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Antimicrobial peptides (AMPs) are believed to be a prominent alternative to the common antibiotics. However, despite decades of research, there are still no good clinical examples of peptide-based antimicrobial drugs for system application. The main reasons are loss of activity in the human body, cytotoxicity, and low selectivity. To overcome these challenges, a well-established structure-function relationship for AMPs is critical. In the present study, we focused on the well-known examples of melittin and magainin to investigate in detail the initial stages of AMP interaction with lipid membranes at low peptide-to-lipid ratio. By combining the patch-clamp technique with the bioelectrochemical method of intramembrane field compensation, we showed that these peptides interact with the membrane in different ways: melittin inserts deeper into the lipid bilayer than magainin. This difference led to diversity in pore formation. While magainin, after a threshold concentration, formed the well-known toroidal pores, allowing the translocation of the peptide through the membrane, melittin probably induced predominantly pure lipidic pores with a very low rate of peptide translocation. Thus, our results shed light on the early stages of peptide-membrane interactions and suggest new insights into the structure-function relationship of AMPs based on the depth of their membrane insertion.

FAK (focal adhesin kinase), a tyrosine kinase, plays an imperative role in cell-cell communication, particularly in cell signaling systems. It is a multi-functional signaling protein, which integrates and transduces signals into cancer cells through growth factor receptors or integrin and its interaction with Paxillin (PAX). The molecular processes by which FAK promotes the development and progression of cancer have progressively established the possible relationship between FAK-PAX complex in many types of cancer. The interaction of FAX and PAX is very important in breast cancer and thus acts as an essential biomarker for drugs, vaccines or peptide inhibitor designing. In this regard, computational approaches, particularly peptide designing to target the binding interface of the interacting partners, would greatly assist the design of peptide inhibitors against various cancer. Accordingly, in this present study, we screened 236 experimentally validated anti-breast cancer peptides using computational drugs repositioning approach to design peptides targeting the FAK-PAX complex. Using protein-peptide docking the binding site for the HP1 was confirmed and a total of 236 anti-breast cancer peptides were screened. Among the 236, only 12 peptides reported a docking score better than the control. From these 12, Magainin with the docking score - 103.8 ± 10.3 kcal/mol, NRC-07 with the docking score - 100.8 ± 16.5 kcal/mol, and Indolicidin with the docking score - 101.7 ± 3.9 kcal/mol, peptides potentially inhibit the FAX-PAX binding. Calculation of protein's motion and FEL revealed the binding and inhibitory behavior. Moreover, binding free energy (MM/GBSA) confirmed that Magainin exhibited the total binding energy - 53.28 kcal/mol, NRC-07 possessed the TBE - 44.16 kcal/mol, and Indolicidin reported the TBE of - 40.48 kcal/mol, thus explaining the inhibitory potential of these peptides. In conclusion, these peptides exhibit strong inhibitory potential and could abrogate the FAK-PAX complex in in vitro models and thus may relieve the burden of breast cancer., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Antimicrobial peptides (AMPs) offer advantages over conventional antibiotics; for example, bacteria develop more resistance to small-molecule antibiotics than to AMPs. The interaction of the AMPs with the lipopolysaccharide (LPS) layer of the Gram-negative bacteria cell envelope is not well understood. A MARTINI model was constructed of a Gram-negative bacterial outer membrane interacting with the AMP Magainin 2. In a 20 μs molecular dynamics (MD) simulation, the AMP diffused to the LPS layer of the cell envelope and remained there, suggesting interactions between the Magainin 2 and the LPS layer, causing the AMP to concentrate at that position. The free energy profile for the insertion of the Magainin 2 into the membrane was also calculated using umbrella sampling, which showed that the AMP positioned such that the cationic side chains of the AMP coordinated to the negatively charged phosphate groups of the LPS layer. These simulations indicate that the AMP Magainin 2 partition into the LPS layer of a bacterial membrane.

