Peptide-membrane interactions and mechanisms of membrane destruction by amphipathic α-helical antimicrobial peptides

Antimicrobial peptides (AMPs) have received considerable interest as a source of new antibiotics with the potential for treatment of multiple-drug resistant infections. An important class of AMPs is composed of linear, cationic peptides that form amphipathic α-helices. Among the most potent of these are the cecropins and synthetic peptides that are hybrids of cecropin and the bee venom peptide, mellitin. Both cecropins and cecropin-mellitin hybrids exist in solution as unstructured monomers, folding into predominantly α-helical structures upon membrane binding with their long helical axis parallel to the bilayer surface. Studies using model membranes have shown that these peptides intercalate into the lipid bilayer just below the level of the phospholipid glycerol backbone in a location that requires expansion of the outer leaflet of the bilayer, and evidence from a variety of experimental approaches indicates that expansion and thinning of the bilayer are common characteristics during the early stages of antimicrobial peptide-membrane interactions. Subsequent disruption of the membrane permeability barrier may occur by a variety of mechanisms, leading ultimately to loss of cytoplasmic membrane integrity and cell death.     

Flexibility is a mechanical determinant of antimicrobial activity for amphipathic cationic α-helical antimicrobial peptides

Antimicrobial peptides (AMPs) are recognized as the potential substitutions for common antibiotics. Flexibility has been demonstrated to be a dominant on antimicrobial activity of an AMP, similar to the structural parameters such as hydrophobicity and hydrophobic moment as well as positive charge. To better understand the effect of flexibility on antimicrobial activity, we herein examined seventy-eight peptides derived from nine different species. Defined as a weighted average of amino acid flexibility indices over whole residue chain of AMP, flexibility index was used to scale the peptide flexibility and indicated to be a reflection of mechanical properties such as tensile and flexural rigidities. The results demonstrated that flexibility index is relevant to but different from other structural properties, may enhance activity against Escherichia coli for stiff clustered peptides or reduce activity against E. coli for flexible clustered peptides, and its optimum occurs at about − 0.5. This effect of flexibility on antimicrobial activity may be involved to the antimicrobial actions, such as stable peptide-bound leaflet formation and sequent stress concentration in target cell membrane, mechanically. The present results provide a new insight in understanding antimicrobial actions and may be useful in seeking for a new structure–activity relationship for cationic and amphipathic α-helical peptides.     

Role of helicity of α-helical antimicrobial peptides to improve specificity

A major barrier to the use of antimicrobial peptides as antibiotics is the toxicity or ability to lyse eukaryotic cells. In this study, a 26-residue amphipathic α-helical antimicrobial peptide A12L/A20L (Ac-KWKSFLKTFKSLK KTVLHTLLKAISS-amide) was used as the framework to design a series of D- and L-diastereomeric peptides and study the relationships of helicity and biological activities of α-helical antimicrobial peptides. Peptide helicity was measured by circular dichroism spectroscopy and demonstrated to correlate with the hydrophobicity of peptides and the numbers of D-amino acid substitutions. Therapeutic index was used to evaluate the selectivity of peptides against prokaryotic cells. By introducing D-amino acids to replace the original L-amino acids on the non-polar face or the polar face of the helix, the hemolytic activity of peptide analogs have been significantly reduced. Compared to the parent peptide, the therapeutic indices were improved of 44-fold and 22-fold against Gram-negative and Grampositive bacteria, respectively. In addition, D- and L-diastereomeric peptides exhibited lower interaction with zwitterionic eukaryotic membrane and showed the significant membrane damaging effect to bacterial cells. Helicity was proved to play a crucial role on peptide specificity and biological activities. By simply replacing the hydrophobic or the hydrophilic amino acid residues on the non-polar or the polar face of these amphipathic derivatives of the parent peptide with D-amino acids, we demonstrated that this method could have excellent potential for the rational design of antimicrobial peptides with enhanced specificity.     

Simulation of voltage-dependent interactions of α-helical peptides with lipid bilayers

Pore formation in lipid bilayers by channel-forming peptides and toxins is thought to follow voltage-dependent insertion of amphipathic α-helices into lipid bilayers. We have developed an approximate potential for use within the CHARMm molecular mechanics program which enables one to simulate voltage-dependent interaction of such helices with a lipid bilayer. Two classes of helical peptides which interact with lipid bilayers have been studied: (a) δ-toxin, a 26 residue channel-forming peptide from Staphylococcus aureus; and (b) synthetic peptides corresponding to the α5 and α7 helices of the pore-forming domain of Bacillus thuringiensis CryIIIA δ-endotoxin. Analysis of δ-toxin molecular dynamics (MD) simulations suggested that the presence of a transbilayer voltage stabilized the inserted location of δ-toxin helices, but did not cause insertion per se. A series of simulations for the α5 and α7 peptides revealed dynamic switching of the α5 helix between a membrane-associated and a membrane-inserted state in response to a transbilayer voltage. In contrast the α7 helix did not exhibit such switching but instead retained a membrane associated state. These results are in agreement with recent experimental studies of the interactions of synthetic α5 and α7 peptides with lipid bilayers.     

