Molecular View by Fourier Transform Infrared Spectroscopy of the Relationship between Lactocin 705 and Membranes: Speculations on Antimicrobial Mechanism

Lactocin 705 is a bacteriocin whose activity depends upon the complementation of two peptides, termed Lac705α and Lac705β. Neither Lac705α nor Lac705β displayed bacteriocin activity by itself when the growth of sensitive cells was monitored. To obtain molecular insights into the lactocin 705 mechanism of action, Fourier transform infrared spectroscopy was used to investigate the interactions of each peptide (Lac705α and Lac705β) with dipalmitoylphosphatidylcholine liposomal membranes. Both peptides show the ability to interact with the zwitterionic membrane but at different bilayer levels. While Lac705α interacts with the interfacial region inducing dehydration, Lac705β peptide interacts with only the hydrophobic core. This paper presents the first experimental evidence that supports the hypothesis that Lac705α and Lac705β peptides could form a transmembrane oligomer. From the obtained results, a mechanism of action of lactocin 705 on membrane systems is proposed. The component Lac705α could induce the dehydration of the bilayer interfacial region, and the Lac705β peptide could insert in the hydrophobic region of the membrane where the peptide has adequate conditions to achieve the oligomerization.     

Differential Roles of the Two-Component Peptides of Lactocin 705 in Antimicrobial Activity

Lactobacillus casei CRL705 produces a class IIb bacteriocin, lactocin 705, which relies on the complementary action of two components, Lac705α and Lac705β. These peptides exert a bactericidal effect on the indicator strain Lactobacillus plantarum CRL691, with an optimal Lac705α/Lac705β peptide ratio of 1 to 4. Electron microscopy studies showed that treated CRL691 cells have their cell wall severely damaged, with mesosome-like membranous formations protruding into their cytoplasm. Although less pronounced, a similar effect was also observed with the Lac705β peptide alone. Furthermore, Lac705β increased the inhibitory action of a diluted supernatant of L. casei CRL705, while Lac705α protected CRL691 cells from inhibition. Both peptides were required to dissipate the proton motive force (Δψ and ΔpH) of CRL691 cells. These data suggested that of the two components of lactocin 705, the Lac705α peptide is responsible for receptor recognition, and the Lac705β peptide is the active component on the cell membrane of CRL691 cells. Received: 12 April 2002 / Accepted: 24 May 2002     

Identification and nucleotide sequence of genes involved in the synthesis of lactocin 705, a two-peptide bacteriocin from Lactobacillus casei CRL 705

The structural gene determinants of lactocin 705, a bacteriocin produced by Lactobacillus casei CRL 705, have been amplified from a plasmid of approximately 35 kb and sequenced. Lactocin 705 is a class IIb bacteriocin, whose activity depends upon the complementation of two peptides (705alpha and 705beta) of 33 amino acid residues each. These peptides are synthesized as precursors with signal sequences of the double-glycine type, which exhibited high identities with the leader peptides of plantaricin S and J from Lactobacillus plantarum, brochocin C from Brochotrix campestris, sakacin P from Lactobacillus sake, and the competence stimulating peptides from Streptococcus gordonii and Streptococcus mitis. However, the two mature bacteriocins 705alpha and 705beta do not show significant similarity to other sequences in the databases.     

Structures and mode of membrane interaction of a short alpha helical lytic peptide and its diastereomer determined by NMR, FTIR, and fluorescence spectroscopy.

