Real-time quantitative analysis of lipid disordering by aurein 1.2 during membrane adsorption, destabilisation and lysis

Effective antimicrobial peptides (AMPs) distinguish between the host and microbial cells, show selective antimicrobial activity and exhibit a fast killing mechanism. Although understanding the structure-function characteristics of AMPs is important, the impact of the peptides on the architecture of membranes with different lipid compositions is also critical in understanding the molecular mechanism and specificity of membrane destabilisation. In this study, the destabilisation of supported lipid bilayers (SLBs) by the AMP aurein 1.2 was quantitatively analysed by dual polarisation interferometry. The lipid bilayers were formed on a planar silicon oxynitride chip, and composed of mixed synthetic lipids, or Escherichiacoli lipid extract. The molecular events leading sequentially from peptide adsorption to membrane lysis were examined in real time by changes in bilayer birefringence (lipid molecular ordering) as a function of membrane-bound peptide mass. Aurein 1.2 bound weakly without any change in membrane ordering at low peptide concentration (5 μM), indicating a surface-associated state without significant perturbation in membrane structure. At 10 μM peptide, marked reversible changes in molecular ordering were observed for all membranes except DMPE/DMPG. However, at 20 μM aurein 1.2, removal of lipid molecules, as determined by mass loss with a concomitant decrease in birefringence during the association phase, was observed for DMPC and DMPC/DMPG SLBs, which indicates membrane lysis by aurein. The membrane destabilisation induced by aurein 1.2 showed cooperativity at a particular peptide/lipid ratio with a critical mass/molecular ordering value. Furthermore, the extent of membrane lysis for DMPC/DMPG was nearly double that for DMPC. However, no lysis was observed for DMPC/DMPG/cholesterol, DMPE/DMPG and E. coli SLBs. The extent of birefringence changes with peptide mass suggested that aurein 1.2 binds to the membrane without inserting through the bilayer and membrane lysis occurs through detergent-like micellisation above a critical P/L ratio. Real-time quantitative analysis of the structural properties of membrane organisation has allowed the membrane destabilisation process to be resolved into multiple steps and provides comprehensive information to determine the molecular mechanism of aurein 1.2 action.     

Selective Interaction of Colistin with Lipid Model Membranes

Although colistin’s clinical use is limited due to its nephrotoxicity, colistin is considered to be an antibiotic of last resort because it is used to treat patients infected with multidrug-resistant bacteria. In an effort to provide molecular details about colistin’s ability to kill Gram-negative (G(?)) but not Gram-positive (G(+)) bacteria, we investigated the biophysics of the interaction between colistin and lipid mixtures mimicking the cytoplasmic membrane of G(+), G(?) bacteria as well as eukaryotic cells. Two different models of the G(?) outer membrane (OM) were assayed: lipid A with two deoxy-manno-octulosonyl sugar residues, and Escherichia coli lipopolysaccharide mixed with dilaurylphosphatidylglycerol. We used circular dichroism and x-ray diffuse scattering at low and wide angle in stacked multilayered samples, and neutron reflectivity of single, tethered bilayers mixed with colistin. We found no differences in secondary structure when colistin was bound to G(?) versus G(+) membrane mimics, ruling out a protein conformational change as the cause of this difference. However, bending modulus K_C perturbation was quite irregular for the G(?) inner membrane, where colistin produced a softening of the membranes at an intermediate lipid/peptide molar ratio but stiffening at lower and higher peptide concentrations, whereas in G(+) and eukaryotic mimics there was only a slight softening. Acyl chain order in G(?) was perturbed similarly to K_C. In G(+), there was only a slight softening and disordering effect, whereas in OM mimics, there was a slight stiffening and ordering of both membranes with increasing colistin. X-ray and neutron reflectivity structural results reveal colistin partitions deepest to reach the hydrocarbon interior in G(?) membranes, but remains in the headgroup region in G(+), OM, and eukaryotic mimics. It is possible that domain formation is responsible for the erratic response of G(?) inner membranes to colistin and for its deeper penetration, which could increase membrane permeability.     

