reorganization of lipid packing and NFO-induced membrane destabilization and lipid extraction. This work provides a structural understanding of Aβ neurotoxicity via membrane interactions and aids the effort in understanding early events in Alzheimer's disease and other neurodegenerative diseases.

Interplay of Hydrophobic and Electrostatic Interactions between Polyoxometalates and Lipid Molecules

The Hofmeister series has long been of both scientific and technological importance because it allows systematic predictions of the interaction between ions and molecules to be made. In this work, three different polyoxometalate (POM) anions and three different lipid monolayers were used to investigate the interplay between electrostatic and hydrophobic interactions. We used three Keggin-type POMs with different charges ([PW12O40]3–, [SiW12O40]4–, and [H2W12O40]6–) to eliminate any effects of morphology and to focus on the effects of the charge and hydrophobicity of the POM anions. The POMs adsorb onto cationic lipid monolayers via electrostatic interactions, while hydrophobic interaction is the dominant factor for adsorption onto anionic lipid monolayers when charge neutralization is provided by cationic counterions. In contrast, the dominant interactions experienced by zwitterionic lipid monolayers can switch between electrostatic interactions for POMs with higher charge density and hydrophobic interact...

Effects of Concentration and Ionization Degree of Anchoring Cationic Polymers on the Lateral Heterogeneity of Anionic Lipid Monolayers.

We employed coarse-grained Monte Carlo simulations to investigate a system composed of cationic polymers and a phosphatidyl-choline membrane monolayer, doped with univalent anionic phosphatidylserine (PS) and tetravalent anionic phosphatidylinositol 4,5-bisphosphate (PIP2) lipid molecules. For this system, we consider the conditions under which multiple cationic polymers can anchor onto the monolayer and explore how the concentration and ionization degree of the polymers affect the lateral rearrangement and fluidity of the negatively charged lipids. Our work shows that the anchoring cationic polymers predominantly bind the tetravalent anionic PIP2 lipids and drag the PIP2 clusters to migrate on the monolayer. The polymer/PIP2 binding is found to be drastically enhanced by increasing the polymer ionization fraction, which causes the PIP2 lipids to form into larger clusters and reduces the mobility of the polymer/PIP2 complexes. As expected, stronger competition effects between anchoring polymers occur at higher polymer concentrations, for which each anchoring polymer partially dissociates from the monolayer and hence sequesters a smaller PIP2 cluster. The desorbed segments of the anchored polymers exhibit a faster mobility on the membrane, whereas the PIP2 clusters are closely restrained by the limited adhering cationic segments of anchoring polymers. We further demonstrate that the PIP2 molecules display a hierarchical mobility in the PIP2 clusters, which is regulated by the synergistic effect between the cationic segments of the polymers. The PS lipids sequester in the vicinity of the polymer/PIP2 complexes if the tetravalent PIP2 lipids cannot sufficiently neutralize the cationic polymers. Finally, we illustrate that the increase in the ionic concentration of the solution weakens the lateral clustering and the mobility heterogeneity of the charged lipids. Our work thus provides a better understanding of the fundamental biophysical mechanism of the concentration gradients and the hierarchical mobility of the anionic lipids in the membrane caused by the cationic polymer anchoring on length and time scales that are generally inaccessible by atomistic models. It also offers insight into the development and design of novel biological applications on the basis of the modulation of signaling lipids.

Monte Carlo study on a complex of cationic polymers and anionic lipid monolayer

Abstract We develop a coarse-grained Monte Carlo model for the anchoring of cationic polymers onto a phosphatidyl-choline (PC) lipid monolayer, doped with univalent phosphatidylserine (PS) and tetravalent phosphatidylinositol 4, 5-bisphosphate (PIP2) anionic lipids. Using this model, we extensively explore the effects of important factors on the structural alterations of the polymers/monolayer complex, including the polymer concentration, the polymer ionization fraction and the ionic concentration of the salt solution. We find the substantial disparity in the scaling of the anchoring/dissociation transition for polymers/monolayer complex and polyelectrolyte/surface system, which demonstrates that the mobile anionic monolayer exerts stronger attraction on cationic polymers than uniformly charged surface, thereby illustrating the significant predominance of PIP2 lipids in the anchoring procedure. In the polymer anchoring regime, increasing the polymer ionization fraction drastically strengthens the attractions between polymers and the monolayer at low polymer concentrations, which in turn results in the transformation of individual polymer chains from a brush-like structure to a pancake-like structure and leads to the enhancement of PIP2 sequestration. Elevating the polymer concentration strengthens the competition of the anchoring between individual polymers, and the fraction of anchored polymers can even saturate at sufficiently high polymer concentrations. At high polymer concentrations, this competition forces both weakly and strongly charged polymers to anchor onto the membrane in the brush-like conformation, thereby sequestering smaller PIP2 clusters. The PS lipids cluster around the polymer/PIP2 complexes when the amount of tetravalent PIP2 lipids is insufficient to neutralize the anchored cationic polymers. We also observe that the monolayer can be overcharged due to the anchoring of polymers at high polymer concentrations. Our work thus approaches an improved understanding of the anchoring processes from a fundamental perspective based on the estimates of the stability and the structural variations of the polymers/monolayer complex with important molecular factors.

