Neutral Membranes Research Articles

Membrane-binding properties of gating modifier and pore-blocking toxins: Membrane interaction is not a prerequisite for modification of channel gating

Many venom peptides are potent and selective inhibitors of voltage-gated ion channels, including channels that are validated therapeutic targets for treatment of a wide range of human diseases. However, the development of novel venom-peptide-based therapeutics requires an understanding of their mechanism of action. In the case of voltage-gated ion channels, venom peptides act either as pore blockers that bind to the extracellular side of the channel pore or gating modifiers that bind to one or more of the membrane-embedded voltage sensor domains. In the case of gating modifiers, it has been debated whether the peptide must partition into the membrane to reach its binding site. In this study, we used surface plasmon resonance, fluorescence spectroscopy and molecular dynamics to directly compare the lipid-binding properties of two gating modifiers (μ-TRTX-Hd1a and ProTx-I) and two pore blockers (ShK and KIIIA). Only ProTx-I was found to bind to model membranes. Our results provide further evidence that the ability to insert into the lipid bilayer is not a requirement to be a gating modifier. In addition, we characterised the surface of ProTx-I that mediates its interaction with neutral and anionic phospholipid membranes and show that it preferentially interacts with anionic lipids.

Membrane-Binding Properties of Gating-Modifier and Pore Blocking Toxins: Membrane Interaction is not a Prerequisite for Modification of Channel Gating

Many venom peptides are potent and selective inhibitors of voltage-gated ion channels, including channels that are validated therapeutic targets for treatment of a wide range of human diseases. However, the development of novel venom-peptide-based therapeutics requires an understanding of their mechanism of action. In the case of voltage-gated ion channels, venom peptides act either as pore blockers that plug the extracellular face of the channel pore or gating modifiers that bind to one or more of the membrane-embedded voltage sensor domains. It has been debated whether membrane partitioning is an intrinsic feature of gating modifier toxins. In this study we used surface plasmon resonance and molecular dynamics to directly compare the lipid binding properties of two gating modifiers (μ-TRTX-Hd1a and ProTx-I) and two pore-blockers (ShK and KIIIA). Of the peptides tested only ProTx-I showed concentration-dependent binding to phospholipid model membranes. Our results provide further evidence that the ability to insert into the lipid bilayer is not a requirement to be a gating modifier. In addition, we have characterised the lipid interaction surface of ProTx1 to neutral and anionic phospholipid membranes.

Analysis of constant tension-induced rupture of lipid membranes using activation energy.

The stretching of biomembranes and lipid membranes plays important roles in various physiological and physicochemical phenomena. Here we analyzed the rate constant kp of constant tension-induced rupture of giant unilamellar vesicles (GUVs) as a function of tension σ using their activation energy Ua. To determine the values of kp, we applied constant tension to a GUV membrane using the micropipette aspiration method and observed the rupture of GUVs, and then analyzed these data statistically. First, we investigated the temperature dependence of kp for GUVs of charged lipid membranes composed of negatively charged dioleoylphosphatidylglycerol (DOPG) and electrically neutral dioleoylphosphatidylcholine (DOPC). By analyzing this result, the values of Ua of tension-induced rupture of DOPG/DOPC-GUVs were obtained. Ua decreased with an increase in σ, supporting the classical theory of tension-induced pore formation. The analysis of the relationship between Ua and σ using the theory on the electrostatic interaction effects on the tension-induced rupture of GUVs provided the equation of Ua including electrostatic interaction effects, which well fits the experimental data of the tension dependence of Ua. A constant which does not depend on tension, U0, was also found to contribute significantly to Ua. The Arrhenius equations for kp using the equation of Ua and the parameters determined by the above analysis fit well to the experimental data of the tension dependence of kp for DOPG/DOPC-GUVs as well as for DOPC-GUVs. On the basis of these results, we discussed the possible elementary processes underlying the tension-induced rupture of GUVs of lipid membranes. These results indicate that the Arrhenius equation using the experimentally determined Ua is useful in the analysis of tension-induced rupture of GUVs.

