Amphipathic Cationic Antimicrobial Peptides Research Articles

Synthesis and antimicrobial activity of aminoalkyl resveratrol derivatives inspired by cationic peptides

Antimicrobial resistance is a global concern, far from being resolved. The need of new drugs against new targets is imminent. In this work, we present a family of aminoalkyl resveratrol derivatives with antibacterial activity inspired by the properties of cationic amphipathic antimicrobial peptides. Surprisingly, the newly designed molecules display modest activity against aerobically growing bacteria but show surprisingly good antimicrobial activity against anaerobic bacteria (Gram-negative and Gram-positive) suggesting specificity towards this bacterial group. Preliminary studies into the action mechanism suggest that activity takes place at the membrane level, while no cross-resistance with traditional antibiotics is observed. Actually, some good synergistic relations with existing antibiotics were found against Gram-negative pathogens. However, some cytotoxicity was observed, despite their low haemolytic activity. Our results show the importance of the balance between positively charged moieties and hydrophobicity to improve antimicrobial activity, setting the stage for the design of new drugs based on these molecules.

Dual Oral Tissue Adhesive Nanofiber Membranes for pH-Responsive Delivery of Antimicrobial Peptides.

Bioadhesive membranes with controllable and reversible underwater adhesion are desirable for several biomedical applications ranging from biosensing, drug/therapeutic delivery, and tissue regeneration. Here, we present dual soft mucosal and hard bone/enamel tissue adhesive nanofiber membranes composed of chitosan and pectin derivatives for pH-controlled delivery of antimicrobial peptides (AMPs) in the oral cavity. Ex vivo testing with porcine esophagus (soft mucosal mimic) indicated a 2-fold increase in the mucoadhesion of chitosan membranes with 0.05 wt % oxidized pectin coating, while the uncoated membranes exhibited 3-4-fold stronger adhesion to hydroxyapatite discs (enamel/hard bone mimic) compared to the coated membranes. The former is attributed to a synergistic interaction of surface nanofiber topography, intermolecular hydrogen bonding, and aldehyde-amine chemistry between surface polar groups and mucosal proteins, while the latter may arise from electrostatic interactions between cationic amines (-NH3+) in chitosan and anionic phosphates (-PO43-) in hydroxyapatite. Further, the dual hard-soft oral tissue adhesive nanofiber membranes loaded with cationic amphipathic AMPs (D-GL13K and IDR-1018) elicited pH-responsive AMP delivery and antimicrobial action comparable to chlorhexidine (CHX) against oral streptococci. Concurrently, the AMP loaded membranes were cytocompatible to both soft epithelial tissue-derived human oral keratinocytes and hard calvarial murine pre-osteoblast cells. We envision these membranes to function as adhesive gingival grafts and guided bone regeneration (GBR) membranes at the hard-soft tissue interface while simultaneously protecting against oral infections.

Cationic Amphipathic Antimicrobial Peptides Perturb the Inner Membrane of Germinated Spores Thus Inhibiting Their Outgrowth

The mode of action of four cationic amphipathic antimicrobial peptides (AMPs) was evaluated against the non-pathogenic, Gram-positive, spore-forming bacterium, Bacillus subtilis. The AMPs were TC19, TC84, BP2, and the lantibiotic Nisin A. TC19 and TC84 were derived from the human thrombocidin-1. Bactericidal peptide 2 (BP2) was derived from the human bactericidal permeability increasing protein (BPI). We employed structured illumination microscopy (SIM), fluorescence microscopy, Alexa 488-labeled TC84, B. subtilis mutants producing proteins fused to the green fluorescent protein (GFP) and single-cell live imaging to determine the effects of the peptides against spores. TC19, TC84, BP2, and Nisin A showed to be bactericidal against germinated spores by perturbing the inner membrane, thus preventing outgrowth to vegetative cells. Single cell live imaging showed that the AMPs do not affect the germination process, but the burst time and subsequent generation time of vegetative cells. Alexa 488-labeled TC84 suggested that the TC84 might be binding to the dormant spore-coat. Therefore, dormant spores were also pre-coated with the AMPs and cultured on AMP-free culture medium during single-cell live imaging. Pre-coating of the spores with TC19, TC84, and BP2 had no effect on the germination process, and variably affected the burst time and generation time. However, the percentage of spores that burst and grew out into vegetative cells was drastically lower when pre-coated with Nisin A, suggesting a novel application potential of this lantibiotic peptide against spores. Our findings contribute to the understanding of AMPs and show the potential of AMPs as eventual therapeutic agents against spore-forming bacteria.