Interaction of the Antimicrobial Peptides Rhesus θ-Defensin and Porcine Protegrin-1 with Anionic Phospholipid Monolayers.

A combination of Langmuir isotherm, Brewster angle microscopy (BAM), and neutron reflectivity studies have been performed to gain insight into the effects on model bacterial cell membranes of the antimicrobial peptides, Rhesus θ-defensin 1 (RTD-1), and porcine protegrin 1 (PG-1). The peptides were interacted with monolayers spread at the air-water interface and prepared from a 3:1 molar mixture of phosphatidylethanolamine and phosphatidylglycerol used to approximate the cell membranes of Gram positive bacteria. The Langmuir film balance measurements show that both peptides perturb the lipid monolayers causing an increase in surface pressure, and the BAM studies show that each results in the formation of small domains within the lipid films, around 5 μm diameter. The overall change in monolayer surface pressure caused by PG-1, however, is a little more pronounced than that due to RTD-1 (+8.5 mN·m(-1) vs +5.5 mN·m(-1)), and the rate of its initial interaction with the monolayer is a little more rapid than that for RTD-1. The neutron reflectivity studies also show differences for PG-1 and RTD-1, with the model fits to these data showing that the more amphiphilic PG-1 becomes fully embedded within the lipid film-causing an extension of the lipid acyl chains but leaving the thickness of the lipid headgroup layer unaffected-while RTD-1 is seen to insert less deeply-causing the same extension of the lipid acyl chains as PG-1 but also causing a significant increase in thickness of the lipid headgroup layer. The various differing effects of the two peptides on anionic lipid monolayers are discussed in the context of their differing hemolytic activities, and their proposed differing propensities to form transmembrane pores.

Spatial Rearrangement and Mobility Heterogeneity of an Anionic Lipid Monolayer Induced by the Anchoring of Cationic Semiflexible Polymer Chains.

We use Monte Carlo simulations to investigate the interactions between cationic semiflexible polymer chains and a model fluid lipid monolayer composed of charge-neutral phosphatidyl-choline (PC), tetravalent anionic phosphatidylinositol 4,5-bisphosphate (PIP2), and univalent anionic phosphatidylserine (PS) lipids. In particular, we explore how chain rigidity and polymer concentration influence the spatial rearrangement and mobility heterogeneity of the monolayer under the conditions where the cationic polymers anchor on the monolayer. We find that the anchored cationic polymers only sequester the tetravalent PIP2 lipids at low polymer concentrations, where the interaction strength between the polymers and the monolayer exhibits a non-monotonic dependence on the degree of chain rigidity. Specifically, maximal anchoring occurs at low polymer concentrations, when the polymer chains have an intermediate degree of rigidity, for which the PIP2 clustering becomes most enhanced and the mobility of the polymer/PIP2 complexes becomes most reduced. On the other hand, at sufficiently high polymer concentrations, the anchoring strength decreases monotonically as the chains stiffen—a result that arises from the pronounced competitions among polymer chains. In this case, the flexible polymers can confine all PIP2 lipids and further sequester the univalent PS lipids, whereas the stiffer polymers tend to partially dissociate from the monolayer and only sequester smaller PIP2 clusters with greater mobilities. We further illustrate that the mobility gradient of the single PIP2 lipids in the sequestered clusters is sensitively modulated by the cooperative effects between anchored segments of the polymers with different rigidities. Our work thus demonstrates that the rigidity and concentration of anchored polymers are both important parameters for tuning the regulation of anionic lipids.