Improved chlorine resistance of polyamide thin-film composite membranes with a terpolymer coating

Abstract Polyamide (PA) thin-film composite (TFC) membranes are easily degraded under the attack of active chlorine in feed water or during chemical washing. In the present work, a random terpolymer of 2-acrylamido-2-methyl propanesulfonic acid, acrylamide and 1-vinylimidazole (P(AMPS-co-Am-co-VI)) was designed as a coating material to improve the chlorine tolerance of PA TFC membrane. The terpolymer was first synthesized via conventional free radical polymerization and then coated onto commercial available PA TFC membrane by dip-coating process. The changes in surface chemistry, morphology, hydrophilicity/hydrophobicity and charge characteristics of the membrane after surface coating were investigated in detail. It was shown that the incorporation of terpolymer coating created a more neutral, hydrophilic, and smooth membrane surface. The chlorine resistance of the membranes was evaluated by comparing the permeation and separation properties of the unmodified and modified membranes after chlorine exposure. And the experimental results indicated that the chlorine tolerance of PA TFC membranes was improved significantly, especially in acid environment. The coating layer prevents the selective layer from the attack of active chlorine as a barrier layer more than a sacrificial layer. The coating material and method developed in this work can facilitate the design and manufacture of highly chlorine resistant TFC membrane for seawater desalination and industrial separation.

Biomarker validity in the critically ill: all must face the (continuous) renal replacement challenge!

We embrace with enthusiasm the recent study by Roderburg et al. [1] identifying osteopontin (OPN) as a valuable early prognostic biomarker in critically ill patients. Of note is that these investigators did not provide details on the incidence of acute kidney injury or the need for renal replacement therapy (RRT) in their patient population. We recently argued that currently used markers of systemic inflammation such as C-reactive protein and procalcitonin are substantially cleared during continuous renal replacement therapy (CRRT) and thus may lose sensitivity to evaluate the degree and evolution of inflammation [2]. OPN is a highly negatively charged protein. Its nascent molecular weight (MW) approximates 32 kDa with slight variations due to post-translational modification or proteolytic cleavage [3]. OPN is not eliminated by slow extended dialysis [4]. This is not surprising, however, because the 5 kDa membrane cutoff point for molecular diffusion with this technique lies largely below the MW of OPN. Theoretically, continuous hemofiltration may remove OPN from the circulation but evidence is lacking. Moreover, CRRT is increasingly performed with novel membranes such as the surface-treated acrylonitrile 69 membrane [5]. Surface treatment implies coating with a polyethylene imine biopolymer resulting in a more neutral membrane surface composed of areas with a high density of positive charges. In addition to being highly biocompatible and permeable, this membrane displays potent adsorptive capacity. More information regarding an eventual significant “loss” of OPN by filtration and/or membrane adsorption during CRRT is imperative before this protein can be accepted as a valid prognostic biomarker.

Interaction study between maltose-modified PPI dendrimers and lipidic model membranes

The influence of maltose-modified poly(propylene imine) (PPI) dendrimers on dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) (3%) liposomes was studied. Fourth generation (G4) PPI dendrimers with primary amino surface groups were partially (open shell glycodendrimers — OS) or completely (dense shell glycodendrimers — DS) modified with maltose residues. As a model membrane, two types of 100nm diameter liposomes were used to observe differences in the interactions between neutral DMPC and negatively charged DMPC/DMPG bilayers. Interactions were studied using fluorescence spectroscopy to evaluate the membrane fluidity of both the hydrophobic and hydrophilic parts of the lipid bilayer and using differential scanning calorimetry to investigate thermodynamic parameter changes. Pulsed-filed gradient NMR experiments were carried out to evaluate common diffusion coefficient of DMPG and DS PPI in D2O when using below critical micelle concentration of DMPG. Both OS and DS PPI G4 dendrimers show interactions with liposomes. Neutral DS dendrimers exhibit stronger changes in membrane fluidity compared to OS dendrimers. The bilayer structure seems more rigid in the case of anionic DMPC/DMPG liposomes in comparison to pure and neutral DMPC liposomes. Generally, interactions of dendrimers with anionic DMPC/DMPG and neutral DMPC liposomes were at the same level. Higher concentrations of positively charged OS dendrimers induced the aggregation process with negatively charged liposomes. For all types of experiments, the presence of NaCl decreased the strength of the interactions between glycodendrimers and liposomes. Based on NMR diffusion experiments we suggest that apart from electrostatic interactions for OS PPI hydrogen bonds play a major role in maltose-modified PPI dendrimer interactions with anionic and neutral model membranes where a contact surface is needed for undergoing multiple H-bond interactions between maltose shell of glycodendrimers and surface membrane of liposome.