Roles of carboxyl groups in the transmembrane insertion of peptides

We have used pHLIP® [pH (low) insertion peptide] to study the roles of carboxyl groups in transmembrane (TM) peptide insertion. pHLIP binds to the surface of a lipid bilayer as a disordered peptide at neutral pH; when the pH is lowered, it inserts across the membrane to form a TM helix. Peptide insertion is reversed when the pH is raised above the characteristic pK_a (6.0). A key event that facilitates membrane insertion is the protonation of aspartic acid (Asp) and/or glutamic acid (Glu) residues, since their negatively charged side chains hinder membrane insertion at neutral pH. In order to gain mechanistic understanding, we studied the membrane insertion and exit of a series of pHLIP variants where the four Asp residues were sequentially mutated to nonacidic residues, including histidine (His). Our results show that the presence of His residues does not prevent the pH-dependent peptide membrane insertion at ∼ pH 4 driven by the protonation of carboxyl groups at the inserting end of the peptide. A further pH drop leads to the protonation of His residues in the TM part of the peptide, which induces peptide exit from the bilayer. We also find that the number of ionizable residues that undergo a change in protonation during membrane insertion correlates with the pH-dependent insertion into the lipid bilayer and exit from the lipid bilayer, and that cooperativity increases with their number. We expect that our understanding will be used to improve the targeting of acidic diseased tissue by pHLIP.     

Oligomerization of Fusogenic Peptides Promotes Membrane Fusion by Enhancing Membrane Destabilization

A key element of membrane fusion reactions in biology is the involvement of specific fusion proteins. In many viruses, the proteins that mediate membrane fusion usually exist as homotrimers. Furthermore, they contain extended triple-helical coiled-coil domains and fusogenic peptides. It has been suggested that the coiled-coil domains present the fusogenic peptide in a conformation or geometry favorable for membrane fusion. To test the hypothesis that trimerization of fusogenic peptide is related to optimal fusion, we have designed and synthesized a triple-stranded coiled-coil X31 peptide, also known as the ccX31, which mimics the influenza virus hemagglutinin fusion peptide in the fusion-active state. We compared the membrane interactive properties of ccX31 versus the monomeric X31 fusogenic peptide. Our data show that trimerization enhances peptide-induced leakage of liposomal contents and lipid mixing. Furthermore, studies using micropipette aspiration of single vesicles reveal that ccX31 decreases lysis tension, τ_lysis, but not area expansion modulus, K_a, of phospholipid bilayers, whereas monomeric X31 peptide lowers both τ_lysis and K_a. Our results are consistent with the hypothesis that oligomerization of fusogenic peptide promotes membrane fusion, possibly by enhancing localized destabilization of lipid bilayers.     

Binding of β-Amyloid (1-42) Peptide to Negatively Charged Phospholipid Membranes in the Liquid-Ordered State: Modeling and Experimental Studies

To explore the initial stages of amyloid β peptide (Aβ42) deposition on membranes, we have studied the interaction of Aβ42 in the monomeric form with lipid monolayers and with bilayers in either the liquid-disordered or the liquid-ordered (L_o) state, containing negatively charged phospholipids. Molecular dynamics (MD) simulations of the system have been performed, as well as experimental measurements. For bilayers in the L_o state, in the absence of the negatively charged lipids, interaction is weak and it cannot be detected by isothermal calorimetry. However, in the presence of phosphatidic acid, or of cardiolipin, interaction is detected by different methods and in all cases interaction is strongest with lower (2.5–5 mol %) than higher (10–20 mol %) proportions of negatively charged phospholipids. Liquid-disordered bilayers consistently allowed a higher Aβ42 binding than L_o ones. Thioflavin T assays and infrared spectroscopy confirmed a higher proportion of β-sheet formation under conditions when higher peptide binding was measured. The experimental results were supported by MD simulations. We used 100 ns MD to examine interactions between Aβ42 and three different 512 lipid bilayers consisting of palmitoylsphingomyelin, dimyristoyl phosphatidic acid, and cholesterol in three different proportions. MD pictures are different for the low- and high-charge bilayers, in the former case the peptide is bound through many contact points to the bilayer, whereas for the bilayer containing 20 mol % anionic phospholipid only a small fragment of the peptide appears to be bound. The MD results indicate that the binding and fibril formation on the membrane surface depends on the composition of the bilayer, and is the result of a subtle balance of many inter- and intramolecular interactions between the Aβ42 and membrane.     

