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.     

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.     

Binding of amphipathic α-helical antimicrobial peptides to lipid membranes: Lessons from temporins B and L

Temporins constitute a family of amphipathic α-helical antimicrobial peptides (AMP) and contain some of the shortest cytotoxic peptides, comprised of only 10-14 residues. General characteristics of temporins parallel those of other AMP, both in terms of structural features and biophysical properties relating to their interactions with membrane lipids, with selective lipid-binding properties believed to underlie the discrimination between target vs host cells. Lipid-binding properties also contribute to the cytotoxicity AMP, causing permeabilization of their target cell membranes. The latter functional property of AMP involves highly interdependent acidic phospholipid-induced conformational changes, aggregation, and formation of toxic oligomers in the membrane. These oligomers are subsequently converted to amyloid-type fibers, as demonstrated for e.g. temporins B and L in our laboratory, and more recently for dermaseptins by Auvynet et al. Amyloid state represents the generic minimum in the folding/aggregation free energy landscape, and for AMP its formation most likely serves to detoxify the peptides, in keeping with the current consensus on mature amyloid being inert and non-toxic. The above scenario is supported by sequence analyses of temporins as well as other amphipathic α-helical AMP belonging to diverse families. Accordingly, sequence comparison identifies ‘conformational switches’, domains with equal probabilities for adopting random coil, α-helical and β-sheet structures. These regions were further predicted also to aggregate and assemble into amyloid β-sheets. Taken together, the lipid-binding properties and structural characterization lend support to the notion that the mechanism of membrane permeabilization by temporins B and L and perhaps of most AMP could be very similar, if not identical, to that of the paradigm amyloid forming cytotoxic peptides, responsible for degenerative cell loss in e.g. prion, Alzheimer's and Parkinson's disease, and type 2 diabetes.     

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.     

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].     

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.     

Synthesis of all-hydrocarbon stapled α-helical peptides by ring-closing olefin metathesis

This protocol provides a detailed procedure for the preparation of stapled α-helical peptides, which have proven their potential as useful molecular probes and as next-generation therapeutics. Two crucial features of this protocol are (i) the construction of peptide substrates containing hindered α-methyl, α-alkenyl amino acids and (ii) the ring-closing olefin metathesis (RCM) of the resulting resin-bound peptide substrates. The stapling systems described in this protocol, namely bridging one or two turns of an α-helix, are highly adaptable to most peptide sequences, resulting in favorable RCM kinetics, helix stabilization and promotion of cellular uptake.     

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.     

Cell fingerprint patterns using designed α-helical peptides to screen for cell-specific toxicity

We conducted cell-based cytotoxicity screening of a 101-membered α-helical peptide library using cell fingerprints (CFPs). The CFP data suggested that there is a relationship between cytotoxicity and peptide characteristics, such as hydrophobicity, charge, and amino acid composition. In spite of the small size of the library used in this study, several peptides demonstrated cell-specific toxicity. The strategy of combining a designed peptide library with CFP thus shows real promise for peptide-based screening with cells.     

A short α-helical antimicrobial peptide with antibacterial selectivity

A 13-residue alpha-helical peptide (K6L5WP), designed from Leu6-->Pro substitution of a hemolytic alpha-helical peptide (K6L6W), exhibited strong antibacterial activity (MIC: 2 to approximately 4 microM against three gram-positives and three gram-negatives) comparable to that of melittin but had no hemolytic activity. Tryptophan fluorescence studies indicated bacterial selectivity of K6L5WP is closely related to the selective interaction with negatively charged phospholipids on the surface of bacterial cells. These results suggested that the central Pro6 in K6L5WP plays an important role in its bacterial cell selectivity. In conclusion, K6L5WP with antibacterial selectivity may serve as an attractive candidate for the development of antimicrobial agents.     

The effects of Leu or Val residues on cell selectivity of α-helical peptides

In this study, the peptides were designed to compare the effect of multiple Leu or Val residues as the hydrophobic side of an α-helical model on their structure, function, and interaction with model membranes. The Leu-rich peptides displayed 4- to 16-fold stronger antimicrobial activity than Val-rich peptides, while Val-containing peptides showed no haemolysis and weak cytotoxicity. The peptides LR and VR showed an α-helical-rich structure under a membranemimicking environment. Different cell selectivity for Leu- or Val-containing peptides correlated with the targeted cell membranes. The Leu-rich peptide LR(W) and Val-rich peptide VR(W) interacted preferentially with negatively charged phospholipids over zwitterionic phospholipids. VR(W) displayed no interaction with zwitterionic phospholipids, which was consistent with its lack of haemolytic activity. The ability of LR to depolarize bacterial cells was much greater than that of VR. Val- and Leu-rich peptides appeared to kill bacteria in a membrane-targeted fashion, with different modes of action. Leu-rich peptides appeared to be active via a membrane-disrupting mode, while Val-rich peptides were active via the formation of small channels.     

Role of α-helical domains in functioning of ATP-dependent Lon protease of Escherichia coli

Homooligomeric ATP-dependent LonA proteases are bifunctional enzymes belonging to the superfamily of AAA⁺ proteins. Their subunits are formed by five successively connected domains, i.e., N-terminal (N), α-helical (HI(CC)), nucleotide-binding (NB), the second α-helical (H), and proteolytic (P) domains. The presence of the inserted HI(CC) domain determines the uniqueness of LonA proteases among the AAA⁺ proteins. The role of the α-helical domains in the LonA protease functioning was studied with an example of E. coli Lon protease (Ec-Lon). The properties of the intact Ec-Lon and its mutant forms, i.e., Lon-R164A and Lon-R542A bearing the substituted arginine residues at the similar positions in the HI(CC) and H domains, were compared. The H domain was shown to play a crucial role in ATP hydrolysis and enzyme binding to the target protein. The HI(CC) domain is not decisive for the manifestation of the catalytic properties of the enzyme. However, it affects the functioning of Lon ATPase and peptidase sites and is involved in maintaining enzyme stability. The participation of the HI(CC) domain in the formation of three-dimensional structures of LonA proteases and/or their complexes with DNA is suggested.     