Reconstitution of Adiposome and Artificial Lipid Droplets

The Lipid droplet (LD) is a cellular organelle that consists of a neutral lipid core with a phospholipid monolayer membrane associated with proteins. The presence of this organelle in organisms ranging from bacteria to humans demonstrates that it is highly conserved and may be the first membrane‐bound organelle in living organisms. Recent progress in LD research demonstrates its important roles in energy homeostasis and metabolic syndromes. However, the mechanisms governing its formation, dynamics, interaction with other cellular organelles, and association with proteins have remained elusive. Therefore, we developed an in vitro system to facilitate the elucidation of these mechanisms, based on the reconstitution of artificial LDs. First, we generated sphere‐shaped structures with a neutral lipid core and phospholipid monolayer membrane by mechanically mixing neutral lipids and phospholipids followed by two‐step purification. Since the artificial lipid vesicle, a double layer phospholipid membrane structure, is termed as “liposome”, we named this monolayer phospholipid membrane‐bound structure “adiposome”. We then recruited LD structure‐like/resident proteins to the adiposome, including the bacterial LD protein MLDS, C. elegans LD protein MDT‐28, or mammalian LD protein ADRP/PLIN2. We term the functional protein‐coated adiposomes, “artificial lipid droplets”. Using this experimental system, different proteins can be recruited to build artificial LDs for myriad biological and medical goals.

Spontaneous adsorption of coiled-coil model peptides K and E to a mixed lipid bilayer.

A molecular description of the lipid-protein interactions underlying the adsorption of proteins to membranes is crucial for understanding, for example, the specificity of adsorption or the binding strength of a protein to a bilayer, or for characterizing protein-induced changes of membrane properties. In this paper, we extend an automated in silico assay (DAFT) for binding studies and apply it to characterize the adsorption of the model fusion peptides E and K to a mixed phospholipid/cholesterol membrane using coarse-grained molecular dynamics simulations. In addition, we couple the coarse-grained protocol to reverse transformation to atomistic resolution, thereby allowing to study molecular interactions with high detail. The experimentally observed differential binding of the peptides E and K to membranes, as well as the increased binding affinity of helical over unstructered peptides, could be well reproduced using the polarizable Martini coarse-grained (CG) force field. Binding to neutral membranes is shown to be dominated by initial binding of the positively charged N-terminus to the phospholipid headgroup region, followed by membrane surface-aligned insertion of the peptide at the interface between the hydrophobic core of the membrane and its polar headgroup region. Both coarse-grained and atomistic simulations confirm a before hypothesized snorkeling of lysine side chains for the membrane-bound state of the peptide K. Cholesterol was found to be enriched in peptide vicinity, which is probably of importance for the mechanism of membrane fusion. The applied sequential multiscale method, using coarse-grained simulations for the slow adsorption process of peptides to membranes followed by backward transformation to atomistic detail and subsequent atomistic simulations of the preformed peptide-lipid complexes, is shown to be a versatile approach to study the interactions of peptides or proteins with biomembranes.

New aspects of the structure and mode of action of the human cathelicidin LL-37 revealed by the intrinsic probe p-cyanophenylalanine.

The human cathelicidin peptide LL-37 is an important effector of our innate immune system and contributes to host defence with direct antimicrobial activity and immunomodulatory properties, and by stimulating wound healing. Its sequence has evolved to confer specific structural characteristics that strongly affect these biological activities, and differentiate it from orthologues of other primate species. In the present paper we report a detailed study of the folding and self-assembly of this peptide in comparison with rhesus monkey peptide RL-37, taking into account the different stages of its trajectory from bulk solution to contact with, and insertion into, biological membranes. Phenylalanine residues in different positions throughout the native sequences of LL-37 and RL-37 were systematically replaced with the non-invasive fluorescent and IR probe p-cyanophenylalanine. Steady-state and time-resolved fluorescence studies showed that LL-37, in contrast to RL-37, forms oligomers with a loose hydrophobic core in physiological solutions, which persist in the presence of biological membranes. Fourier transform IR and surface plasmon resonance studies also indicated different modes of interaction for LL-37 and RL-37 with anionic and neutral membranes. This correlated with a distinctly different mode of bacterial membrane permeabilization, as determined using a flow cytometric method involving impermeant fluorescent dyes linked to polymers of defined sizes.