Lipid-Mediated Interactions between the Antimicrobial Peptides Magainin 2 and PGLa in Bilayers

A synergistic enhancement of activities has been described for the amphipathic cationic antimicrobial peptides magainin 2 and PGLa when tested in antimicrobial assays or in biophysical experiments using model membranes. In the presence of magainin 2, PGLa changes from an in-planar alignment parallel to the membrane surface to a more transmembrane orientation when investigated in membranes made from fully saturated PC or PC/PG, but not when one of the fatty acyl chains is unsaturated. Such lipid-mediated changes in the membrane topology of polypeptide domains could provide an interesting mechanism for the regulation of membrane proteins. Here we investigated the PGLa topology in a wide variety of membranes made of saturated or unsaturated PE, PC, and/or PG using 15N solid-state NMR spectroscopy. In contrast to predictions made by previous models the data show that membrane curvature has only a minor effect on PGLa realignment. Furthermore, using 2H solid-state NMR spectroscopy of deuterated phospholipid fatty acyl chains the order parameters of the lipids were investigated in the presence of PGLa, magainin, or equimolar peptide mixtures. Both peptides cause a pronounced decrease in the order parameters when oriented parallel to the membrane surface, an effect that reverts when PGLa flips into transmembrane alignments. Taken together, these data are suggestive that the magainin-induced disordering of fatty acyl chains provides an important driving force for PGLa realignment.

Bactericidal activity of amphipathic cationic antimicrobial peptides involves altering the membrane fluidity when interacting with the phospholipid bilayer

BackgroundAmphipathic cationic antimicrobial peptides (AMPs) TC19 and TC84, derived from the major AMPs of human blood platelets, thrombocidins, and Bactericidal Peptide 2 (BP2), a synthetic designer peptide showed to perturb the membrane of Bacillus subtilis. We aimed to determine the means by which the three AMPs cause membrane perturbation in vivo using B. subtilis and to evaluate whether the membrane alterations are dependent on the phospholipid composition of the membrane. MethodsPhysiological analysis was employed using Alexa Fluor 488 labelled TC84, various fluorescence dyes, fluorescent microscopy techniques and structured illumination microscopy. ResultsTC19, TC84 and BP2 created extensive fluidity domains in the membrane that are permeable, thus facilitating the entering of the peptides and the leakage of the cytosol. The direct interaction of the peptides with the bilayer create the fluid domains. The changes caused in the packing of the phospholipids lead to the delocalization of membrane bound proteins, thus contributing to the cell's destruction. The changes made to the membrane appeared to be not dependent on the composition of the phospholipid bilayer. ConclusionsThe distortion caused to the fluidity of the membrane by the AMPs is sufficient to facilitate the entering of the peptides and leakage of the cytosol. General significanceHere we show in vivo that cationic AMPs cause “membrane leaks” at the site of membrane insertion by altering the organization and fluidity of the membrane. Our findings thus contribute to the understanding of the membrane perturbation characteristic of cationic AMPs.

Synthetic antimicrobial peptides delocalize membrane bound proteins thereby inducing a cell envelope stress response

BackgroundThree amphipathic cationic antimicrobial peptides (AMPs) were characterized by determining their effect on Gram-positive bacteria using Bacillus subtilis strain 168 as a model organism. These peptides were TC19 and TC84, derivatives of thrombocidin-1 (TC-1), the major AMPs of human blood platelets, and Bactericidal Peptide 2 (BP2), a synthetic designer peptide based on human bactericidal permeability increasing protein (BPI). MethodsTo elucidate the possible mode of action of the AMPs we performed a transcriptomic analysis using microarrays. Physiological analyses were performed using transmission electron microscopy (TEM), fluorescence microscopy and various B. subtilis mutants that produce essential membrane bound proteins fused to green fluorescent protein (GFP). ResultsThe transcriptome analysis showed that the AMPs induced a cell envelope stress response (cell membrane and cell wall). The cell membrane stress response was confirmed with the physiological observations that TC19, TC84 and BP2 perturb the membrane of B. subtilis. Using B. subtilis mutants, we established that the cell wall stress response is due to the delocalization of essential membrane bound proteins involved in cell wall synthesis. Other essential membrane proteins, involved in cell membrane synthesis and metabolism, were also delocalized due to alterations caused by the AMPs. ConclusionsWe showed that peptides TC19, TC84 and BP2 perturb the membrane causing essential proteins to delocalize, thus preventing the possible repair of the cell envelope after the initial interference with the membrane. General significanceThese AMPs show potential for eventual clinical application against Gram-positive bacterial cells and merit further application-oriented investigation.