Effects of palmitoylation on dynamics and phospholipid-bilayer-perturbing properties of the N-terminal segment of pulmonary surfactant protein SP-C as shown by 2H-NMR

It has been proposed that palmitoylation of the N-terminal segment of surfactant protein SP-C is important for maintaining association of pulmonary surfactant complexes with interfacial films compressed to high pressures at the end of expiration. In this study, we examined surfactant membrane models containing palmitoylated and nonpalmitoylated synthetic peptides, based on the N-terminal SP-C sequence, in dipalmitoylphosphatidylcholine (DPPC)/egg phosphatidylglycerol (7:3, w/w) by ²H-NMR. Perturbations of lipid properties by the peptide versions were compared in samples containing chain- and headgroup-deuterated lipid (DPPC-d₆₂ and DPPC-d₄ respectively). Also, deuterated peptide palmitate chains were compared with those of DPPC in otherwise identical lipid-protein mixtures. Palmitoylated peptide increased average DPPC-d₆₂ chain orientational order slightly, particularly for temperatures spanning gel and liquid crystalline coexistence, implying penetration of palmitoylated peptide into ordered membrane. In contrast, the nonpalmitoylated peptide had a small disordering effect in this temperature range. Both peptide versions perturbed DPPC-d₄ headgroup orientation similarly, suggesting little effect of palmitoylation on the largely electrostatic peptide-headgroup interaction. Deuterated acyl chains attached to the SP-C N-terminal segment displayed a qualitatively different distribution of chain order, and lower average order, than DPPC-d₆₂ in the same membranes. This likely reflects local perturbation of lipid headgroup spacing by the peptide portion interacting with the bilayer near the peptide palmitate chains. This study suggests that SP-C-attached acyl chains could be important for coupling of lipid and protein motions in surfactant bilayers and monolayers, especially in the context of ordered phospholipid structures such as those potentially formed during exhalation, when stabilization of the respiratory surface by surfactant is the most crucial.     

Membranotropic activity of optical isomers of the neuropeptide kyotorphin and a cardiotonic agent, suphan

On modeled monolayer phospholipid (formed from azolectin) membranes, we studied the surface activity of optical isomers of a dipeptide, kyotorphin (Tyr-Arg), and a cardiotonic agent, suphan (N-succinyltryptophan potassium salt). It was found that the membranotropic activity of four studied isomers of kyotorphin is distributed in the order: LL>DL≈LD>DD, and two isomers (by tryptophan) of suphan as LL>DL. The data obtained suggest that the primary mechanism underlying binding of kyotorphin and suphan to the plasma membrane can be considered based on interaction of their molecules with the molecules of membrane phospholipids. Binding of molecules of kyotorphin and suphan by the lipid matrix to the plasma membrane and/or their incorporation into the matrix is a result of the above interaction.     

Membrane interactions in small fast-tumbling bicelles as studied by 31P NMR

Small fast-tumbling bicelles are ideal for studies of membrane interactions at molecular level; they allow analysis of lipid properties using solution-state NMR. In the present study we used ³¹P NMR relaxation to obtain detailed information on lipid head-group dynamics. We explored the effect of two topologically different membrane-interacting peptides on bicelles containing either dimyristoylphosphocholine (DMPC), or a mixture of DMPC and dimyristoylphosphoglycerol (DMPG), and dihexanoylphosphocholine (DHPC). KALP21 is a model transmembrane peptide, designed to span a DMPC bilayer and dynorphin B is a membrane surface active neuropeptide. KALP21 causes significant increase in bicelle size, as evidenced by both dynamic light scattering and ³¹P T₂ relaxation measurements. The effect of dynorphin B on bicelle size is more modest, although significant effects on T₂ relaxation are observed at higher temperatures. A comparison of ³¹P T₁ values for the lipids with and without the peptides showed that dynorphin B has a greater effect on lipid head-group dynamics than KALP21, especially at elevated temperatures. From the field-dependence of T₁ relaxation data, a correlation time describing the overall lipid motion was derived. Results indicate that the positively charged dynorphin B decreases the mobility of the lipid molecules  – in particular for the negatively charged DMPG – while KALP21 has a more modest influence. Our results demonstrate that while a transmembrane peptide has severe effects on overall bilayer properties, the surface bound peptide has a more dramatic effect in reducing lipid head-group mobility. These observations may be of general importance for understanding peptide–membrane interactions.     