The interaction of many lytic cationic antimicrobial peptides with their target cells involves electrostatic interactions, hydrophobic effects, and the formation of amphipathic secondary structures, such as alpha helices or beta sheets. We have shown in previous studies that incorporating approximately 30%d-amino acids into a short alpha helical lytic peptide composed of leucine and lysine preserved the antimicrobial activity of the parent peptide, while the hemolytic activity was abolished. However, the mechanisms underlying the unique structural features induced by incorporating d-amino acids that enable short diastereomeric antimicrobial peptides to preserve membrane binding and lytic capabilities remain unknown. In this study, we analyze in detail the structures of a model amphipathic alpha helical cytolytic peptide KLLLKWLL KLLK-NH2 and its diastereomeric analog and their interactions with zwitterionic and negatively charged membranes. Calculations based on high-resolution NMR experiments in dodecylphosphocholine (DPCho) and sodium dodecyl sulfate (SDS) micelles yield three-dimensional structures of both peptides. Structural analysis reveals that the peptides have an amphipathic organization within both membranes. Specifically, the alpha helical structure of the L-type peptide causes orientation of the hydrophobic and polar amino acids onto separate surfaces, allowing interactions with both the hydrophobic core of the membrane and the polar head group region. Significantly, despite the absence of helical structures, the diastereomer peptide analog exhibits similar segregation between the polar and hydrophobic surfaces. Further insight into the membrane-binding properties of the peptides and their depth of penetration into the lipid bilayer has been obtained through tryptophan quenching experiments using brominated phospholipids and the recently developed lipid/polydiacetylene (PDA) colorimetric assay. The combined NMR, FTIR, fluorescence, and colorimetric studies shed light on the importance of segregation between the positive charges and the hydrophobic moieties on opposite surfaces within the peptides for facilitating membrane binding and disruption, compared to the formation of alpha helical or beta sheet structures.     

Lactococcin G is a potassium ion-conducting, two-component bacteriocin.

Lactococcin G is a novel lactococcal bacteriocin whose activity depends on the complementary action of two peptides, termed alpha and beta. Peptide synthesis of the alpha and beta peptides yielded biologically active lactococcin G, which was used in mode-of-action studies on sensitive cells of Lactococcus lactis. Approximately equivalent amounts of both peptides were required for optimal bactericidal effect. No effect was observed with either the alpha or beta peptide in the absence of the complementary peptide. The combination of alpha and beta peptides (lactococcin G) dissipates the membrane potential (delta omega), and as a consequence cells release alpha-aminoisobutyrate, a non-metabolizable alanine analog that is accumulated through a proton motive-force dependent mechanism. In addition, the cellular ATP level is dramatically reduced, which results in a drastic decrease of the ATP-driven glutamate uptake. Lactococcin G does not form a proton-conducting pore, as it has no effect on the transmembrane pH gradient. Dissipation of the membrane potential by uncouplers causes a slow release of potassium (rubidium) ions. However, rapid release of potassium was observed in the presence of lactococcin G. These data suggest that the bactericidal effect of lactococcin G is due to the formation of potassium-selective channels by the alpha and beta peptides in the target bacterial membrane.     

Biological and molecular characterization of a two-peptide lantibiotic produced by Lactococcus lactis IFPL105

The lactic acid bacterium Lactococcus lactis IFPL105 secretes a broad spectrum bacteriocin produced from the 46 kb plasmid pBAC105. The bacteriocin was purified to homogeneity by ionic and hydrophobic exchange and reverse-phase chromatography. Bacteriocin activity required the complementary action of two distinct peptides (alpha and beta) with average molecular masses of 3322 and 2848 Da, respectively. The genes encoding the two peptides were cloned and sequenced and were found to be identical to the ltnAB genes from plasmid pMRC01 of L. lactis DPC3147. LtnA and LtnB contain putative leader peptide sequences similar to the known 'double glycine' type. The predicted amino acid sequence of mature LtnA and LtnB differed from the amino acid content determined for the purified alpha and beta peptides in the residues serine, threonine, cysteine and alanine. Post-translational modification, and the formation of lanthionine or methyllanthionine rings, could partly explain the difference. Hybridization experiments showed that the organization of the gene cluster in pBAC105 responsible for the production of the bacteriocin is similar to that in pMRC01, which involves genes encoding modifying enzymes for lantibiotic biosynthesis and dual-function transporters. In both cases, the gene clusters are flanked by IS946 elements, suggesting an en bloc transposition. The findings from the isolation and molecular characterization of the bacteriocin provide evidence for the lantibiotic nature of the two peptides.     

A novel lactococcal bacteriocin whose activity depends on the complementary action of two peptides.