Peptide binding to membranes is common and fundamental in biochemistry and biophysics and critical for applications ranging from drug delivery to the treatment of bacterial infections. However, it is largely unclear, from a theoretical point of view, what peptides of different sequences and structures share in the membrane-binding and insertion process. In this work, we analyze three prototypical membrane-binding peptides (α-helical magainin, PGLa, and β-hairpin tachyplesin) during membrane binding, using molecular details provided by Markov state modeling and microsecond-long molecular dynamics simulations. By leveraging both geometric and data-driven collective variables that capture the essential physics of the amphiphilic and cationic peptide-membrane interactions, we reveal how the slowest kinetic process of membrane binding is the dynamic rolling of the peptide from an attached to a fully bound state. These results not only add fundamental knowledge of the theory of how peptides bind to biological membranes but also open new avenues to study general peptides in more complex environments for further applications.

Membrane disrupting antimicrobial peptides (AMPs) are often amphipathic peptides that interact directly with lipid bilayers. AMPs are generally thought to interact mostly with lipid head groups, but it is less clear how the lipid alkyl chain length and saturation modulate interactions with membranes. Here, we used native mass spectrometry to measure the stoichiometry of three different AMPs-LL-37, indolicidin, and magainin-2-in lipid nanodiscs. We also measured the activity of these AMPs in unilamellar vesicle leakage assays. We found that LL-37 formed specific hexamer complexes but with different intermediates and affinities that depended on the bilayer thickness. LL-37 was also most active in lipid bilayers containing longer, unsaturated lipids. In contrast, indolicidin incorporated to a higher degree into more fluid lipid bilayers but was more active with bilayers with thinner, less fluid lipids. Finally, magainin-2 incorporated to a higher degree into bilayers with longer, unsaturated alkyl chains and showed more activity in these same conditions. Together, these data show that higher amounts of peptide incorporation generally led to higher activity and that AMPs tend to incorporate more into longer unsaturated lipid bilayers. However, the activity of AMPs was not always directly related to amount of peptide incorporated., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The histidine-rich peptides of the LAH4 family were designed using cationic antimicrobial peptides such as magainin and PGLa as templates. The LAH4 amphipathic helical sequences exhibit a multitude of interesting biological properties such as antimicrobial activity, cell penetration of a large variety of cargo and lentiviral transduction enhancement. The parent peptide associates with lipid bilayers where it changes from an orientation along the membrane interface into a transmembrane configuration in a pH-dependent manner. Here we show that LAH4 adopts a transmembrane configuration in fully saturated DMPC membranes already at pH 3.5, i.e. much below the pK a of the histidines whereas the transition pH in POPC correlates closely with histidine neutralization. In contrast in POPG membranes the in-planar configuration is stabilized by about one pH unit. The differences in pH can be converted into energetic contributions for the in-plane to transmembrane transition equilibrium, where the shift in the transition pH due to lipid saturation corresponds to energies which are otherwise obtained by the exchange of several cationic with hydrophobic residues. A similar dependence on lipid saturation has also been observed when the PGLa and magainin antimicrobial peptides interact within lipid bilayers suggesting that the quantitative evaluation presented in this paper also applies to other membrane polypeptides., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Nanoparticles (NPs) promise a huge potential for clinical diagnostic and therapeutic applications. However, nano-bio (e.g., the NP-cell membrane) interactions and underlying mechanisms are still largely elusive. In this study, two types of congeneric peptides, namely PGLa and magainin 2 (MAG2), with similar membrane activities were employed as model ligands for NP decoration, and the diffusion behaviours (including both translation and rotation) of the ligand-decorated NPs on a lipid bilayer membrane were studied via molecular dynamics simulations. It was found that, although both PGLa- and MAG2-coated NPs showed alternatively "hopping" and "jiggling" diffusions, the PGLa-coated ones had an enhanced circling at the hopping stage, while a much confined circling at the jiggling stage. In contrast, the MAG2-coated NPs demonstrated constant circling tendencies throughout the diffusion process. Such differences in the coupling between translational and rotational dynamics of these two types of NPs are ascribed to the different ligand-lipid interactions of PGLa and MAG2, in which the PGLa ligands prefer to vertically insert into the membrane, while MAG2 tends to lie flat on the membrane surface. Our results are helpful for the understanding the underlying associations between the NP motions and their interfacial membrane interactions, and shed light on the possibility of regulating NP behaviours on a cellular surface for better biomedical uses.