Lytic cell death induced by melittin bypasses pyroptosis but induces NLRP3 inflammasome activation and IL-1\u03b2 release

The nucleotide-binding domain and leucine-rich repeat-containing receptor with a pyrin domain 3 (NLRP3) inflammasome is a sensor for different types of infections and alterations of homeostatic parameters, including abnormally high levels of the extracellular nucleotide ATP or crystallization of different metabolites. All NLRP3 activators trigger a similar intracellular pathway, where a decrease in intracellular K+ concentration and permeabilization of plasma membrane are key steps. Cationic amphipathic antimicrobial peptides and peptide toxins permeabilize the plasma membrane. In fact, some of them have been described to activate the NLRP3 inflammasome. Among them, the bee venom antimicrobial toxin peptide melittin is known to elicit an inflammatory reaction via the NLRP3 inflammasome in response to bee venom. Our study found that melittin induces canonical NLRP3 inflammasome activation by plasma membrane permeabilization and a reduction in the intracellular K+ concentration. Following melittin treatment, the apoptosis-associated speck-like protein, an adaptor protein with a caspase recruitment domain (ASC), was necessary to activate caspase-1 and induce IL-1β release. However, cell death induced by melittin prevented the formation of large ASC aggregates, amplification of caspase-1 activation, IL-18 release and execution of pyroptosis. Therefore, melittin-induced activation of the NLRP3 inflammasome results in an attenuated inflammasome response that does not result in caspase-1 dependent cell death.

NMR Structural Studies of Antimicrobial Peptides: LPcin Analogs

Lactophoricin (LPcin), a component of proteose peptone (113–135) isolated from bovine milk, is a cationic amphipathic antimicrobial peptide consisting of 23 amino acids. We designed a series of N- or C-terminal truncated variants, mutated analogs, and truncated mutated analogs using peptide-engineering techniques. Then, we selected three LPcin analogs of LPcin-C8 (LPcin-YK1), LPcin-T2WT6W (LPcin-YK2), and LPcin-T2WT6W-C8 (LPcin-YK3), which may have better antimicrobial activities than LPcin, and successfully expressed them in E. coli with high yield. We elucidated the 3D structures and topologies of the three LPcin analogs in membrane environments by conducting NMR structural studies. We investigated the purity of the LPcin analogs and the α-helical secondary structures by performing 1H-15N 2D HSQC and HMQC-NOESY liquid-state NMR spectroscopy using protein-containing micelle samples. We measured the 3D structures and tilt angles in membranes by conducting 15N 1D and 2D 1H-15N SAMMY type solid-state NMR spectroscopy with an 800 MHz in-house-built 1H-15N double-resonance solid-state NMR probe with a strip-shield coil, using protein-containing large bicelle samples aligned and confirmed by molecular-dynamics simulations. The three LPcin analogs were found to be curved α-helical structures, with tilt angles of 55–75° for normal membrane bilayers, and their enhanced activities may be correlated with these topologies.

Optimized Expression and Characterization of Antimicrobial Peptides, LPcin Analogs

Lactophoricin (LPcin or LPcin‐I), a cationic amphipathic antimicrobial peptide consisting of 23 amino acids, is a part of proteose peptone (113–135) isolated from bovine milk. The 23 amino acids of LPcin are relatively long for industrial uses as antimicrobial peptides. Therefore, a series of N‐ or C‐terminal truncated variant mutated analogs and truncated mutated analogs were designed using peptide engineering techniques to investigate the structure–activity relationships. Then, three analogs of LPcin‐C8 [(LPcin‐YK1), LPcin‐T2WT6W (LPcin‐YK2), and LPcin‐T2WT6W‐C8 (LPcin‐YK3)] were optimized to express with high yield and characterized for their structural studies. Successful expression of these three LPcin analogs in Escherichia coli system was accomplished through the formation of a fusion protein with higher yield. The peptides were purified using several biophysical techniques and finally isolated using reversed‐phase preparative HPLC. The purity, molecular mass, and the secondary structure of the purified and isolated LPcin analogs were characterized by polyacrylamide gel electrophoresis, mass spectrometry, and circular dichroism spectrometry. The secondary structure of LPcin and its three analog peptides clearly showed α‐helical characteristics in membrane environments and random‐coil conformation in water. Consequently, our results so far have shown that the three LPcin analogs could become potent antimicrobial peptides like LPcin as an alternative of new antibiotics in the near future.