A Study of How Chelating Agents Interact with Neutral Lipid Membranes

Chelating agents are used in a range of areas, from treating metal poisoning in medicine to removing unwanted heavy metal ions from a given solution in biochemistry. Specifically, ethylenediaminetetraacetic acid (EDTA) is widely used in biochemistry and other areas of science to sequester polyvalent cations such as Ca2+ and Fe3+ from solutions. Small angle x-ray scattering [1] has shown that low concentrations (mM range) of EDTA in solution introduce a phase coexistence in homogeneous 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) multilamellar vesicles. This suggests that there are interactions taking place between EDTA and the lipid head group. We investigate the interaction of EDTA with lipids in the presence of various cations, using 1H NMR. A discussion of the interactions present between EDTA and lipids will be formulated based on observed proton chemical shifts and relaxation rates.[1] Johnson et al, Langmuir 2014.

Methyl-branched lipids promote the membrane adsorption of α-synuclein by enhancing shallow lipid-packing defects.

Alpha-synuclein (AS) is a synaptic protein that is directly involved in Parkinson's disease due to its tendency to form protein aggregates. Since AS aggregation can be dependent on the interactions between the protein and the cell plasma membrane, elucidating the membrane binding properties of AS is of crucial importance to establish the molecular basis of AS aggregation into toxic fibrils. Using a combination of in vitro reconstitution experiments based on Giant Unilamellar Vesicles (GUVs), confocal microscopy and all-atom molecular dynamics simulations, we have investigated the membrane binding properties of AS, with a focus on the relative contribution of hydrophobic versus electrostatic interactions. In contrast with previous observations, we did not observe any binding of AS to membranes containing the ganglioside GM1, even at relatively high GM1 content. AS, on the other hand, showed a stronger affinity for neutral flat membranes consisting of methyl-branched lipids. To rationalize these results, we used all-atom molecular dynamics simulations to investigate the influence of methyl-branched lipids on interfacial membrane properties. We found that methyl-branched lipids promote the membrane adsorption of AS by creating shallow lipid-packing defects to a larger extent than polyunsaturated and monounsaturated lipids. Our findings suggest that methyl-branched lipids may constitute a remarkably adhesive substrate for peripheral proteins that adsorb on membranes via hydrophobic insertions.

Correlated Motion and Complex Formation of Lipid-Raft Components Analyzed by High-Resolution Secondary Ion Mass Spectrometry

It is widely believed that certain membrane lipids and membrane-anchored proteins associate to form clusters with emergent function. While extensive data on phase diagrams are available for a range of lipid compositions, these are most commonly visualized by the partitioning of dyes between different phases. We have been developing the use of secondary ion mass spectrometry imaging to directly visualize molecules of interest within supported lipid bilayers (Lozano et al. JACS 2013). Specifically, we want to address the question which neutral membrane components associate with the well-known lipid-raft marker ganglioside GM1, which has a single negative charge, after GM1 is reorganized by an in-plane electric field on a patterned supported bilayer (SLB). NanoSIMS imaging was used to generate molecule-specific concentration profiles of several SLB compositions following electrophoresis: GM1/DOPC, GM1/CHOL/DOPC, and GM1/CHOL/PSM/DOPC. In a control SLB sample without GM1 it is observed that CHOL, PSM, and DOPC do not reorganize by membrane electrophoresis. However, NanoSIMS images clearly illustrate that PSM and CHOL associate with GM1 as it is transported toward the positive electrode while DOPC is displaced in the opposite direction towards the negative electrode. Further analysis of the concentration profiles for the co-diffusing components (GM1/CHOL/PSM) suggest the formation of a 4:2:1 PSM:GM1:CHOL stoichiometric complex. These results demonstrate that both cholesterol and sphingomyelin do tend to associate with GM1 and we might speculate that similar behavior would be observed for GPI or myristolyated proteins.

Implicit Membrane Investigation of the Stability of Antimicrobial Peptide β-Barrels and Arcs.

Previous simulations showed that the β-hairpin antimicrobial peptide (AMP) protegrin-1 can form stable octameric β-barrels and tetrameric arcs (half barrels) in both implicit and explicit membranes. Here, we extend this investigation to several AMPs of similar structure: tachyplesin, androctonin, polyphemusin, gomesin, and the retrocyclin θ-defensin. These peptides form short β-hairpins stabilized by 2-3 disulfide bonds. We also examine synthetic β-sheet peptides selected from a combinatorial library for their ability or inability to form pores in lipid membranes. When heptameric, octameric, and decameric β-barrels and tetrameric arcs of these peptides were embedded in pre-formed neutral or anionic lipid pores (i.e., pores in neutral or anionic membranes, respectively), a variety of behaviors and membrane binding energies were observed. Due to the cationic charge of the peptides, more favorable transfer energies and more stable binding were observed in anionic than neutral pores. The synthetic peptides bound very strongly and formed stable barrels and arcs in both neutral and anionic pores. The natural AMPs exhibited unfavorable or marginally favorable binding energy and kinetic stability in neutral pores, consistent with the lower hemolytic activity of some of them compared with protegrin-1. Binding to anionic pores was more favorable, but significant distortions of the barrel or arc structures were sometimes noted. These results are discussed in light of the available experimental data. The diversity of behaviors obtained makes it unlikely that the barrel and arc mechanisms are valid for the entire family of β-hairpin AMPs.