The antimicrobial peptide (AMP) magainin 2 induces nanopores in the lipid membranes of giant unilamellar vesicles (GUVs), as observed by the leakage of water-soluble fluorescent probes from the inside to the outside of GUVs through the pores. However, molecular transport through a single nanopore has not been investigated in detail yet and is studied in the present work by simulation. A single pore was designed in the membrane of a GUV using computer-aided design software. Molecular transport, from the outside to the inside of GUV through the nanopore, of various fluorescent probes such as calcein, Texas-Red Dextran 3000 (TRD-3k), TRD-10k and TRD-40k was then simulated. The effect of variation in GUV size (diameter) was also investigated. A single exponential growth function was fitted to the time course of the fluorescence intensity inside the GUV and the corresponding rate constant of molecular transport was calculated, which decreases with an increase in the size of fluorescent probe and also with an increase in the size of GUV. The rate constant found by simulation agrees reasonably well with reported experimental results for inside-to-outside probe leakage. Based on Fick's law of diffusion an analytical treatment is developed for the rate constant of molecular transport that supports the simulation results. These investigations contribute to a better understanding of the mechanism of pore formation using various membrane-active agents in the lipid membranes of vesicles and the biomembranes of cells.

We addressed the onset of synergistic activity of the two well-studied antimicrobial peptides magainin 2 (MG2a) and PGLa using lipid-only mimics of Gram-negative cytoplasmic membranes. Specifically, we coupled a joint analysis of small-angle x-ray and neutron scattering experiments on fully hydrated lipid vesicles in the presence of MG2a and L18W-PGLa to all-atom and coarse-grained molecular dynamics simulations. In agreement with previous studies, both peptides, as well as their equimolar mixture, were found to remain upon adsorption in a surface-aligned topology and to induce significant membrane perturbation, as evidenced by membrane thinning and hydrocarbon order parameter changes in the vicinity of the inserted peptide. These effects were particularly pronounced for the so-called synergistic mixture of 1:1 (mol/mol) L18W-PGLa/MG2a and cannot be accounted for by a linear combination of the membrane perturbations of two peptides individually. Our data are consistent with the formation of parallel heterodimers at concentrations below a synergistic increase of dye leakage from vesicles. Our simulations further show that the heterodimers interact via salt bridges and hydrophobic forces, which apparently makes them more stable than putatively formed antiparallel L18W-PGLa and MG2a homodimers. Moreover, dimerization of L18W-PGLa and MG2a leads to a relocation of the peptides within the lipid headgroup region as compared to the individual peptides. The early onset of dimerization of L18W-PGLa and MG2a at low peptide concentrations consequently appears to be key to their synergistic dye-releasing activity from lipid vesicles at high concentrations., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Combining two known antimicrobial peptides (AMPs) into a hybrid peptide is one promising avenue in the design of agents with increased antibacterial activity. However, very few previous studies have considered the effect of creating a hybrid from one AMP that permeabilizes membranes and another AMP that acts intracellularly after translocating across the membrane. Moreover, very few studies have systematically evaluated the order of parent peptides or the presence of linkers in the design of hybrid AMPs. Here, we use a combination of antibacterial measurements, cellular assays and semi-quantitative confocal microscopy to characterize the activity and mechanism for a library of sixteen hybrid peptides. These hybrids consist of permutations of two primarily membrane translocating peptides, buforin II and DesHDAP1, and two primarily membrane permeabilizing peptides, magainin 2 and parasin. For all hybrids, the permeabilizing peptide appeared to dominate the mechanism, with hybrids primarily killing bacteria through membrane permeabilization. We also observed increased hybrid activity when the permeabilizing parent peptide was placed at the N-terminus. Activity data also highlighted the potential value of considering AMP cocktails in addition to hybrid peptides. Together, these observations will guide future design efforts aiming to design more active hybrid AMPs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The transbilayer asymmetry of the biomembrane is generated due to the differences in lipid and protein compositions between two leaflets, which plays important roles in physiological functions. However, transbilayer asymmetry can also be originated due to a nonequal number of lipids or proteins in each leaflet, which has not been well recognized. Therefore, to shed light on this field, here we generated theoretical models for the effect of transbilayer asymmetry originated from the differences in the number of lipids and peptides in each leaflet on the state of lipid bilayers. The first model described the effect of asymmetric lipid distribution on the state of lipid bilayers. We obtained theoretical equations for the fractional change in area per lipid in both leaflets as a function of the ratio of the number of lipids in each leaflet, which agreed with the molecular dynamics simulation results quantitatively. Results indicated that tensions in both leaflets are opposite in direction, and their magnitude is the same. We also performed experiments on the effect of lipid insertion in the outer leaflet on the fractional change in area per lipid. These results agreed quantitatively with the values predicted by the above model. The second model described the effect of asymmetric distribution of peptides on the state of lipid bilayers. We obtained theoretical equations for the area per lipid in both leaflets as a function of the surface concentration of peptides located only in the outer leaflet, which agreed with the results of the antimicrobial peptide magainin 2-induced area change.