A lactococcal bacteriocin, termed lactococcin G, was purified to homogeneity by a simple four-step purification procedure that includes ammonium sulfate precipitation, binding to a cation exchanger and octyl-Sepharose CL-4B, and reverse-phase chromatography. The final yield was about 20%, and nearly a 7,000-fold increase in the specific activity was obtained. The bacteriocin activity was associated with three peptides, termed alpha 1, alpha 2, and beta, which were separated by reverse-phase chromatography. Judging from their amino acid sequences, alpha 1 and alpha 2 were the same gene product. Differences in their configurations presumably resulted in alpha 2 having a slightly lower affinity for the reverse-phase column than alpha 1 and a reduced bacteriocin activity when combined with beta. Bacteriocin activity required the complementary action of both the alpha and the beta peptides. When neither alpha 1 nor beta was in excess, about 0.3 nM alpha 1 and 0.04 nM beta induced 50% growth inhibition, suggesting that they might interact in a 7:1 or 8:1 ratio. As judged by the amino acid sequence, alpha 1 has an isoelectric point of 10.9, an extinction coefficient of 1.3 x 10(4) M-1 cm-1, and a molecular weight of 4,346 (39 amino acid residues long). Similarly, beta has an isoelectric point of 10.4, an extinction coefficient of 2.4 x 10(4) M-1 cm-1, and a molecular weight of 4110 (35 amino acid residues long). Molecular weights of 4,376 and 4,109 for alpha 1 and beta, respectively, were obtained by mass spectrometry. The N-terminal halves of both the alpha and beta peptides may form amphiphilic alpha-helices, suggesting that the peptides are pore-forming toxins that create cell membrane channels through a "barrel-stave" mechanism. The C-terminal halves of both peptides consist largely of polar amino acids.     

Correlation of three-dimensional structures with the antibacterial activity of a group of peptides designed based on a nontoxic bacterial membrane anchor

To understand the functional differences between a nontoxic membrane anchor corresponding to the N-terminal sequence of the Escherichia coli enzyme IIA(Glc) and a toxic antimicrobial peptide aurein 1.2 of similar sequence, a series of peptides was designed to bridge the gap between them. An alteration of a single residue of the membrane anchor converted it into an antibacterial peptide. Circular dichroism spectra indicate that all peptides are disordered in water but helical in micelles. Structures of the peptides were determined in membrane-mimetic micelles by solution NMR spectroscopy. The quality of the distance-based structures was improved by including backbone angle restraints derived from a set of chemical shifts ((1)H(alpha), (15)N, (13)C(alpha), and (13)C(beta)) from natural abundance two-dimensional heteronuclear correlated spectroscopy. Different from the membrane anchor, antibacterial peptides possess a broader and longer hydrophobic surface, allowing a deeper penetration into the membrane, as supported by intermolecular nuclear Overhauser effect cross-peaks between the peptide and short chain dioctanoyl phosphatidylglycerol. An attempt was made to correlate the NMR structures of these peptides with their antibacterial activity. The activity of this group of peptides does not correlate exactly with helicity, amphipathicity, charge, the number of charges, the size of the hydrophobic surface, or hydrophobic transfer free energy. However, a correlation is established between the peptide activity and membrane perturbation potential, which is defined by interfacial hydrophobic patches and basic residues in the case of cationic peptides. Indeed, (31)P solid state NMR spectroscopy of lipid bilayers showed that the extent of lipid vesicle disruption by these peptides is proportional to their membrane perturbation potential.     

Structure, topology, and tilt of cell-signaling peptides containing nuclear localization sequences in membrane bilayers determined by solid-state NMR and molecular dynamics simulation studies