Peptide derived from the lipid binding domain of Group IB human pancreatic phospholipase A2 possesses antibacterial activity

Group 1B human pancreatic secretory phospholipase A₂ (hp-sPLA₂), a digestive enzyme synthesized by pancreatic acinar cells and present in pancreatic juice, do not have antibacterial activity towards Escherichia coli. Our earlier results suggest that the N-terminal first ten amino acid residues of hp-sPLA₂ constitute major portion of the membrane binding domain of full-length enzyme and is responsible for the precise orientation of enzyme on the membrane surface by inserting into the lipid bilayers (Pande et al. (2006) Biochemistry, 45,12436–12447). In this study we report the antibacterial properties of a peptide (AVWQFRKMIK-CONH₂; N10 peptide), which corresponds to the N-terminal first ten amino acid residues of hp-sPLA₂, against E. coli. Full-length hp-sPLA₂, which contains this peptide sequence as N-terminal α-helix, did not showed detectable antibacterial activity. Presence of physiological concentration of salt or preincubation of N10 peptide with soluble anionic polymer inhibits the antibacterial activity indicating the importance of electrostatic interaction in binding of peptide to bacterial membrane. Addition of peptide resulted in destabilization of outer as well as inner cytoplasmic membrane of E. coli suggesting bacterial membranes to be the main target of action. N10 peptide exhibits strong synergism with lysozyme and potentiates the antibacterial activity of lysozyme. The peptide was inactive against human erythrocyte. Our result shows for the first time that a peptide fragment of hp-sPLA₂ possesses antibacterial activity towards E. coli and at subinhibitory concentration and can potentiate the antibacterial activity of membrane active enzyme. These observations suggest that N10 peptide may play an important role in the antimicrobial activity of pancreatic juice.     

Protein Partitioning into Ordered Membrane Domains: Insights from Simulations

Cellular membranes are laterally organized into domains of distinct structures and compositions by the differential interaction affinities between various membrane lipids and proteins. A prominent example of such structures are lipid rafts, which are ordered, tightly packed domains that have been widely implicated in cellular processes. The functionality of raft domains is driven by their selective recruitment of specific membrane proteins to regulate their interactions and functions; however, there have been few general insights into the factors that determine the partitioning of membrane proteins between coexisting liquid domains. In this work, we used extensive coarse-grained and atomistic molecular dynamics simulations, potential of mean force calculations, and conceptual models to describe the partitioning dynamics and energetics of a model transmembrane domain from the linker of activation of T cells. We find that partitioning between domains is determined by an interplay between protein-lipid interactions and differential lipid packing between raft and nonraft domains. Specifically, we show that partitioning into ordered domains is promoted by preferential interactions between peptides and ordered lipids, mediated in large part by modification of the peptides by saturated fatty acids (i.e., palmitoylation). Ordered phase affinity is also promoted by elastic effects, specifically hydrophobic matching between the membrane and the peptide. Conversely, ordered domain partitioning is disfavored by the tight molecular packing of the lipids therein. The balance of these dominant drivers determines partitioning. In the case of the wild-type linker of activation of T cells transmembrane domain, these factors combine to yield enrichment of the peptide at L_o/L_d interfaces. These results define some of the general principles governing protein partitioning between coexisting membrane domains and potentially explain previous disparities among experiments and simulations across model systems.     