Background: Gamma aminobutyric acid (GABA), an inhibitory neurotransmitter, is produced via decarboxylation of l-glutamate through the glutamic acid decarboxylase (GAD) enzyme. The synchronic action of GABA-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) enzymes convert the GABA metabolite into succinate. Given this background, our research was aimed at probing the effect of Magainin II, on the activity of GABA shunt metabolizing enzymes., Methods: Male NIH mice were administered peripherally by Magainin II (50 μg/kg body weight) and saline solution (%0.9 (w/v)) as the control vehicle. At different time intervals, the mice were sacrificed to evaluate the effect of Magainin II injection on the GABA shunt pathway. The activity of hypothalamic GAD, GABA-T and SSADH enzymes were determined using relevant enzyme assays., Results: Magainin II effectively enhanced the activity of GAD, by %90, 24 h after injection, while quenching the activities of GABA-T and SSADH by %43 and %71, respectively. In vitro models also revealed the direct but reversible interaction between the peptide and each of the individual enzymes of GABA shunt pathway., Conclusion: This study confirms the probable role of Magainin II in increasing the GABA content of the mouse hypothalamus. This property might candidate the peptide as a novel agent for improving the symptoms of many GABA dependent psychiatric disorders., (Copyright © 2018. Published by Elsevier Inc.)