Cell-signaling peptides have been extensively used to transport functional molecules across the plasma membrane into living cells. These peptides consist of a hydrophobic sequence and a cationic nuclear localization sequence (NLS). It has been assumed that the hydrophobic region penetrates the hydrophobic lipid bilayer and delivers the NLS inside the cell. To better understand the transport mechanism of these peptides, in this study, we investigated the structure, orientation, tilt of the peptide relative to the bilayer normal, and the membrane interaction of two cell-signaling peptides, SA and SKP. Results from CD and solid-state NMR experiments combined with molecular dynamics simulations suggest that the hydrophobic region is helical and has a transmembrane orientation with the helical axis tilted away from the bilayer normal. The influence of the hydrophobic mismatch, between the hydrophobic length of the peptide and the hydrophobic thickness of the bilayer, on the tilt angle of the peptides was investigated using thicker POPC and thinner DMPC bilayers. NMR experiments showed that the hydrophobic domain of each peptide has a tilt angle of 15 +/- 3 degrees in POPC, whereas in DMPC, 25 +/- 3 degree and 30 +/- 3 degree tilts were observed for SA and SKP peptides, respectively. These results are in good agreement with molecular dynamics simulations, which predict a tilt angle of 13.3 degrees (SA in POPC), 16.4 degrees (SKP in POPC), 22.3 degrees (SA in DMPC), and 31.7 degrees (SKP in DMPC). These results and simulations on the hydrophobic fragment of SA or SKP suggest that the tilt of helices increases with a decrease in bilayer thickness without changing the phase, order, and structure of the lipid bilayers.     

Interaction of mutant influenza virus hemagglutinin fusion peptides with lipid bilayers: probing the role of hydrophobic residue size in the central region of the fusion peptide.

The amino-terminal region of the membrane-anchored subunit of influenza virus hemagglutinin, the fusion peptide, is crucial for membrane fusion of this virus. The peptide is extruded from the interior of the protein and inserted into the lipid bilayer of the target membrane upon induction of a conformational change in the protein by low pH. Although the effects of several mutations in this region on the fusion behavior and the biophysical properties of the corresponding peptides have been studied, the structural requirements for an active fusion peptide have still not been defined. To probe the sensitivity of the fusion peptide structure and function to small hydrophobic perturbations in the middle of the hydrophobic region, we have individually replaced the alanine residues in positions 5 and 7 with smaller (glycine) or bulkier (valine) hydrophobic residues and measured the extent of fusion mediated by these hemagglutinin constructs as well as some biophysical properties of the corresponding synthetic peptides in lipid bilayers. We find that position 5 tolerates a smaller and position 7 a larger hydrophobic side chain. All peptides contained segments of alpha-helical (33-45%) and beta-strand (13-16%) conformation as determined by CD and ATR-FTIR spectroscopy. The order parameters of the peptide helices and the lipid hydrocarbon chains were determined from measurements of the dichroism of the respective infrared absorption bands. Order parameters in the range of 0.0-0.6 were found for the helices of these peptides, which indicate that these peptides are most likely aligned with their alpha-helices at oblique angles to the membrane normal. Some (mostly fusogenic) peptides induced significant increases of the order parameter of the lipid hydrocarbon chains, suggesting that the lipid bilayer becomes more ordered in the presence of these peptides, possibly as a result of dehydration at the membrane surface.     

Isolation, partial characterization, and mode of action of Acidocin J1132, a two-component bacteriocin produced by Lactobacillus acidophilus JCM 1132.

Lactobacillus acidophilus JCM 1132 produces a heat-stable, two-component bacteriocin designated acidocin J1132 that has a narrow inhibitory spectrum. Maximum production of acidocin J1132 in MRS broth was detected at pH 5.0. Acidocin J1132 was purified by ammonium sulfate precipitation and sequential cation exchange and reversed-phase chromatographies. Acidocin J1132 activity was associated with two components, termed alpha and beta. On the basis of N-terminal amino acid sequencing and the molecular masses of the alpha and beta components, it is interpreted that the compounds differ by an additional glycine residue in the beta component. Both alpha and beta had inhibitory activity, and an increase in activity by the complementary action of the two components was observed. Acidocin J1132 is bactericidal and dissipates the membrane potential and the pH gradient in sensitive cells, which affect such proton motive force-dependent processes as amino acid transport. Acidocin J1132 also caused efflux of preaccumulated amino acid taken up via a unidirectional ATP-driven transport system. Secondary structure prediction revealed the presence of an amphiphilic alpha-helix region that could form hydrophilic pores. These results suggest that acidocin J1132 is a pore-forming bacteriocin that creates cell membrane channels through the "barrel-stave" mechanism.     