Peptide-membrane binding is not enough to explain bioactivity: A case study

Membrane-active peptides are a promising class of antimicrobial and anticancer therapeutics. For this reason, their molecular mechanisms of action are currently actively investigated. By exploiting Electron Paramagnetic Resonance, we study the membrane interaction of two spin-labeled analogs of the antimicrobial and cytotoxic peptide trichogin GA IV (Tri), with opposite bioactivity: Tri(Api⁸), able to selectively kill cancer cells, and Tri(Leu⁴), which is completely nontoxic. In our attempt to determine the molecular basis of their different biological activity, we investigate peptide impact on the lateral organization of lipid membranes, peptide localization and oligomerization, in the zwitter-ionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) model membrane We show that, despite their divergent bioactivity, both peptide analogs (i) are membrane-bound, (ii) display a weak tendency to oligomerization, and (iii) do not induce significant lipid rearrangement. Conversely, literature data show that the parent peptide trichogin, which is cytotoxic without any selectivity, is strongly prone to dimerization and affects the reorganization of POPC membranes. Its dimers are involved in the rotation around the peptide helix, as observed at cryogenic temperatures in the millisecond timescale. Since this latter behavior is not observed for the inactive Tri(Leu⁴), we propose that for short-length peptides as trichogin oligomerization and molecular motions are crucial for bioactivity, and membrane binding alone is not enough to predict or explain it. We envisage that small changes in the peptide sequence that affect only their ability to oligomerize, or their molecular motions inside the membrane, can tune the peptide activity on membranes of different compositions.     

Antimicrobial activity and membrane selective interactions of a synthetic lipopeptide MSI-843

Lipopeptide MSI-843 consisting of the nonstandard amino acid ornithine (Oct-OOLLOOLOOL-NH₂) was designed with an objective towards generating non-lytic short antimicrobial peptides, which can have significant pharmaceutical applications. Octanoic acid was coupled to the N-terminus of the peptide to increase the overall hydrophobicity of the peptide. MSI-843 shows activity against bacteria and fungi at micromolar concentrations. It permeabilizes the outer membrane of Gram-negative bacterium and a model membrane mimicking bacterial inner membrane. Circular dichroism investigations demonstrate that the peptide adopts α-helical conformation upon binding to lipid membranes. Isothermal titration calorimetry studies suggest that the peptide binding to membranes results in exothermic heat of reaction, which arises from helix formation and membrane insertion of the peptide. ²H NMR of deuterated-POPC multilamellar vesicles shows the peptide-induced disorder in the hydrophobic core of bilayers. ³¹P NMR data indicate changes in the lipid head group orientation of POPC, POPG and Escherichia colitotal lipid bilayers upon peptide binding. Results from ³¹P NMR and dye leakage experiments suggest that the peptide selectively interacts with anionic bilayers at low concentrations (up to 5 mol%). Differential scanning calorimetry experiments on DiPOPE bilayers and ³¹P NMR data from E.coli total lipid multilamellar vesicles indicate that MSI-843 increases the fluid lamellar to inverted hexagonal phase transition temperature of bilayers by inducing positive curvature strain. Combination of all these data suggests the formation of a lipid-peptide complex resulting in a transient pore as a plausible mechanism for the membrane permeabilization and antimicrobial activity of the lipopeptide MSI-843.     

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.     

Effect of the aminoacid composition of model α-helical peptides on the physical properties of lipid bilayers and peptide conformation: a molecular dynamics simulation

The interaction of a model Lys flanked α-helical peptides K₂-X₂₄-K₂, (X = A,I,L,L+A,V) with lipid bilayers composed of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) both, in a gel and in a liquid-crystalline state, has been studied by molecular dynamics simulations. It has been shown that these peptides cause disordering of the lipid bilayer in the gel state but only small changes have been monitored in a liquid-crystalline state. The peptides affect ordering of the surrounding lipids depending on the helix stability which is determined by amino acid side chains – their volume, shape, etc. We have shown that the helix does not keep the linear shape in all simulations but often bends or breaks. During some simulations with a very small difference between hydrophobic length of peptide and membrane thickness the peptide exhibits negligible tilt. At the same time changes in peptide conformations during simulations resulted in appearance of superhelix.     