Reduction of Helical Content by Insertion of a Disulfide Bond Leads to an Antimicrobial Peptide with Decreased Hemolytic Activity

CAPs off to the S-S bond: Application of a strategy involving the introduction of intramolecular disulfide bonds to amphipathic cationic antimicrobial peptides (CAPs) led to decreased α-helicity and hydrophobicity. The mutant peptides were observed to have significantly increased (250-fold) minimum hemolytic concentrations while maintaining MIC values against E. coli, affording more therapeutically selective CAPs.

Atomic-Resolution Three Dimensional Structures and Membrane Locations of Antimicrobial Piscidin 1 and Piscidin 3 in Aligned Lipid Bilayers: A Solid-State NMR and Molecular Dynamics Investigation

Piscidin, an amphipathic cationic antimicrobial peptide (AMP) active against a broad range of pathogens including multidrug-resistant bacteria and HIV-1, belongs to a large family of vital host-defense peptides that interact, at least initially, with negatively-charged microbial membranes in order to perform their function. While two piscidin isoforms, piscidin 1 (p1) and piscidin 3 (p3), are highly homologous, they display unequal antimicrobial and hemolytic effects. As a way to identify factors optimizing specific molecular interactions directly related to their mode of membrane activity, we have investigated p1 and p3 bound to lipid membranes that mimic bacterial membranes. Previously, we used solid-state NMR on 15N-labeled peptides to demonstrate that membrane-bound p1 and p3 adopt an alpha-helical structure and lie in the plane of hydrated lipid bilayers where they experience fast dynamics. Our recent analysis of two-dimensional solid-state NMR data has lead to the first atomic resolution three-dimensional backbone structures of p1 and p3 bound to aligned lipid bilayers. Structural calculations based on the NMR data and molecular dynamics simulations have been performed to yield a refined structure and membrane location for each peptide. We will explain how our atomic-level investigation of the structure, dynamics, and bilayer location of piscidin provides new insights into its mode of action and therefore allows us better to understand how AMPs disrupt bacterial membranes and induce cell death. The long term goal is to derive common principles that could facilitate the design of pharmaceuticals with enhanced antibacterial activity and lower toxicity on mammalian cells.

Antimicrobial Peptides Activate the Rcs Regulon through the Outer Membrane Lipoprotein RcsF

Salmonella enterica species are exposed to envelope stresses due to their environmental and infectious lifestyles. Such stresses include amphipathic cationic antimicrobial peptides (CAMPs), and resistance to these peptides is an important property for microbial virulence for animals. Bacterial mechanisms used to sense and respond to CAMP-induced envelope stress include the RcsFCDB phosphorelay, which contributes to survival from polymyxin B exposure. The Rcs phosphorelay includes two inner membrane (IM) proteins, RcsC and RcsD; the response regulator RcsB; the accessory coregulator RcsA; and an outer membrane bound lipoprotein, RcsF. Transcriptional activation of the Rcs regulon occurred within minutes of exposure to CAMP and during the first detectable signs of CAMP-induced membrane disorder. Rcs transcriptional activation by CAMPs required RcsF and preservation of its two internal disulfide linkages. The rerouting of RcsF to the inner membrane or its synthesis as an unanchored periplasmic protein resulted in constitutive activation of the Rcs regulon and RcsCD-dependent phosphorylation. These findings suggest that RcsFCDB activation in response to CAMP-induced membrane disorder is a result of a change in structure or availability of RcsF to the IM signaling constituents of the Rcs phosphorelay.