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 15 N 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 2 H 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., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Antimicrobial peptides (AMPs) are alternatives to conventional antibiotics against multi-drug resistant bacteria with low potential for developing microbial resistance. The design of such molecules requires understanding of the mechanisms of action, particularly the interaction with bacteria cell membranes. In this work, we determine the mechanism responsible for the higher activity against Escherichia coli of the C-terminal lysine dimer of magainin 2, (MG2) 2 K, in comparison to the monomeric peptide magainin 2 (MG2). Langmuir monolayers and vesicles made with the E. coli lipid extract were used to address the two possible states for the peptide-membrane interaction, namely the "binding state" and "pore state", respectively. The incorporation of MG2 and (MG2) 2 K in lipid monolayers at the air-water interface caused slight differences in surface pressure isotherms and polarization-modulated infrared reflection absorption (PM-IRRAS) spectra, and therefore the difference in activity is not associated with the binding state. In contrast, large differences were observed in the leakage experiments where (MG2) 2 K was shown to disrupt the large unilamellar vesicles to a much higher extent owing to efficient pore formation. The binding and penetration of MG2 and (MG2) 2 K were also probed with molecular dynamics (MD) simulations for bilayers made with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine:1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE:POPG). (MG2) 2 K forms disordered toroidal pores at a significant lower concentration than for MG2. In summary, the combination of experimental and computational simulation results indicated that the "pre-assembling state" of (MG2) 2 K dimer leads to a reduced number of molecules and shorter time being required to kill E. coli., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aligned CW-EPR membrane protein samples provide additional topology interactions that are absent from conventional randomly dispersed samples. These samples are aptly suited to studying antimicrobial peptides because of their dynamic peripheral topology. In this study, four consecutive substitutions of the model antimicrobial peptide magainin 2 were synthesized and labeled with the rigid TOAC spin label. The results revealed the helical tilts to be 66° ± 5°, 76° ± 5°, 70° ± 5°, and 72° ± 5° for the TOAC substitutions H7, S8, A9, and K10 respectively. These results are consistent with previously published literature. Using the EPR (electron paramagnetic resonance) mechanical alignment technique, these substitutions were used to critically assess the topology and surface orientation of the peptide with respect to the membrane. This methodology offers a rapid and simple approach to investigate the structural topology of antimicrobial peptides., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Antibiotic residues can cause antibiotic resistance in livestock and their food safety-related issues have increased the consumer demand for products lacking these residues. Hence, developing safe and effective antibiotic alternatives is important to the animal feed industry. With their strong antibacterial actions, antimicrobial peptides have potential as antibiotic alternatives., Results: We investigated the antibacterial and immunomodulatory activities and the mechanisms of action of an antimicrobial peptide. The hybrid antimicrobial peptide magainin II-cecropin B (Mag II-CB) gene was transformed into the medicinal Cordyceps militaris fungus. Recombinant Mag II-CB exhibited broad-spectrum antibacterial activity in vitro and its antibacterial and immunomodulatory functions were evaluated in BALB/c mice infected with Escherichia coli (ATCC 25922). Histologically, Mag II-CB ameliorated E. coli-related intestinal damage and maintained the integrity of the intestinal mucosal barrier by up-regulating tight junction proteins (zonula occludens-1, claudin-1 and occludin). The intestinal microbial flora was positively modulated in the Mag II-CB-treated mice infected with E. coli. Mag II-CB treatment also supported immune functioning in the mice by regulating their plasma immunoglobulin and ileum secreted immunoglobulin A levels, by attenuating their pro-inflammatory cytokine levels, and by elevating their anti-inflammatory cytokines levels. Moreover, directly feeding the infected mice with the C. militaris mycelium producing Mag II-CB further proofed the antibacterial and immunomodulatory functions of recombinant hybrid antimicrobial peptide., Conclusion: Our findings suggest that both purified recombinant AMPs and C. militaris mycelium producing AMPs display antibacterial and immunomodulatory activities in mice. And C. militaris producing AMPs has the potential to become a substitute to antibiotics as a feed additive for livestock in future.

The potential of antimicrobial peptides (AMPs) as an effective therapeutic alternative to classic and current antibiotics has encouraged studies to understand how they interact with the bacterial membrane. Here we describe how to detect, by circular dichroism (CD), the secondary structures of two antimicrobial peptides, magainin 2 and cecropin A, in the presence of E. coli bacterial cells.