Purification and genetic characterization of plantaricin NC8, a novel coculture-inducible two-peptide bacteriocin from Lactobacillus plantarum NC8

A new, coculture-inducible two-peptide bacteriocin named plantaricin NC8 (PLNC8) was isolated from Lactobacillus plantarum NC8 cultures which had been induced with Lactococcus lactis MG1363 or Pediococcus pentosaceus FBB63. This bacteriocin consists of two distinct peptides, named alpha and beta, which were separated by C(2)-C(18) reverse-phase chromatography and whose complementary action is necessary for full plantaricin NC8 activity. N-terminal sequencing of both purified peptides showed 28 and 34 amino acids residues for PLNC8 alpha and PLNC8 beta, respectively, which showed no sequence similarity to other known bacteriocins. Mass spectrometry analysis showed molecular masses of 3,587 Da (alpha) and 4,000 Da (beta). The corresponding genes, designated plNC8A and plNC8B, were sequenced, and their nucleotide sequences revealed that both peptides are produced as bacteriocin precursors of 47 and 55 amino acids, respectively, which include N-terminal leader sequences of the double-glycine type. The mature alpha and beta peptides contain 29 and 34 amino acids, respectively. An open reading frame, orfC, which encodes a putative immunity protein was found downstream of plNC8B and overlapping plNC8A. Upstream of the putative -35 region of plNC8B, two direct repeats of 9 bp were identified, which agrees with the consensus sequence and structure of promoters of class II bacteriocin operons whose expression is dependent on an autoinduction mechanism.     

Purification and partial amino acid sequence of plantaricin 1.25ααα and 1.25βββ, two bacteriocins produced by Lactobacillus plantarum TMW1.25

Two bacteriocins produced by Lactobacillus plantarum TMW1.25 have been purified by a four-step purification procedure, including ammonium sulphate precipitation and cation-exchange chromatography followed by hydrophobic-interaction chromatography on octyl sepharose. The final purification was performed by repeated reversed-phase chromatography steps which yielded two bacteriocin fractions designated plantaricin 1.25 alpha and plantaricin 1.25 beta. The molecular masses of the peptides in these fractions were 5979 and 5203 Da, respectively. Combination of the fractions did not have any synergistic effects on bacteriocin activity, indicating that they each contain a one-peptide bacteriocin. The major peptide in the alpha fraction was blocked at its N-terminus, and a partial sequence (25 residues) could only be obtained after cleavage with CNBr. This sequence did not show clear homologies with known bacteriocins. The beta peptide has been sequenced almost completely and consists, presumably, of 53 residues. This peptide displayed strong homology to the known N-terminal part of brevicin 27 produced by Lactobacillus brevis SB27. The results showed that the beta peptide contains as many as six consecutive lysine residues at the N-terminus.     

Bilayer localization of membrane-active peptides studied in biomimetic vesicles by visible and fluorescence spectroscopies.

Depth of bilayer penetration and effects on lipid mobility conferred by the membrane-active peptides magainin, melittin, and a hydrophobic helical sequence KKA(LA)7KK (denoted KAL), were investigated by colorimetric and time-resolved fluorescence techniques in biomimetic phospholipid/poly(diacetylene) vesicles. The experiments demonstrated that the extent of bilayer permeation and peptide localization within the membrane was dependent upon the bilayer composition, and that distinct dynamic modifications were induced by each peptide within the head-group environment of the phospholipids. Solvent relaxation, fluorescence correlation spectroscopy and fluorescence quenching analyses, employing probes at different locations within the bilayer, showed that magainin and melittin inserted close to the glycerol residues in bilayers incorporating negatively charged phospholipids, but predominant association at the lipid-water interface occurred in bilayers containing zwitterionic phospholipids. The fluorescence and colorimetric analyses also exposed the different permeation properties and distinct dynamic influence of the peptides: magainin exhibited the most pronounced interfacial attachment onto the vesicles, melittin penetrated more into the bilayers, while the KAL peptide inserted deepest into the hydrophobic core of the lipid assemblies. The solvent relaxation results suggest that decreasing the lipid fluidity might be an important initial factor contributing to the membrane activity of antimicrobial peptides.     