Binding of the Cationic Peptide (KL)4K to Lipid Monolayers at the Air-Water Interface: Effect of Lipid Headgroup Charge, Acyl Chain Length, and Acyl Chain Saturation.

The binding of the cationic peptide (KL)4K to monolayers of different anionic lipids was determined by adsorption experiments. The chemical structure of the anionic phospholipids was changed in different ways. First, the hydrophobic region of phosphatidylglycerols was altered by elongation of the acyl chain length. Second, an unsaturated chain was introduced. Third, lipids with negatively charged headgroups of different chemical structure were compared. (KL)4K itself shows no surface activity and does not bind to monolayers of zwitterionic lipids. Analysis of (KL)4K binding to anionic lipid monolayers reveals a competition between two binding processes: (i) incorporation of the peptide into the acyl chain region (surface pressure increase) and (ii) electrostatic interaction screening the negative charges with reduction of charge repulsion (surface pressure decrease due to monolayer condensation). The lipid acyl chain length and the chemical structure of the headgroup have minor effects on the binding properties. However, a strong dependence on the phase state of the monolayer was observed. In the liquid-expanded (LE) phase, the fluid monolayer provides enough space, so that peptide insertion due to hydrophobic interactions dominates. For monolayers in the liquid-condensed (LC) phase, peptide binding followed by monolayer condensation is the main effect.

Interactions of Two Fragments of the Human Antimicrobial Peptide LL-37 with Zwitterionic and Anionic Lipid Monolayers

Abstract The interactions of two fragments of the human antimicrobial LL-37 (LL-32 and LL-20) with lipid monolayers at the soft liquid/air interface have been characterized. To model the interaction with mammalian cell membranes, lipid monolayers composed of the zwitterionic DPPC and DOPC were used. To investigate the interaction with bacterial cell membranes, lipid monolayers of anionic DPPG and POPG were used. DPPC and DPPG exhibit a first-order phase transition from the disordered liquid to the ordered condensed state, whereas POPG and DOPC monolayers are in the fluid disordered state at all surface pressures studied. Therefore, the influence of the monolayer phase state on peptide-lipid interactions can be studied. To obtain insight into the peptide structure and their influence on phospholipid membranes, film balance measurements were coupled with surface sensitive Infrared Reflection-Absorption Spectroscopy (IRRAS). The results were compared to CD measurements in bulk. LL-32 is more surface active and can better intercalate into lipid monolayers than LL-20. Even though LL-32 has no cell-selectivity, our results show how the peptide interacts differently with zwitterionic compared to anionic membrane models. The interaction with DPPC monolayers is based on simple intercalation of the peptides between the lipid molecules. However, the peptides bind in a two-step process to DPPG monolayers, which results in a fluidization of the lipid film. This can be related to a membrane thinning.

Susceptibility of sheep, human, and pig erythrocytes to haemolysis by the antimicrobial peptide Modelin 5

Modelin-5-CONH2, a synthetic antimicrobial peptide, was used to gain an insight into species-selective haemolytic activity. The peptide displayed limited haemolytic activity against sheep (12%), human (2%), and pig (2%) erythrocytes. Our results show that Modelin-5-CONH2 had a disordered structure in the presence of vesicles formed from sheep, human, and pig erythrocyte lipid extract (<26% helical) yet folded to form helices in the presence of a phosphatidylcholine (PC) membrane interface (e.g. >42% in the presence of 1,2-dimyristoyl-sn-glycero-3-phosphocholine). Monolayer studies showed a strong correlation between anionic lipid content and monolayer insertion and lysis inducing surface pressure changes of 9.17 mN m(-1) for 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine compared with PC monolayers, which induced pressure changes of ca. 3 mN m(-1). The presence of cholesterol in the membrane is shown to increase the packing density as the PC:sphingomyelin (SM) ratio increases so preventing the peptide from forming a stable association with the membrane. The data suggests that the key driver for membrane interaction for Modelin-5-CONH2 is the anionic lipid attraction. However, the key factors in the species-specific haemolysis level for this peptide are the differing packing densities which are influenced by the SM:PC:cholesterol ratio.