Magainin 2 (MAG2) and PGLa are two α-helical antimicrobial peptides found in the skin of the African frog Xenopus laevis. They act by permeabilizing bacterial membranes and exhibit an exemplary synergism. Here, we determined the detailed molecular alignment and dynamical behavior of MAG2 in oriented lipid bilayers by using 2 H-NMR on Ala-d 3 -labeled peptides, which yielded orientation-dependent quadrupolar splittings of the labels. The amphiphilic MAG2 helix was found to lie flat on the membrane surface in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC)/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), as expected, with a tilt angle close to 90°. This orientation fits well with all-atom molecular-dynamics simulations of MAG2 performed in DMPC and DMPC/DMPG. In the presence of an equimolar amount of PGLa, the NMR analysis showed that MAG2 becames tilted at an angle of 120°, and its azimuthal rotation angle also changes. Since this interaction was found to occur in a concentration range where the peptides per se do not interact with their own type, we propose that MAG2 forms a stable heterodimer with PGLa. Given that the PGLa molecules in the complex are known to be flipped into a fully upright orientation, with a helix tilt close to 180°, they must make up the actual transmembrane pore. We thus suggest that the two negative charges on the C-terminus of the obliquely tilted MAG2 peptides neutralize some of the cationic groups on the upright PGLa helices. This would stabilize the assembly of PGLa into a toroidal pore with an overall reduced charge density, which could explain the mechanism of synergy., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Peptidomic analysis of norepinephrine-stimulated skin secretions of the tetraploid frog Xenopus fraseri Boulenger, 1905 (Pipidae) led to identification of 13 host-defense peptides. The primary structures of the peptides demonstrate that they belong to the magainin (3 peptides), peptide glycine-leucine-amide, PGLa (4 peptides), and xenopsin-precursor fragment, XPF (2 peptides) families, first identified in Xenopus laevis, together with caerulein precursor fragment-related peptides, CPF-RP (4 peptides), first identified in Silurana tropicalis. In addition, the secretions contain a molecular variant of xenopsin displaying the substitution Arg(4)→Lys compared with X. laevis xenopsin and peptide glycine-tyrosine-amide (PGYa) (GRIIPIYPEFERVFA KKVYPLY.NH2) whose function is unknown. The most potent antimicrobial peptide identified is CPF-RP-F1 (GFGSVLGKALKFGANLL.NH2) with MIC=12.5μM against Staphylococcus aureus and 50μM against Escherichia coli. On the basis of similarities in morphology and advertisement calls, X. fraseri has been placed in a species group that includes the octoploids Xenopus amieti and Xenopus andrei, and the tetraploid Xenopus pygmaeus. Cladistic analyses based upon the primary structures of magainin, PGLa, and CPF-RP peptides support a close evolutionary relationship between X. fraseri, X. amieti and X. andrei but suggest a more distant relationship with X. pygmaeus., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Certain antibiotic peptides are thought to permeabilize membranes of pathogens by effects that are also observed for simple detergents, such as membrane thinning and disordering, asymmetric bilayer expansion, toroidal pore formation, and micellization. Here we test the hypothesis that such peptides act additively with detergents when applied in parallel. Additivity is defined analogously to a fractional inhibitory concentration index of unity, and the extent and mechanism of leakage is measured by the fluorescence lifetime-based vesicle leakage assay using calcein-loaded vesicles. Good additivity was found for the concerted action of magainin 2, the fungicidal lipopeptide class of surfactins from Bacillus subtilis QST713, and the detergent octyl glucoside, respectively, with the detergent C12EO8. Synergistic or superadditive action was observed for fengycins from B. subtilis, as well as the detergent CHAPS, when combined with C12EO8. The results illustrate two mechanisms of synergistic action: First, maximal leakage requires an optimum degree of heterogeneity in the system that may be achieved by mixing a graded with an all-or-none permeabilizer. (The optimal perturbation should be focused to certain defect structures, yet not to the extent that some vesicles are not affected at all.) Second, a cosurfactant may enhance the bioavailability of a poorly soluble peptide. The results are important for understanding the concerted action of membrane-permeabilizing compounds in biology as well as for optimizing formulations of such antimicrobials for medical applications or crop protection., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Poly(ethylene glycol) (PEG)-grafted magainin 2 and tachyplesin I were simulated with lipid bilayers. In the simulations of PEGylated magainin 2 and tachyplesin I in water, both peptides are wrapped by PEG chains. The α-helical structure of PEGylated magainin 2 is broken in water, while the β-sheet of PEGylated tachyplesin I keeps stable, similar to the structural behavior of unPEGylated peptides, in agreement with experiments. Simulations of PEGylated peptides with lipid bilayers show that PEG chains block the electrostatic interaction between cationic residues of peptides and anionic phosphates of lipids, leading to the less binding of the peptide to the bilayer surface, which is observed more significantly for magainin 2 than for tachyplesin I. Since the random-coiled magainin 2 can be more completely covered by PEGs than does the β-sheet tachyplesin I, the PEGylation effect on the decreased binding is larger for magainin 2, showing the dependence of PEGylation on the peptide structure. These simulation findings qualitatively support the experimental observation of the different extents of the reduced membrane-permeabilizing activity for PEGylated magainin 2 and tachyplesin I.

High-resolution structure elucidation has been challenging for the large group of host-defense peptides that form helices on or within membranes but do not manifest a strong folding propensity in aqueous solution. Here we report the crystal structure of an analogue of the widely studied host-defense peptide magainin 2. Magainin 2 (S8A, G13A, G18A) is a designed variant that displays enhanced antibacterial activity relative to the natural peptide. The crystal structure of magainin 2 (S8A, G13A, G18A), obtained for the racemic form, features a dimerization mode that has previously been proposed to play a role in the antibacterial activity of magainin 2 and related peptides.