Bilayer conformation of fusion peptide of influenza virus hemagglutinin: a molecular dynamics simulation study

Unraveling the conformation of membrane-bound viral fusion peptides is essential for understanding how those peptides destabilize the bilayer topology of lipids that is important for virus-cell membrane fusion. Here, molecular dynamics (MD) simulations were performed to investigate the conformation of the 20 amino acids long fusion peptide of influenza hemagglutinin of strain X31 bound to a dimyristoyl phosphatidylcholine (DMPC) bilayer. The simulations revealed that the peptide adopts a kinked conformation, in agreement with the NMR structures of a related peptide in detergent micelles. The peptide is located at the amphipathic interface between the headgroups and hydrocarbon chains of the lipid by an energetically favorable arrangement: The hydrophobic side chains of the peptides are embedded into the hydrophobic region and the hydrophilic side chains are in the headgroup region. The N-terminus of the peptide is localized close to the amphipathic interface. The molecular dynamics simulations also revealed that the peptide affects the surrounding bilayer structure. The average hydrophobic thickness of the lipid phase close to the N-terminus is reduced in comparison with the average hydrophobic thickness of a pure dimyristoyl phosphatidylcholine bilayer.     

Differential scanning calorimetry and X-ray diffraction studies of the specificity of the interaction of antimicrobial peptides with membrane-mimetic systems

Interest in biophysical studies on the interaction of antimicrobial peptides and lipids has strongly increased because of the rapid emergence of antibiotic-resistant bacterial strains. An understanding of the molecular mechanism(s) of membrane perturbation by these peptides will allow a design of novel peptide antibiotics as an alternative to conventional antibiotics. Differential scanning calorimetry and X-ray diffraction studies have yielded a wealth of quantitative information on the effects of antimicrobial peptides on membrane structure as well as on peptide location. These studies clearly demonstrated that antimicrobial peptides show preferential interaction with specific phospholipid classes. Furthermore, they revealed that in addition to charge-charge interactions, membrane curvature strain and hydrophobic mismatch between peptides and lipids are important parameters in determining the mechanism of membrane perturbation. Hence, depending on the molecular properties of both lipid and peptide, creation of bilayer defects such as phase separation or membrane thinning, pore formation, promotion of nonlamellar lipid structures or bilayer disruption by the carpet model or detergent-like action, may occur. Moreover, these studies suggest that these different processes may represent gradual steps of membrane perturbation. A better understanding of the mutual dependence of these parameters will help to elucidate the molecular mechanism of membrane damage by antimicrobial peptides and their target membrane specificity, keys for the rationale design of novel types of peptide antibiotics.     

The effect of a beta‐lactamase inhibitor peptide on bacterial membrane structure and integrity: a comparative study

Co‐administration of beta‐lactam antibiotics and beta‐lactamase inhibitors has been a favored treatment strategy against beta‐lactamase‐mediated bacterial antibiotic resistance, but the emergence of beta‐lactamases resistant to current inhibitors necessitates the discovery of novel non‐beta‐lactam inhibitors. Peptides derived from the Ala46–Tyr51 region of the beta‐lactamase inhibitor protein are considered as potent inhibitors of beta‐lactamase; unfortunately, peptide delivery into the cell limits their potential. The properties of cell‐penetrating peptides could guide the design of beta‐lactamase inhibitory peptides. Here, our goal is to modify the peptide with the sequence RRGHYY that possesses beta‐lactamase inhibitory activity under in vitro conditions. Inspired by the work on the cell‐penetrating peptide pVEC, our approach involved the addition of the N‐terminal hydrophobic residues, LLIIL, from pVEC to the inhibitor peptide to build a chimera. These residues have been reported to be critical in the uptake of pVEC. We tested the potential of RRGHYY and its chimeric derivative as a beta‐lactamase inhibitory peptide on Escherichia coli cells and compared the results with the action of the antimicrobial peptide melittin, the beta‐lactam antibiotic ampicillin, and the beta‐lactamase inhibitor potassium clavulanate to get mechanistic details on their action. Our results show that the addition of LLIIL to the N‐terminus of the beta‐lactamase inhibitory peptide RRGHYY increases its membrane permeabilizing potential. Interestingly, the addition of this short stretch of hydrophobic residues also modified the inhibitory peptide such that it acquired antimicrobial property. We propose that addition of the hydrophobic LLIIL residues to the peptide N‐terminus offers a promising strategy to design novel antimicrobial peptides in the battle against antibiotic resistance. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Membrane perturbation by the antimicrobial peptide PMAP-23: A fluorescence and molecular dynamics study