Influence of Arenicin on Phase Transitions and Ordering of Lipids in 2D Model Membranes

Antimicrobial peptides (AMPs) are important effector molecules of the innate immune system of all species. AMPs are highly selective and can be used as lead structures for the development of new drugs complementing standard antibiotic therapies. Understanding the crucial parameters of peptide-membrane interactions is necessary for elucidation of the molecular mechanisms of action. Phospholipid monolayers, as simple 2D models of the membrane surface, can be effectively used for studies of peptide-membrane interactions. The present study is focused on the recently discovered peptide arenicin-1 (Ar-1), which possesses antibacterial and antifungal activities. A linear derivative with serine residues instead of cysteines (C/S-Ar-1) was additionally used to investigate the influence of the AMP on the phase behavior of lipid monolayers at the air/liquid interface. Using the Langmuir balance technique and IRRAS allows us to conclude that both original and modified arenicins reveal a strong influence on the phase transition of anionic phospholipids (fluidization of the lipid hydrocarbon chains), whereas the thermodynamic properties of the zwitterionic phospholipid layers are not affected. A strong effect of the modified peptide on the ordering of negatively charged phospholipids at the air-water interface compared to zwitterionic phospholipids has been observed using GIXD measurements, supported by IRRAS simulations for the spectral range corresponding to the lipid hydrocarbon chains. At lateral pressures above 30 mN/m, both peptides are squeezed out from zwitterionic lipid monolayers, but remains attached to and partly incorporated in anionic lipid monolayers. This study points at the importance of the interplay between hydrophobic and electrostatic interactions for the membrane disruption by AMPs.

Membrane-Mediated Neuroprotection by Curcumin from Amyloid-β-Peptide-Induced Toxicity

Amyloid-β peptide (Aβ)-membrane interactions have been implicated in the formation of toxic oligomers that permeabilize membranes, allowing an influx of calcium ions and triggering cell death in the pathogenesis of Alzheimer's disease (AD). Curcumin, a small dietary polyphenolic molecule, has been shown to reduce Aβ-induced toxicity and AD pathology. We investigate here the effect of curcumin on Aβ40-induced toxicity in cultured human neuroblastoma SH-SY5Y cells and test a novel neuroprotection mechanism in which curcumin reduces Aβ-membrane interactions and attenuates Aβ-induced membrane disruptions. Predominantly monomeric Aβ40 exerts toxicity toward SH-SY5Y cells and has been shown to insert spontaneously into anionic lipid monolayers at the air/water interface, resulting in the misfolding and assembly of Aβ into β-sheet-enriched oligomers. Concomitantly, membrane morphology and lipid packing are disrupted. Curcumin dose-dependently ameliorates Aβ-induced neurotoxicity and reduces either the rate or extent of Aβ insertion into anionic lipid monolayers. Moreover, curcumin reduces Aβ-induced dye leakage from lipid-bilayer-covered, dye-loaded, porous silica microspheres. Because curcumin neither affects the inherent surface activity of Aβ nor modifies the membrane properties, it reduces Aβ insertion by directly attenuating Aβ-membrane interactions and reducing Aβ-induced membrane disruption. Although the exact molecular mechanism of curcumin's membrane protective effect remains unclear, this effect could in part contribute to curcumin's neuroprotective effect with respect to Aβ-induced toxicity. Our work reveals a novel molecular mechanism by which curcumin reduces Aβ-related pathology and toxicity and suggests a therapeutic strategy for preventing or treating AD by targeting the inhibition of Aβ-induced membrane disruption.

Interaction of Tau Protein with Model Lipid Membranes Induces Tau Structural Compaction and Membrane Disruption

The misfolding and aggregation of the intrinsically disordered, microtubule-associated tau protein into neurofibrillary tangles is implicated in the pathogenesis of Alzheimer's disease. However, the mechanisms of tau aggregation and toxicity remain unknown. Recent work has shown that anionic lipid membranes can induce tau aggregation and that membrane permeabilization may serve as a pathway by which protein aggregates exert toxicity, suggesting that the plasma membrane may play dual roles in tau pathology. This prompted our investigation to assess tau's propensity to interact with membranes and to elucidate the mutually disruptive structural perturbations the interactions induce in both tau and the membrane. We show that although highly charged and soluble, the full-length tau (hTau40) is also highly surface active, selectively inserts into anionic DMPG lipid monolayers and induces membrane morphological changes. To resolve molecular-scale structural details of hTau40 associated with lipid membranes, X-ray and neutron scattering techniques are utilized. X-ray reflectivity indicates hTau40s presence underneath a DMPG monolayer and penetration into the lipid headgroups and tailgroups, whereas grazing incidence X-ray diffraction shows that hTau40 insertion disrupts lipid packing. Moreover, both air/water and DMPG lipid membrane interfaces induce the disordered hTau40 to partially adopt a more compact conformation with density similar to that of a folded protein. Neutron reflectivity shows that tau completely disrupts supported DMPG bilayers while leaving the neutral DPPC bilayer intact. Our results show that hTau40s strong interaction with anionic lipids induces tau structural compaction and membrane disruption, suggesting possible membrane-based mechanisms of tau aggregation and toxicity in neurodegenerative diseases.