Conductance modulation of charged lipid bilayer using electrolyte-gated graphene-field effect transistor.

Graphene is an attention-grabbing material in electronics, physics, chemistry, and even biology because of its unique properties such as high surface-area-to-volume ratio. Also, the ability of graphene-based materials to continuously tune charge carriers from holes to electrons makes them promising for biological applications, especially in lipid bilayer-based sensors. Furthermore, changes in charged lipid membrane properties can be electrically detected by a graphene-based electrolyte-gated graphene field effect transistor (GFET). In this paper, a monolayer graphene-based GFET with a focus on the conductance variation caused by membrane electric charges and thickness is studied. Monolayer graphene conductance as an electrical detection platform is suggested for neutral, negative, and positive electric-charged membrane. The electric charge and thickness of the lipid bilayer (QLP and LLP) as a function of carrier density are proposed, and the control parameters are defined. Finally, the proposed analytical model is compared with experimental data which indicates good overall agreement.

Dye-release assay for investigation of antimicrobial peptide activity in a competitive lipid environment.

A dye-release method for investigating the effect of a competitive lipid environment on the activity of two membrane-disrupting antimicrobial peptides (AMP), maculatin 1.1 and aurein 1.2, is presented. The results support the general conclusion that AMP have greater affinity for negatively charged membranes, for example bacterial membranes, than for the neutral membrane surface found in eukaryotic cells, but only within a competitive lipid environment. Indeed, in a single-model membrane environment, both peptides were more potent against neutral vesicles than against charged vesicles. The approach was also used to investigate the effect of pre-incubating the peptides in a neutral lipid environment then introducing charged lipid vesicles. Maculatin was shown to migrate from the neutral lipid bilayers, where pores had already formed, to the charged membrane bilayers. This result was also observed for charged-to-charged bilayers but, interestingly, not for neutral-to-neutral lipid interfaces. Aurein was able to migrate from either lipid environment, indicating weaker binding to lipid membranes, and a different molecular mechanism for lysis of lipid bilayers. Competitive lipid environments could be used to assess other critical conditions that modulate the activity of membrane peptides or proteins.

Passive and Iontophoretic Transport of Fluorides across Enamel In Vitro

Passive and iontophoretic transport of fluoride from three fluoride sources, NaF, sodium monofluorophosphate (MFP), and SnF2 solutions, across bovine enamel was investigated to (1) determine the characteristics of the intrinsic barrier of enamel for ion transport, (2) examine the feasibility of iontophoretically enhanced transport of fluoride across enamel, and (3) identify the transport mechanisms involved in enamel iontophoresis. Conductivity experiments were performed with bovine enamel specimens in side-by-side diffusion cells to evaluate the electrical and barrier properties of the enamel with electrolytes of different ion sizes and under different ion concentrations and pH conditions in vitro. Transport experiments of the enamel were performed in the diffusion cells with the NaF, MFP, and SnF2 solutions. The conductivity results showed that the enamel specimens behaved as a neutral membrane or that of low pore charge density. Cathodal iontophoresis significantly enhanced the delivery of fluoride ions across the enamel from the solutions over passive transport, consistent with Nernst-Planck theory and the direct field effect (i.e., electrophoresis) as the dominant flux-enhancing mechanism. The enamel demonstrated significant transport hindrance for the ions, and the effective pore radii of the transport pathways in the enamel were found to be approximately 0.7-0.9 nm.

Interaction of the rattlesnake toxin crotamine with model membranes.