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.     

Development of helix-stabilized antimicrobial peptides composed of lysine and hydrophobic α,α-disubstituted α-amino acid residues

Lysine-based amphipathic nonapeptides, including homochiral peptides [Ac-(l-Lys-l-Lys-Xaa)₃-NH₂ (Xaa = Gly, Ala, Aib, Ac₅c, or Ac₆c) and Ac-(d-Lys-d-Lys-Aib)₃-NH₂], a heterochiral peptide [Ac-(l-Lys-d-Lys-Aib)₃-NH₂], and a racemic mixture of diastereomeric peptides [Ac-(rac-Lys-rac-Lys-Aib)₃-NH₂] were designed and synthesized to investigate the relationship between their preferred secondary structures and their antimicrobial activity. Peptide 5, [Ac-(l-Lys-l-Lys-Ac₆c)₃-NH₂] formed a stable α-helical structure and exhibited strong activity against Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa).     

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.     

Functional analysis of an α-helical antimicrobial peptide derived from a novel mouse defensin-like gene

Gene-encoded antimicrobial peptides (AMPs) are an essential component of the innate immune system in many species. Analysis of β-defensin gene expression in mouse tissue using primers that were specific for conserved sequences located outside of the β-defensin translated region identified a novel small gene. The novel gene had an open reading frame of 114 bp and encoded a predicted protein of 37 amino acid residues. A search of the genome database revealed that the gene locus and the sequence of exon 1 of this novel gene were similar to subgroup 1 mouse β-defensins. A small peptide, K17 (FSPQMLQDIIEKKTKIL), derived from the amino acid sequence of this novel gene was synthesized. Circular dichroism (CD) spectroscopic analysis of chemically synthesized peptide demonstrated that the peptide exhibited random coil conformation in aqueous solution, but the peptide adopted helical conformation in the presence of trifluoroethanol or sodium dodecyl sulfate, a membrane-mimicking environment. The peptide exhibited bactericidal activity against Salmonella enterica serovar Typhimurium (Gram negative) and Staphylococcus aureus (Gram positive); it was not cytotoxic in cultures of mammalian cells or hemolytic in cultures of erythrocytes. These results suggested that K17 may be a candidate therapeutic for the treatment of bacterial infection.     

Solution NMR studies of amphibian antimicrobial peptides: Linking structure to function?

The high-resolution three-dimensional structure of an antimicrobial peptide has implications for the mechanism of its antimicrobial activity, as the conformation of the peptide provides insights into the intermolecular interactions that govern the binding to its biological target. For many cationic antimicrobial peptides the negatively charged membranes surrounding the bacterial cell appear to be a main target. In contrast to what has been found for other classes of antimicrobial peptides, solution NMR studies have revealed that in spite of the wide diversity in the amino acid sequences of amphibian antimicrobial peptides (AAMPs), they all adopt amphipathic α-helical structures in the presence of membrane-mimetic micelles, bicelles or organic solvent mixtures. In some cases the amphipathic AAMP structures are directly membrane-perturbing (e.g. magainin, aurein and the rana-box peptides), in other instances the peptide spontaneously passes through the membrane and acts on intracellular targets (e.g. buforin). Armed with a high-resolution structure, it is possible to relate the peptide structure to other relevant biophysical and biological data to elucidate a mechanism of action. While many linear AAMPs have significant antimicrobial activity of their own, mixtures of peptides sometimes have vastly improved antibiotic effects. Thus, synergy among antimicrobial peptides is an avenue of research that has recently attracted considerable attention. While synergistic relationships between AAMPs are well described, it is becoming increasingly evident that analyzing the intermolecular interactions between these peptides will be essential for understanding the increased antimicrobial effect. NMR structure determination of hybrid peptides composed of known antimicrobial peptides can shed light on these intricate synergistic relationships. In this work, we present the first NMR solution structure of a hybrid peptide composed of magainin 2 and PGLa bound to SDS and DPC micelles. The hybrid peptide adopts a largely helical conformation and some information regarding the inter-helix organization of this molecule is reported. The solution structure of the micelle associated MG2-PGLa hybrid peptide highlights the importance of examining structural contributions to the synergistic relationships but it also demonstrates the limitations in the resolution of the currently used solution NMR techniques for probing such interactions. Future studies of antimicrobial peptide synergy will likely require stable isotope-labeling strategies, similar to those used in NMR studies of proteins.     

Synthesis of α-aspartyl-containing cyclic peptides

Summary α-Aspartyl-containing cyclic pentapeptides were synthesised in high yields using a strategy that maintained fluorenylmethyl protection on the aspartic acid side chain during chain assembly, resin cleavage and cyclisation of the linear precursors. Tetra-n-butylammonium fluoride treatment of the fluorenylmethyl-protected cyclic peptides catalysed imide formation, whereas piperidine-induced deprotection resulted in good yields of the target cyclic peptides.     

Intramolecular distortion of the α-helical structure of polypeptides

Broadening of the infrared amide A, amide I and amide II bands of α-helical polypeptides has been observed for thermodynamically unstable α-helices. This spectroscopic fact can be explained now by the geometrical distortions of the backbone of the helical structure. Two models for distorted helices which include regular or irregular distortions of the angles of internal rotation of the main polypeptide chain have been considered. It is pointed out that the instability of α-helix is associated with irregular distortions of the polypeptide backbone.