Effect of hydrocarbon stapling on the properties of α-helical antimicrobial peptides isolated from the venom of hymenoptera

The impact of inserting hydrocarbon staples into short α-helical antimicrobial peptides lasioglossin III and melectin (antimicrobial peptides of wild bee venom) on their biological and biophysical properties has been examined. The stapling was achieved by ring-closing olefin metathesis, either between two S-2-(4′-pentenyl) alanine residues (S ₅) incorporated at i and i + 4 positions or between R-2-(7′-octenyl) alanine (R ₈) and S ₅ incorporated at the i and i + 7 positions, respectively. We prepared several lasioglossin III and melectin analogs with a single staple inserted into different positions within the peptide chains as well as analogs with double staples. The stapled peptides exhibited a remarkable increase in hemolytic activity, while their antimicrobial activities decreased. Some single stapled peptides showed a higher resistance against proteolytic degradation than native ones, while the double stapled analogs were substantially more resistant. The CD spectra of the singly stapled peptides measured in water showed only a slightly better propensity to form α-helical structure when compared to native peptides, whereas the doubly stapled analogs exhibited dramatically enhanced α-helicity.     

The mode of α-synuclein binding to membranes depends on lipid composition and lipid to protein ratio

Interactions of the presynaptic protein α-synuclein with membranes are involved in its physiological action as well as in the pathological misfolding and aggregation related to Parkinsons's disease. We studied the conformation and orientation of α-synuclein bound to model vesicular membranes using multiparametric response polarity-sensitive fluorescent probes together with CD and EPR measurements. At low lipid to α-synuclein ratio the protein binds membranes through its N-terminal domain. When lipids are in excess, the α-helical content and the role of the C-terminus in binding increase. Highly rigid membranes also induce a greater α-helical content and a lower polarity of the protein microenvironment.     

Effect of disulphide bond position on salt resistance and LPS-neutralizing activity of α-helical homo-dimeric model antimicrobial peptides

To investigate the effects of disulphide bond position on the salt resistance and lipopolysaccharide (LPS)-neutralizing activity of α-helical homo-dimeric antimicrobial peptides (AMPs), we synthesized an α-helical model peptide (K6L4W1) and its homo-dimeric peptides (di-K(6)L(4)W(1)-N, di-K(6)L(4)W(1)-M, and di-K(6)L(4)W(1)-C) with a disulphide bond at the N-terminus, the central position, and the C-terminus of the molecules, respectively. Unlike (6)L(4)W(1) and di-K(6)L(4)W(1)-M, the antimicrobial activity of di-K(6)L(4)W(1)-N and di-K(6)L(4)W(1)-C was unaffected by 150 mM NaCl. Both di-K(6)L(4)W(1)-N and di-K(6)L(4)W(1)-C caused much greater inhibitory effects on nitric oxide (NO) release in LPS-induced mouse macrophage RAW 264.7 cells, compared to di-K(6)L(4)W(1)-M. Taken together, our results indicate that the presence of a disulphide bond at the N- or C-terminus of the molecule, rather than at the central position, is more effective when designing salt-resistant α-helical homo-dimeric AMPs with potent antimicrobial and LPS-neutralizing activities. [BMB reports 2011; 44(11): 747-752].     

Endotoxin-neutralizing activity and mechanism of action of a cationic α-helical antimicrobial octadecapeptide derived from α-amylase of rice