Interaction of a peptide corresponding to the loop domain of the S2 SARS-CoV virus protein with model membranes

The severe acute respiratory syndrome coronavirus (SARS-CoV) envelope spike (S) glycoprotein is responsible for the fusion between the membranes of the virus and the target cell. In the case of the S2 domain of protein S, it has been found a highly hydrophobic and interfacial domain flanked by the heptad repeat 1 and 2 regions; significantly, different peptides pertaining to this domain have shown a significant leakage effect and an important plaque formation inhibition, which, similarly to HIV-1 gp41, support the role of this region in the fusion process. Therefore, we have carried out a study of the binding and interaction with model membranes of a peptide corresponding to segment 1073–1095 of the SARS-CoV S glycoprotein, peptide SARS_L in the presence of different membrane model systems, as well as the structural changes taking place in both the lipid and the peptide induced by the binding of the peptide to the membrane. Our results show that SARS_L strongly partitions into phospholipid membranes and organizes differently in lipid environments, displaying membrane activity modulated by the lipid composition of the membrane. These data would support its role in SARS-CoV mediated membrane fusion and suggest that the region where this peptide resides could be involved in the merging of the viral and target cell membranes.     

Reversible Effects of Peptide Concentration and Lipid Composition on H-Ras Lipid Anchor Clustering

Dynamic clusters of lipid-anchored Ras proteins are important for high-fidelity signal transduction in cells. The average size of Ras nanoclusters was reported to be independent of protein expression levels, and cholesterol depletion is commonly used to test the raft-preference of nanoclusters. However, whether protein concentration and membrane domain stability affect Ras clustering in a reversible manner is not well understood. We used coarse-grained molecular dynamics simulations to examine the reversibility of the effects of peptide and cholesterol concentrations as well as a lipid domain-perturbing nanoparticle (C₆₀) on the dynamics and stability of H-Ras lipid-anchor nanoclusters. By comparing results from these simulations with previous observations from the literature, we show that effects of peptide/cholesterol concentrations on the dynamics and stability of H-Ras peptide nanoclusters are reversible. Our results also suggest a correlation between the stabilities of lipid domains and Ras nanoclusters, which is supported by our finding that C₆₀ penetrates into the liquid-disordered domain of the bilayer, destabilizing lipid domains and thereby the stability of the nanoclusters.     

Designed Fluorescent Probes Reveal Interactions between Amyloid-β(1-40) Peptides and GM1 Gangliosides in Micelles and Lipid Vesicles

A hallmark of the common Alzheimer's disease (AD) is the pathological conversion of its amphiphatic amyloid-β (Aβ) peptide into neurotoxic aggregates. In AD patients, these aggregates are often found to be tightly associated with neuronal G_M1 ganglioside lipids, suggesting an involvement of G_M1 not only in aggregate formation but also in neurotoxic events. Significant interactions were found between micelles made of newly synthesized fluorescent G_M1 gangliosides labeled in the polar headgroup or the hydrophobic chain and Aβ(1-40) peptide labeled with a BODIPY-FL-C1 fluorophore at positions 12 and 26, respectively. From an analysis of energy transfer between the different fluorescence labels and their location in the molecules, we were able to place the Aβ peptide inside G_M1 micelles, close to the hydrophobic-hydrophilic interface. Large unilamellar vesicles composed of a raftlike G_M1/bSM/cholesterol lipid composition doped with labeled G_M1 at various positions also interact with labeled Aβ peptide tagged to amino acids 2 or 26. A faster energy transfer was observed from the Aβ peptide to bilayers doped with 581/591-BODIPY-C₁₁-G_M1 in the nonpolar part of the lipid compared with 581/591-BODIPY-C₅-G_M1 residing in the polar headgroup. These data are compatible with a clustering process of G_M1 molecules, an effect that not only increases the Aβ peptide affinity, but also causes a pronounced Aβ peptide penetration deeper into the lipid membrane; all these factors are potentially involved in Aβ peptide aggregate formation due to an altered ganglioside metabolism found in AD patients.     

Bordetella parapertussis PagP Mediates the Addition of Two Palmitates to the Lipopolysaccharide Lipid A