Structural contributions to the intracellular targeting strategies of antimicrobial peptides

The interactions of cationic amphipathic antimicrobial peptides (AMPs) with anionic biological membranes have been the focus of much research aimed at improving the activity of such compounds in the search for therapeutic leads. However, many of these peptides are thought to have other polyanions, such as DNA or RNA, as their ultimate target. Here a combination of fluorescence and circular dichroism (CD) spectroscopies has been used to assess the structural properties of amidated versions of buforin II, pleurocidin and magainin 2 that support their varying abilities to translocate through bacterial membranes and bind to double stranded DNA. Unlike magainin 2 amide, a prototypical membrane disruptive AMP, buforin II amide adopts a poorly helical structure in membranes closely mimicking the composition of Gram negative bacteria, such as Escherichia coli, and binds to a short duplex DNA sequence with high affinity, ultimately forming peptide–DNA condensates. The binding affinities of the peptides to duplex DNA are shown to be related to the structural changes that they induce. Furthermore, CD also reveals the conformation of the bound peptide buforin II amide. In contrast with a synthetic peptide, designed to adopt a perfect amphipathic α-helix, buforin II amide adopts an extended or polyproline II conformation when bound to DNA. These results show that an α-helix structure is not required for the DNA binding and condensation activity of buforin II amide.

Structure and Mode of Action of Microplusin, a Copper II-chelating Antimicrobial Peptide from the Cattle Tick Rhipicephalus (Boophilus) microplus

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single alpha-helical globular domain, microplusin consists of five alpha-helices: alpha1 (residues Gly-9 to Arg-21), alpha2 (residues Glu-27 to Asn-40), alpha3 (residues Arg-44 to Thr-54), alpha4 (residues Leu-57 to Tyr-64), and alpha5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.

High-Resolution Structure of Piscidin in Aligned Lipid Bilayers: Implications for Antimicrobial Mode of Action

Piscidin is an amphipathic cationic antimicrobial peptide that belongs to a large family of versatile host-defense peptides, which interact, at least initially, with cell membranes in order to perform their function. In the research presented here, we characterize the secondary structure and dynamics of piscidin in order to identify factors optimizing specific molecular interactions that are directly related to its function and mode of action. Our long term goal is to identify common principles that will facilitate the design of pharmaceuticals with broad-spectrum antibacterial activity, minimum induction of bacterial resistance, and low toxicity for mammalian cells.Previously, we demonstrated that membrane-bound piscidin 1 (p1) adopts an alpha-helical structure, which lies in the plane of the bilayer and experiences fast motions. Here, high-resolution solid-state NMR spectra have been obtained from multiply 15N-backbone-labeled p1 aligned in hydrated lipid bilayers. Analysis of data from twenty sites of this 22-mer reveals two helical segments separated by a kink at Gly13. This kink may help one portion of the peptide insert more deeply in the hydrophobic lipid bilayer, which may be related to the mechanism of membrane disruption. To characterize water exposure, hydrogen-deuterium exchange experiments have been performed on 15N-backbone labeled samples. In addition, solid-state NMR was applied to 15N-side-chain-labeled His-17 p1 to titrate this side chain, which resides at the interface between the hydrophilic and hydrophobic domains of p1. Overall, our atomic-level investigation of the structure, dynamics, and water exposure of membrane-bound piscidin provides new insights into its mode of action and thus help understand how antimicrobial peptides recognize membranes and initiate their activities on microbial cells.

Immobilization Reduces the Activity of Surface-Bound Cationic Antimicrobial Peptides with No Influence upon the Activity Spectrum

Early studies of immobilized peptides mainly focused upon the relationship between structural properties and the activity of soluble and surface-tethered sequences. The intention of this study was to analyze the influence of immobilization parameters upon the activity profile of peptides. Resin beads (TentaGel S NH(2), HypoGel 400 NH(2), and HypoGel 200 NH(2)) with polyethylene glycol spacers of different lengths were rendered antimicrobial by linkage of an amphipathic model KLAL peptide and magainin-derived MK5E. Standard solid-phase peptide synthesis, thioalkylation, and ligation strategies were used to immobilize the peptides at the C and N termini and via different side-chain positions. Depending upon the resin capacity and the coupling strategies, peptide loading ranged between 0.1 and 0.25 micromol/mg for C-terminally and around 0.03 micromol/mg for N-terminally and side-chain-immobilized peptides. Tethering conserved the activity spectra of the soluble peptides at reduced concentrations. The resin-bound peptides were antimicrobial toward Escherichia coli and Bacillus subtilis in the millimolar range compared to the results seen with micromolar concentrations of the free peptides. B. subtilis was more susceptible than E. coli. The antimicrobial activity distinctly decreased with reduction of the spacer length. Slight differences in the antimicrobial effect of KLAL and MK5E bound at different chain positions on TentaGel S NH(2) suggest that the activity is less dependent upon the position of immobilization. Soluble KLAL was active toward red blood cells, whereas MK5E was nonhemolytic at up to about 400 microM. Resin-induced hemolysis hampered the determination of the hemolytic effect of the immobilized peptides. TentaGel S NH(2)-bound peptides enhanced the permeability of the POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline) and mixed POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPC/POPG) bilayers used to model the charge properties of the biological targets. The results suggest that surface immobilization of the cationic amphipathic antimicrobial peptides does not influence the membrane-permeabilizing mode of action. Peptide insertion into the target membrane and likely the exchange of membrane-stabilizing bivalent cations contribute to the antimicrobial effect. In conclusion, reasonable antimicrobial activity of surface-bound peptides requires the optimization of the coupling parameters, with the length of the spacer and the amount of target-accessible peptide being the most important factors.