We have previously reported that AmyI-1-18, an octadecapeptide derived from α-amylase (AmyI-1) of rice, is a novel cationic α-helical peptide that exhibited antimicrobial activity against human pathogens, including Porphyromonas gingivalis, Pseudomonas aeruginosa, Propionibacterium acnes, Streptococcus mutans, and Candida albicans. In this study, to further investigate the potential functions of AmyI-1-18, we examined its inhibitory ability against the endotoxic activities of lipopolysaccharides (LPSs, smooth and Rc types) and lipid A from Escherichia coli. AmyI-1-18 inhibited the production of endotoxin-induced nitric oxide (NO), an inflammatory mediator, in mouse macrophages (RAW264) in a concentration-dependent manner. The results of a chromogenic Limulus amebocyte lysate assay illustrated that the ability [50% effective concentration (EC₅₀): 0.17 μM] of AmyI-1-18 to neutralize lipid A was similar to its ability (EC₅₀: 0.26 μM) to neutralize LPS, suggesting that AmyI-1-18 specifically binds to the lipid A moiety of LPS. Surface plasmon resonance analysis of the interaction between AmyI-1-18 and LPS or lipid A also suggested that AmyI-1-18 directly binds to the lipid A moiety of LPS because the dissociation constant (K_D) of AmyI-1-18 with lipid A is 5.6 × 10⁻¹⁰ M, which is similar to that (4.3 × 10⁻¹⁰ M) of AmyI-1-18 with LPS. In addition, AmyI-1-18 could block the binding of LPS-binding protein to LPS, although its ability was less than that of polymyxin B. These results suggest that AmyI-1-18 expressing antimicrobial and endotoxin-neutralizing activities is useful as a safe and potent host defense peptide against pathogenic Gram-negative bacteria in many fields of healthcare.     

Dimeric structures of α-synuclein bind preferentially to lipid membranes

There is substantial evidence which implicates α-synuclein and its ability to aggregate and bind vesicle membranes as critical factors in the development of Parkinson's disease. In order to investigate the interaction between α-synuclein wild type (Wt) and its familial mutants, A53T and A30P with lipid membranes, we developed a novel lipid binding assay using surface enhanced laser desorption/ionisation-time of flight-mass spectrometry (SELDI-TOF MS). Wt and A53T exhibited similar lipid binding profiles; monomeric species and dimers bound with high relative affinity to the lipid surface, the latter of which exhibited preferential binding. Wt and A53T trimers and tetramers were also detected on the lipid surface. A30P exhibited a unique lipid binding profile; monomeric A30P bound with a low relative affinity, however, the dimeric species of A30P exhibited a higher binding ability. Larger order A30P oligomers were not detected on the lipid surface. Tapping mode atomic force microscopy (AFM) imaging was conducted to further examine the α-synuclein-lipid interaction. AFM analysis revealed Wt and its familial mutants can penetrate lipid membranes or disrupt the lipid and bind the hydrophobic alkyl self-assembled monolayer (SAM) used to form the lipid layer. The profile of these studied proteins revealed the presence of ‘small features’ consistent with the presence of monomeric and dimeric forms of the protein. These data collectively indicate that the dimeric species of Wt and its mutants can bind and cause membrane perturbations.     

Self-assembly of amphipathic β-sheet peptides: insights and applications

Amphipathic peptides composed of alternating polar and nonpolar residues have a strong tendency to self-assemble into one-dimensional, amyloid-like fibril structures. Fibrils derived from peptides of general (XZXZ)(n) sequence in which X is hydrophobic and Z is hydrophilic adopt a putative β-sheet bilayer. The bilayer configuration allows burial of the hydrophobic X side chain groups in the core of the fibril and leaves the polar Z side chains exposed to solvent. This architectural arrangement provides fibrils that maintain high solubility in water and has facilitated the recent exploitation of self-assembled amphipathic peptide fibrils as functional biomaterials. This article is a critical review of the development and application of self-assembling amphipathic peptides with a focus on the fundamental insight these types of peptides provide into peptide self-assembly phenomena.     

Anticancer mechanisms of action of two small amphipathic β2,2-amino acid derivatives derived from antimicrobial peptides

We have recently discovered that small antimicrobial β^2,2-amino acid derivatives (Mw < 500) also display activity against cancer cells. To explore their drug potential, we have presently investigated the mechanisms of action of two derivatives BAA-1 (IC₅₀ 8.1 μg/ml) and BAA-2 (IC₅₀ 3.8 μg/ml) on Ramos human Burkitt's lymphoma cells. Studies using annexin-V-FITC/propidium iodide staining and flow cytometry revealed essential mechanistic differences, which was confirmed by screening for active caspases, investigation of mitochondrial membrane potential, and electron microscopy studies. Our results indicated that BAA-1 killed Ramos cells by destabilizing the cell membrane, whereas BAA-2 caused apoptosis by the mitochondrial-mediated pathway.     