Bordetella bronchiseptica PagP (PagP_BB) is a lipid A palmitoyl transferase that is required for resistance to antibody-dependent complement-mediated killing in a murine model of infection. B. parapertussis contains a putative pagP homolog (encoding B. parapertussis PagP [PagP_BPa]), but its role in the biosynthesis of lipid A, the membrane anchor of lipopolysaccharide (LPS), has not been investigated. Mass spectrometry analysis revealed that wild-type B. parapertussis lipid A consists of a heterogeneous mixture of lipid A structures, with penta- and hexa-acylated structures containing one and two palmitates, respectively. Through mutational analysis, we demonstrate that PagP_BPa is required for the modification of lipid A with palmitate. While PagP_BB transfers a single palmitate to the lipid A C-3′ position, PagP_BPa transfers palmitates to the lipid A C-2 and C-3′ positions. The addition of two palmitate acyl chains is unique to B. parapertussis. Mutation of pagP_BPa resulted in a mutant strain with increased sensitivity to antimicrobial peptide killing and decreased endotoxicity, as evidenced by reduced proinflammatory responses via Toll-like receptor 4 (TLR4) to the hypoacylated LPS. Therefore, PagP-mediated modification of lipid A regulates outer membrane function and may be a means to modify interactions between the bacterium and its human host during infection.     

Coarse-grained simulations of the membrane-active antimicrobial Peptide maculatin 1.1

Maculatin 1.1 (M1.1) is a membrane-active antimicrobial peptide (AMP) from an Australian tree frog that forms a kinked amphipathic α-helix in the presence of a lipid bilayer or bilayer-mimetic environment. To help elucidate its mechanism of membrane-lytic activity, we performed a total of ∼8 μs of coarse-grained molecular dynamics (CG-MD) simulations of M1.1 in the presence of zwitterionic phospholipid membranes. Several systems were simulated in which the peptide/lipid ratio was varied. At a low peptide/lipid ratio, M1.1 adopted a kinked, membrane-interfacial location, consistent with experiment. At higher peptide/lipid ratios, we observed spontaneous, cooperative membrane insertion of M1.1 peptide aggregates. The minimum size for formation of a transmembrane (TM) aggregate was just four peptides. The absence of a simple and well-defined central channel, along with the exclusion of lipid headgroups from the aggregates, suggests that a pore-like model is an unlikely explanation for the mechanism of membrane lysis by M1.1. We also performed an extended 1.25 μs simulation of the permeabilization of a complete liposome by multiple peptides. Consistent with the simpler bilayer simulations, formation of monomeric interfacial peptides and TM peptide clusters was observed. In contrast, major structural changes were observed in the vesicle membrane, implicating induced membrane curvature in the mechanism of active antimicrobial peptide lysis. This contrasted with the behavior of the nonpore-forming model peptide WALP23, which inserted into the vesicle to form extended clusters of TM α-helices with relatively little perturbation of bilayer properties.     

Membrane interactions of the anuran antimicrobial peptide HSP1-NH2: Different aspects of the association to anionic and zwitterionic biomimetic systems

Studies have suggested that antimicrobial peptides act by different mechanisms, such as micellisation, self-assembly of nanostructures and pore formation on the membrane surface. This work presents an extensive investigation of the membrane interactions of the 14 amino-acid antimicrobial peptide hylaseptin P1-NH₂ (HSP1-NH₂), derived from the tree-frog Hyla punctata, which has stronger antifungal than antibacterial potential. Biophysical and structural analyses were performed and the correlated results were used to describe in detail the interactions of HSP1-NH₂ with zwitterionic and anionic detergent micelles and phospholipid vesicles. HSP1-NH₂ presents similar well-defined helical conformations in both zwitterionic and anionic micelles, although NMR spectroscopy revealed important structural differences in the peptide N-terminus. ²H exchange experiments of HSP1-NH₂ indicated the insertion of the most N-terminal residues (1–3) in the DPC-d₃₈ micelles. A higher enthalpic contribution was verified for the interaction of the peptide with anionic vesicles in comparison with zwitterionic vesicles. The pore formation ability of HSP1-NH₂ (examined by dye release assays) and its effect on the size and surface charge as well as on the lipid acyl chain ordering (evaluated by Fourier-transform infrared spectroscopy) of anionic phospholipid vesicles showed membrane disruption even at low peptide-to-phospholipid ratios, and the effect increases proportionately to the peptide concentration. On the other hand, these biophysical investigations showed that a critical peptide-to-phospholipid ratio around 0.6 is essential for promoting disruption of zwitterionic membranes. In conclusion, this study demonstrates that the binding process of the antimicrobial HSP1-NH₂ peptide depends on the membrane composition and peptide concentration.