Role of the Side Chains in the Activity of Cyclic N-Substituted Glycine Oligomers

Non-natural mimics of antimicrobial peptides (AMPs) can be designed to display chemical moieties analogous to the active side chains of natural peptides, while its abiotic backbone provides protection from proteolytic degradation. A discrete conformational structure of a molecule is commonly accepted to be another prerequisite for potent antimicrobial activity. N-substituted glycine oligomers (peptoids) are among the most promising candidates as potential anti-infectious agents due to their inherent ease of structural optimization. One of the approaches to rigidify the structure of peptoids is to introduce a covalent limitation and make them cyclic. In the present work we investigate the role of side chains of three cyclic peptoids in their interactions with model bacterial and cell membranes. The outer leaflets of Gram-positive and Gram-negative bacterial membranes were modeled with DPPG and Lipid A-Kdo2 monolayers, respectively. The lipid monolayers at the air-liquid interface were studied using constant-pressure insertion assays, epifluorescence microscopy (EFM), synchrotron X-ray reflectivity (XR) and grazing incident-angle diffraction (GID). We have found that both the electrostatic and hydrophobic forces play important roles in defining interactions of the peptoids with anionic lipid monolayers. Peptoids’ aryl side chains are responsible for the hydrophobic interactions and serve as determinants for the differential activity of the peptidomimetics. Our data suggest that bulkier groups promote an enhanced peptoid insertion into the bacterial membrane monolayer mimics. This agrees well with the higher antibacterial activity displayed by these compounds, indicating that bulkier groups, indeed, help peptoids in adopting rigid bioactive conformation.

Adsorption behaviors and structural transitions of organic cations on an anionic lipid monolayer at the air–water interface

We studied the adsorption behaviors of organic cations, malachite green (MG), on an anionic dipalmitoylphosphatidylglycerol (DPPG) monolayer at the air–water interface for different thermodynamic phases of the lipid monolayer. Simultaneous monitoring of both the surface pressure and the optical second harmonic generation intensity, combined with the fluorescence microscopy and X-ray reflectivity measurement, gave detailed structural information on the adsorbed MG, showing three distinct types of adsorption behaviors depending on the initial phase of the monolayer. We provided plausible molecular models for the vertical position and orientation of MG in each phase: penetration into the DPPG monolayer with a vertical one direction at a fluid phase; vertically but oppositely paired orientations at a more condensed phase; parallel adsorption at the interface near the lipid headgroup region at a highly packed condensed phase. This result can be usefully expanded for the understanding of the penetration mechanism and conformational change of biological adsorbates at the cellular membrane.

Eudragit E100 surface activity and lipid interactions

Eudragit E100 (E100) is a cationic methacrylate polymer that interacts with viral and cell membranes. We studied the effect of pH, ionic strength and the presence of lipid monolayers on the surface activity of the polymer. E100 forms stable monolayers at the air–water interface, either by spreading or when added into the subphase. This behavior is highly influenced by the pH and saline concentration of the subphase. At pH 5 or higher, the adsorption of the polymer to the air–water interface begins immediately after its injection into the subphase, while at pH below 5 E100 remains in the subphase with a particularly slow adsorption to the interface. In addition, low ionic strength (10 mM) in the subphase results in a fast adsorption of the polymer to the interface, even at pH under 5. On the other hand, in the presence of non-ionic (cholesterol) or anionic (monosialoganglioside) lipid monolayers, E100 shows a fast adsorption to the interface, [comma] reaching surface pressures of 25 and 36 mN m −1, respectively. However, E100 barely interacts with monolayers of a zwitterionic lipid (hydrogenated soy lecithin) with a cut-off pressure of 11 mN m −1. The interaction of E100 with GM1 micelles in the subphase reduces its surface activity. Altogether these results show that E100 can effectively penetrate into model membranes and that its amphipathic character is largely dependent on the chemical composition of the aqueous environment and the lipid composition of the membrane.