Differential activity of lytic α-helical peptides on lactobacilli and lactobacilli-derived liposomes

Eukaryotic antimicrobial peptides (AMPs) interact with plasma membrane of bacteria, fungi and eukaryotic parasites. Noteworthy, Lactobacillus delbrueckii subsp. lactis (CIDCA 133) and L. delbrueckii subsp. bulgaricus (CIDCA 331) show different susceptibility to human beta-defensins (β-sheet peptides). In the present work we extended the study to α-helical peptides from anuran amphibian (Aurein 1.2, Citropin 1.1 and Maculatin 1.1). We studied the effect on whole bacteria and liposomes formulated with bacterial lipids through growth kinetics, flow cytometry, leakage of liposome content and studies of peptide insertion in lipid monolayers.Growth of strain CIDCA 331 was dramatically inhibited in the presence of all three peptides and minimal inhibitory concentrations were lower than those for strain CIDCA 133. Flow cytometry revealed that AMPs lead to the permeabilization of bacteria.In addition, CIDCA 331-derived liposomes showed high susceptibility, leading to content leakage and structural disruption. Accordingly, peptide insertion in lipid monolayers demonstrated spontaneous interaction of AMPs with CIDCA 331 lipids. In contrast, lipids monolayers from strain CIDCA 133 were less susceptible.Summarizing we demonstrate that the high resistance of the probiotic strain CIDCA 133 to AMPs extends to α helix peptides Aurein, Citropin and Maculatin. This behavior could be ascribed in part to differences in membrane composition. These findings, along with the previously demonstrated resistance to β defensins from human origin, suggest that strain CIDCA 133 is well adapted to host innate immune effectors from both mammals and amphibians thus indicating conserved mechanisms of interaction with key components of the innate immune system.     

Comparison of protein and lipid composition of the human platelet α-granule membranes and glycerol lysis membranes

Platelet glycerol lysis membranes and α-granule membranes were compared with respect to protein and lipid composition. Crossed immunoelectrophoresis using antibodies against whole platelets, and sodium dodecyl sulphate polyacrylamide gel electrophoresis, revealed the presence of the glycoproteins IIb and IIIa, myosin and an antigen termed G4 in both membrane fractions. The glycoproteins Ia, Ib and IIIb, in addition to β₂-microglobulin and actin, appeared specific for the glycerol lysis membranes, whereas two antigens, termed G8 and G18, were observed only in the α-granule membranes. The localization of glycoprotein IIa was inconclusive. Comparison with the surface-located proteins revealed that the glycerol lysis membranes represented a reasonable approximation to a plasma membrane preparation. Radioactively labelled immunoprecipitates obtained after crossed immunoelectrophoresis of ¹²⁵I-labelled platelets were cut out and applied to sodium dodecyl sulphate electrophoresis on polyacrylamide slab gels. Autoradiography of the dried gels revealed that antigen G4 represented a protein with an average molecular weight of 146 000 in its unreduced state and 132 000 in its reduced state. Antigen G18 represented a protein of molecular weight 130 000–135 000 in the reduced as well as unreduced state. Quantitation of protein and lipids showed that the α-granule membranes contained about one-third as much cholesterol and 2-times as much protein in relation to phospholipids as compared to the glycerol lysis membranes. No significant difference between the two membrane preparations was found as regards the composition of their phospholipids.     

Dynamics and orientation of amphipathic peptides in solution and bound to membranes: a steady-state and time-resolved fluorescence study of staphylococcal δ-toxin and its synthetic analogues

The environment of both the hydrophilic and hydrophobic sides of alpha-helical delta-toxin are probed by tryptophanyl (Trp) fluorescence, when self-association occurs in solution and on binding to membranes. The fluorescence parameters of staphylococcal delta-toxin (Trp15 on the polar side of the amphipathic helix) and synthetic analogues with single Trp at position 5 or 16 (on the apolar side) were studied. The time-resolved fluorescence decays of the peptides in solution show that the local environment of their single Trp is always heterogeneous. Although the self-association degree increases with concentration, as shown by fluorescence anisotropy decays, the lifetimes (and their statistical weight) of Trp16 do not change, contrary to what is observed for Trp15. The first step of self-association is then driven by hydrophobic interactions between apolar sides of alpha-helices, whilst further oligomerization involves their polar side (Trp15) via electrostatic interactions. This is supported by dissociation induced by salt. For all self-associated peptides, the polarity of the Trp microenvironment was not significantly modified upon binding to phospholipid vesicles, as indicated by the small shifts of the fluorescence emission spectra and lifetime values. However, the relative populations of the lifetime classes vary with bound-peptide density similar to the rates of their global motions in bilayers or smaller particles. Quenching experiments by water or lipid-soluble compounds show changes of the orientation of membrane-inserted peptides, from probably dimers lying flat at the interface at low peptide density, to oligomers spanning the membrane and inducing membrane fragmentation at high peptide density.     