Crotamine is one of the main constituents of the venom of the South American rattlesnake Crotalus durissus terrificus. A common gene ancestry and structural similarity with the antimicrobial β-defensins (identical disulfide bond pattern and highly positive net charge) suggested potential antimicrobial activities for this snake toxin. Although crotamine demonstrated low activity against both Gram-positive and Gram-negative bacteria, a pronounced antifungal activity was observed against Candida spp., Trichosporon spp., and Cryptococcus neoformans. Crotamine's selective antimicrobial properties, with no observable hemolytic activity, stimulated us to evaluate the potential applications of this polypeptide as an antiyeast or candicidal agent for medical and industrial application. Aiming to understand the mechanism(s) of action underlying crotamine antimicrobial activity and its selectivity for fungi, we present herein studies using membrane model systems (i.e., large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs), with different phospholipid compositions. We show here that crotamine presents a higher lytic activity on negatively charged membranes compared with neutral membranes, with or without cholesterol or ergosterol content. The vesicle burst was not preceded by membrane permeabilization as is generally observed for pore forming peptides. Although such a property of disrupting lipid membranes is very important to combat multiresistant fungi, no inhibitory activity was observed for crotamine against biofilms formed by several Candida spp. strains, except for a limited effect against C. krusei biofilm.

Biophysical Implications of Sphingosine Accumulation in Membrane Properties at Neutral and Acidic pH

Sphingosine (Sph) is a simple lipid involved in the regulation of several biological processes. When accumulated in the late endosomal/lysosomal compartments, Sph causes changes in ion signaling and membrane trafficking, leading to the development of Niemann-Pick disease type C. Little is known about Sph interaction with other lipids in biological membranes; however, understanding the effect of Sph in the physical state of membranes might provide insights into its mode of action. Using complementary established fluorescence approaches, we show that Sph accumulation leads to the formation of Sph-enriched gel domains in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and POPC/sphingomyelin (SM)/cholesterol (Chol) model membranes. These domains are more easily formed in membrane models mimicking the neutral pH plasma membrane environment (PM) as compared to the acidic lysosomal membrane environment (LM), where higher Sph concentrations (or lower temperatures) are required. Electrophoretic light scattering measurements further revealed that in PM-raft models (POPC/SM/Chol), Sph is mainly neutral, whereas in LM models, the positive charge of Sph leads to electrostatic repulsion, reducing the Sph ability to form gel domains. Thus, formation of Sph-enriched domains in cellular membranes might be strongly regulated by Sph charge.

Synaptotagmin 1 and Ca2+ drive trans SNARE zippering

Synaptotagmin 1 (Syt1) is a major Ca2+-sensor that evokes neurotransmitter release. Here we used site-specific fluorescence resonance energy transfer (FRET) assay to investigate the effects of Syt1 on SNAREpin assembly. C2AB, a soluble version of Syt1, had virtually no stimulatory effect on the rate of the FRET at N-terminus of SNARE complex both with and without Ca2+, indicating C2AB does not interfere with the initial nucleation of SNARE assembly. However, C2AB-Ca2+ accelerated the FRET rate significantly at membrane proximal region, indicating C2AB-Ca2+ promotes the transition from a partially assembled SNARE complex to the fusion-competent SNAREpin. Similar enhancement was also observed at the end of the transmembrane domain of SNARE proteins. The stimulatory effect disappeared if there was no membrane or only neutral membrane present.

Comparison of reversible membrane destabilisation induced by antimicrobial peptides derived from Australian frogs

The membrane destabilising properties of the antimicrobial peptides (AMP) aurein 1.2, citropin 1.1, maculatin 1.1 and caerin 1.1, have been studied by dual polarisation interferometry (DPI). The overall process of peptide induced membrane destabilisation was examined by the changes in bilayer order as a function of membrane-bound peptide mass per unit area and revealed three different modes of action. Aurein 1.2 was the only peptide that significantly destabilised the neutral membrane (DMPC), while all four peptides induced destabilisation of the negatively charged membrane (DMPC/DMPG). On DMPC, citropin 1.1, maculatin 1.1 and caerin 1.1 bound irreversibly at low concentrations but caused a reversible drop in the bilayer order. In contrast to DMPC/DMPG, these three peptides caused a mass drop at the higher concentrations, which may correspond to insertion and bilayer expansion. The critical level of bound peptide necessary to induce membrane destabilisation (peptide:lipid ratio) was determined and correlated with peptide structure. As the most lytic peptide, aurein 1.2 adsorbed strongly prior to dissolution of the bilayer. In contrast, the binding of citropin 1.1, maculatin 1.1 and caerin 1.1 needed to reach a critical level prior to insertion into the membrane and incremental expansion and disruption. Our results demonstrate that sequential events can be monitored in real-time under fluidic conditions to elucidate the complex molecular mechanism of AMP action. In particular, the analysis of birefringence in real time allows the description of a detailed mechanistic model of the impact of peptides on the membrane bilayer order. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.