Several bioactive peptides exert their biological function by interacting with cellular membranes. Structural data on their location inside lipid bilayers are thus essential for a detailed understanding of their mechanism of action. We propose here a combined approach in which fluorescence spectroscopy and molecular dynamics (MD) simulations were applied to investigate the mechanism of membrane perturbation by the antimicrobial peptide PMAP-23. Fluorescence spectra, depth-dependent quenching experiments, and peptide-translocation assays were employed to determine the location of the peptide inside the membrane. MD simulations were performed starting from a random mixture of water, lipids and peptide, and following the spontaneous self-assembly of the bilayer. Both experimental and theoretical data indicated a peptide location just below the polar headgroups of the membrane, with an orientation essentially parallel to the bilayer plane. These findings, together with experimental results on peptide-induced leakage from large and giant vesicles, lipid flip-flop and peptide exchange between vesicles, support a mechanism of action consistent with the “carpet” model. Furthermore, the atomic detail provided by the simulations suggested the occurrence of an additional, more specific and novel mechanism of bilayer destabilization by PMAP-23, involving the unusual insertion of charged side chains into the hydrophobic core of the membrane.     

Binding of pediocin PA-1 with anionic lipid induces model membrane destabilization

To obtain molecular insights into the action mode of antimicrobial activity of pediocin PA-1, the interactions between this bacteriocin and dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylglycerol (DMPG) model membranes have been investigated in D(2)O at pD 6 by Fourier transform infrared spectroscopy. The interactions were monitored with respect to alteration of the secondary structure of pediocin, as registered by the amide I' band, and phospholipid conformation, as revealed by the methylene nu(s)(CH(2)) and carbonyl nu(C;O) stretching vibrations. The results show that no interaction between pediocin and DMPC occurs. By contrast, pediocin undergoes a structural reorganization in the presence of DMPG. Upon heating, pediocin self-aggregates, which is not observed for this pD in aqueous solution. The gel-to-crystalline phase transition of DMPG shifts to higher temperatures with a concomitant dehydration of the interfacial region. Our results indicate that pediocin is an extrinsic peptide and that its action mechanism may lie in a destabilization of the cell membrane.     

Peptide entry inhibitors of enveloped viruses: The importance of interfacial hydrophobicity

There are many peptides known that inhibit the entry of enveloped viruses into cells, including one peptide that is successfully being used in the clinic as a drug. In this review, we discuss the discovery, antiviral activity and mechanism of action of such peptides. While peptide entry inhibitors have been discovered by a wide variety of approaches (structure-based, accidental, intentional, rational and brute force) we show here that they share a common physical chemical property: they are at least somewhat hydrophobic and/or amphipathic and have a propensity to interact with membrane interfaces. We propose that this propensity drives a shared mechanism of action for many peptide entry inhibitors, involving direct interactions with viral and cellular membranes, as well as interactions with the complex hydrophobic protein/lipid interfaces that are exposed, at least transiently, during virus–cell fusion. By interacting simultaneously with the membrane interfaces and other critical hydrophobic surfaces, we hypothesize that peptide entry inhibitors can act by changing the physical chemistry of the membranes, and the fusion protein interfaces bridging them, and by doing so interfere with the fusion of cellular and viral membranes. Based on this idea, we propose that an approach that focuses on the interfacial hydrophobicity of putative entry inhibitors could lead to the efficient discovery of novel, broad-spectrum viral entry inhibitors. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.