Free energy landscapes of two model peptides: α-helical and β-hairpin peptides explored with Brownian dynamics simulation

We applied an atomistic Brownian dynamics (BD) simulation with multiple time step method for the folding simulation of a 13-mer α-helical peptide and a 12-mer β-hairpin peptide, giving successful folding simulations. In this model, the driving energy contribution towards folding came from both electrostatic and van der Waals interactions for the α-helical peptide and from van der Waals interactions for the β-hairpin peptide. Although, many non-native structures having the same or lower energy than that of native structure were observed, the folded states formed the most populated cluster when the structures obtained by the BD simulations were subjected to the cluster analysis based on distance-based root mean square deviation of side-chains between different structures. This result indicates that we can predict the native structures from conformations sampled by BD simulation.     

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.     

Anticancer mechanisms of action of two small amphipathic β(2,2)-amino acid derivatives derived from antimicrobial peptides

We have recently discovered that small antimicrobial β(2,2)-amino acid derivatives (Mw<500) also display activity against cancer cells. To explore their drug potential, we have presently investigated the mechanisms of action of two derivatives BAA-1 (IC(50) 8.1μg/ml) and BAA-2 (IC(50) 3.8μg/ml) on Ramos human Burkitt's lymphoma cells. Studies using annexin-V-FITC/propidium iodide staining and flow cytometry revealed essential mechanistic differences, which was confirmed by screening for active caspases, investigation of mitochondrial membrane potential, and electron microscopy studies. Our results indicated that BAA-1 killed Ramos cells by destabilizing the cell membrane, whereas BAA-2 caused apoptosis by the mitochondrial-mediated pathway.     

Partition of antimicrobial D-L-α-cyclic peptides into bacterial model membranes

Fluorescence spectroscopy is used to characterize the partition of three second-generation D,L-α-cyclic peptides to two lipid model membranes. The peptides have proven antimicrobial activity, particularly against Gram positive bacteria, and the model membranes are formed of either with 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DMPG) or its mixture with 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), at a molar ratio of (1:1). The peptide's intrinsic fluorescence was used in the Steady State and/or Time Resolved Fluorescence Spectroscopy experiments, showing that the peptides bind to the membranes, and the extent of their partition is thereof quantified. The peptide-induced membrane leakage was followed using an encapsulated fluorescent dye.Overall, the partition is mainly driven by electrostatics, but also involves hydrophobic interactions. The introduction of a hydrocarbon tail in one of the residues of the parent peptide, CPR, adjacent to the tryptophan (Trp) residue, significantly improves the partition of the modified peptides, CPRT10 and CPRT14, to both membrane systems. Further, we show that the length of the tail is the main distinguishing factor for the extension of the partition process.The parent peptide induces very limited leakage, at odds with the peptides with tail, that promote fast leakage, increasing in most cases with peptide concentration, and being almost complete for the highest peptide concentration and negatively charged membranes.Overall, the results help the unravelling of the antimicrobial action of these peptides and are well in line with their proven high antimicrobial activity.     

Binding of phospholipids to β-Lactoglobulin and their transfer to lipid bilayers

The bovine milk lipocalin, β-Lactoglobulin (β-LG), has been associated with the binding and transport of small hydrophobic and amphiphilic compounds, whereby it is proposed to increase their bioavailability. We have studied the binding of the fluorescent phospholipid-derivative, NBD-didecanoylphosphatidylethanolamine (NBD-diC₁₀PE) to β-LG by following the increase in amphiphile fluorescence upon binding to the protein using established methods. The equilibrium association constant, K_B, was (1.2 ± 0.2) × 10⁶ M^− 1 at 25 °C, pH 7.4 and I = 0.15 M. Dependence of K_B on pH and on the monomer-dimer equilibrium of β-LG gave insight on the nature of the binding site which is proposed to be the hydrophobic calyx formed by the β-barrel in the protein. The monomer-dimer equilibrium of β-LG was re-assessed using fluorescence anisotropy of Tryptophan. The equilibrium constant for dimerization, K_D, was (7.0 ± 1.5) × 10⁵ M^− 1 at 25 °C, pH 7.4, and 0.15 M ionic strength. The exchange of NBD-diC₁₀PE between β-LG and POPC lipid bilayers was followed by the change in NBD fluorescence. β-LG was shown to be a catalyst of phospholipid exchange between lipid bilayers, the mechanism possibly involving adsorption of the protein at the bilayer surface.