Structural investigation on the adsorption of the MARCKS peptide on anionic lipid monolayers – effects beyond electrostatic

Abstract The presence of charged lipids in the cell membrane constitutes the background for the interaction with numerous membrane proteins. As a result, the valence of the lipids plays an important role concerning their lateral organization in the membrane and therefore the very manner of this interaction. This present study examines this aspect, particularly regarding to the interaction of the anionic lipid DPPS with the highly basic charged effector domain of the MARCKS protein, examined in monolayer model systems. Film balance, fluorescence microscopy and X-ray reflection/diffraction measurements were used to study the behavior of DPPS in a mixture with DPPC for its dependance on the presence of MARCKS (151–175). In the mixed monolayer, both lipids are completely miscible therefore DPPS is incorporated in the ordered crystalline DPPC domains as well. The interaction of MARCKS peptide with the mixed monolayer leads to the formation of lipid/peptide clusters causing an elongation of the serine group of the DPPS up to 7 Å in direction to surface normal into the subphase. The large cationic charge of the peptide pulls out the serine group of the interface which simultaneously causes an elongation of the phosphodiester group of the lipid fraction too. The obtained results were used to compare the interaction of MARCKS peptide with the polyvalent PIP 2 in mixed monolayers. On this way we surprisingly find out, that the relative small charge difference of the anionic lipids causes a significant different interaction with MARCKS (151–175). The lateral arrangement of the anionic lipids depends on their charge values and determines the diffusion of the electrostatic binding clusters within the membrane.

Effect of Point Mutations on the Secondary Structure and Membrane Interaction of Antimicrobial Peptide Anoplin

Anoplin (GLLKRIKTLL-NH(2)) is the smallest linear α-helical antimicrobial peptide found naturally to date. Antibacterial and hemolytic properties of anoplin depend strongly on physicochemical properties. Two anoplin derivatives, anoplin-8K (Ano8K, GLLKTIKKLL-NH(2)) and anoplin-1K5 V8K (Ano1K5 V8K, KLLKVIKLL-NH(2)), were found to have increased bacterial and low hemolytic activity. In the present work physicochemical properties of these three peptides were studied by UV resonance Raman (UVRR) spectroscopy, Langmuir-Blodgett monolayer technique, and carboxyfluorescein (CF) leakage assay. UVRR data indicated that all three peptides adopt predominantly unordered conformation in aqueous buffer solution. In membrane-mimicking trifluoroethanol, the α-helical content increases for all three peptides with Ano1K5 V8K having the highest α-helix percentage, followed by Ano8K and anoplin. Critical micelle concentrations were found to be similar for all three peptides, and the saturation pressure decreases in the sequence Ano1K5 V8K, anoplin, Ano8K. Critical pressure of insertion was found to be greater for anionic lipid monolayer DPPG than for zwitterionic lipid DPPC indicating preferential adsorption of all three peptides to DPPG. Finally, membrane lytic activities of all three peptides toward various model lipid vesicles were compared through CF leakage assay. Overall the data indicate that antimicrobial activity of anoplin increases with charge, whereas membrane lytic activity correlates with peptides helicity and amphipathicity.

Toxin Insertion Into Lipid Monolayers at the Air/Water Interface is Promoted by the Cognate Receptor Protein

Bacterial surfaces mediate many interactions important for health. We describe the production and analysis of a membrane protein containing lipid monolayer, at the air-water interface. Monolayers were formed by spreading a solution of outer membrane protein F (OmpF) / DPPG vesicles at the air-liquid interface. Monolayer structure was analysed parallel and perpendicular to the interface using surface pressure isotherms (SP), Brewster angle microscopy (BAM), see figure, and neutron reflectometry (NR). The domain structure of an OmpF containing monolayer was distinct from that of the protein free monolayer over all surface pressures. OmpF is the receptor for the antibacterial toxin colicin N (ColN) and, by an unknown mechanism, enables the toxin to translocate across the bacterial outer membrane barrier in order to kill E. coli cells. SP and NR data indicated that OmpF enabled ColN to penetrate deeply into the anionic lipid monolayer, and BAM showed a more selective interaction than that seen for lipid monolayers. This suite of methods including selective deuteration can thus provide membrane penetration data for a range of receptor-mediated translocation events in biology where resolution in multiple axes is required.