Antimicrobial Peptides and their Potential as Oral Therapeutic Agents

Dental caries (tooth decay) and periodontal diseases are the most prevalent bacterial infectious diseases of mankind, together affecting almost the entire population of the world. Both diseases are caused by oral bacteria that exist as components of a polymicrobial biofilm, known as dental plaque, on the tooth surface. The control of specific types of bacteria and/or total numbers of bacteria in dental plaque could lead to prevention or resolution of disease. Antimicrobial peptides isolated from a wide range of natural sources have been known for over 30 years yet little progress had been made in the therapeutic application of these peptides. This is due in part to the characteristics, including susceptibility to proteolysis, of the cationic amphipathic antimicrobial peptides that form the majority of peptides discovered to date. Bovine milk is a readily available source of a range of bioactive peptides. We have isolated and characterized a novel anionic antimicrobial peptide, Kappacin, from bovine milk. Antibacterial activity of the peptide is increased when it is complexed with zinc ions. We have demonstrated that a Kappacin:Zn2+ preparation is able to suppress the growth of oral cariogenic bacteria in a biofilm. The Kappacin:Zn2+ antibacterial complex may have potential as an additive to oral care products and other delivery vehicles for the control of oral disease.

Investigating molecular recognition and biological function at interfaces using piscidins, antimicrobial peptides from fish

We studied amidated and non-amidated piscidins 1 and 3, amphipathic cationic antimicrobial peptides from fish, to characterize functional and structural similarities and differences between these peptides and better understand the structural motifs involved in biological activity and functional diversity among amidated and non-amidated isoforms. Antimicrobial and hemolytic assays were carried out to assess their potency and toxicity, respectively. Site-specific high-resolution solid-state NMR orientational restraints were obtained from 15N-labeled amidated and non-amidated piscidins 1 and 3 in the presence of hydrated oriented lipid bilayers. Solid-state NMR and circular dichroism results indicate that the peptides are α-helical and oriented parallel to the membrane surface. This orientation was expected since peptide–lipid interactions are enhanced at the water–bilayer interface for amphipathic cationic antimicrobial peptides. 15N solid-state NMR performed on oriented samples demonstrate that piscidin experiences fast, large amplitude backbone motions around an axis parallel to the bilayer normal. Under the conditions tested here, piscidin 1 was confirmed to be more antimicrobially potent than piscidin 3 and antimicrobial activity was not affected by amidation. In light of functional and structural similarities between piscidins 1 and 3, we propose that their topology and fast dynamics are related to their mechanism of action.

High-Field NMR Studies of Molecular Recognition and Structure−Function Relationships in Antimicrobial Piscidins at the Water−Lipid Bilayer Interface

High magnetic field solid-state NMR was performed on amphipathic cationic antimicrobial peptides from fish to characterize their secondary structure and orientation in hydrated phospholipid bilayers. High-resolution distance and orientational restraints on 13C- and 15N-labeled amidated piscidins 1 and 3 provided site-specific information establishing alpha-helicity and an orientation parallel to the membrane surface. Few membrane-bound natural peptides with this topology have been structurally studied at high resolution in the presence of hydrated lipid bilayers. This orientation was foreseen since the partitioning of amphipathic cationic antimicrobial peptides at the water-bilayer interface allows for favorable peptide-lipid interactions, and it may be related to the mechanism of action. The enhanced resolution obtained at 900 MHz evidences a determinant advantage of ultra-high-field NMR for the structural determination of multiple-labeled peptides and proteins.