Antimicrobial or host defense peptides are innate immune regulators found in all multicellular organisms. Many of them fold into membrane-bound α-helices and function by causing cell wall disruption in microorganisms. Herein we probe the possibility and functional implications of antimicrobial antagonism mediated by complementary coiled-coil interactions between antimicrobial peptides and de novo designed antagonists: anti-antimicrobial peptides. Using sequences from native helical families such as cathelicidins, cecropins, and magainins we demonstrate that designed antagonists can co-fold with antimicrobial peptides into functionally inert helical oligomers. The properties and function of the resulting assemblies were studied in solution, membrane environments, and in bacterial culture by a combination of chiroptical and solid-state NMR spectroscopies, microscopy, bioassays, and molecular dynamics simulations. The findings offer a molecular rationale for anti-antimicrobial responses with potential implications for antimicrobial resistance.

Poration of bacterial membranes by antimicrobial peptides such as magainin 2 is a significant activity performed by innate immune systems. Pore formation by soluble forms of amyloid proteins such as islet amyloid polypeptide (IAPP) is implicated in cell death in amyloidoses. Similarities in structure and poration activity of these two systems suggest a commonality of mechanism. Here, we investigate and compare the mechanisms by which these peptides induce membrane leakage and bacterial cell death through the measurement of liposome leakage kinetics and bacterial growth inhibition. For both systems, leakage occurs through the nucleation-dependent formation of stable membrane pores. Remarkably, we observe IAPP and magainin 2 to be fully cross-cooperative in the induction of leakage and inhibition of bacterial growth. The effects are dramatic, with mixtures of these peptides showing activities >100-fold greater than simple sums of the activities of individual peptides. Direct protein-protein interactions cannot be the origin of cooperativity, as IAPP and its enantiomer D-IAPP are equally cross-cooperative. We conclude that IAPP and magainin 2 induce membrane leakage and cytotoxicity through a shared, cross-cooperative, tension-induced poration mechanism.

PGLa and magainin 2 (MAG2) are amphiphilic antimicrobial peptides from frog skin with known synergistic activity. The orientation of the two helices in membranes was studied using solid-state (15)N-NMR, for each peptide alone and for a 1:1 mixture of the peptides, in a range of different lipid systems. Two types of orientational behavior emerged. 1), In lipids with negative spontaneous curvature, both peptides remain flat on the membrane surface, when assessed both alone and in a 1:1 mixture. 2), In lipids with positive spontaneous curvature, PGLa alone assumes a tilted orientation but inserts into the bilayer in a transmembrane alignment in the presence of MAG2, whereas MAG2 stays on the surface or gets only slightly tilted, when observed both alone and in the presence of PGLa. The behavior of PGLa alone is identical to that of another antimicrobial peptide, MSI-103, in the same lipid systems, indicating that the curvature-dependent helix orientation is a general feature of membrane-bound peptides and also influences their synergistic intermolecular interactions., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Understanding how antimicrobial peptides (AMPs) interact with human cells is important to the development of antimicrobial agents as well as anticancer drugs. However, little is known about the mechanisms by which AMPs bind to cells and exert cytotoxicity. Negatively charged gangliosides on the cell surface are a potential target for cell binding. In this study, we investigated the interaction of F5W-magainin 2 (MG) with gangliosides in detail. MG was colocalized with gangliosides on HeLa cells, indicating that gangliosides act as a receptor for MG. MG also bound to gangliosides in model membranes. The affinity increased with the number of negatively charged sialic acid residues. Physicochemical studies revealed that MG interacts with the monosialoganglioside GM1 differently from the typical bacterial anionic phospholipid phosphatidylglycerol. MG bound to GM1 more strongly than to phosphatidylglycerol, and the binding isotherm for GM1 could be analyzed by the Langmuir equation assuming charge neutralization. This is in contrast to the binding of AMPs to phosphatidylglycerol-containing bilayers, which has been described by the electrostatic attraction-surface partitioning model. Fluorescence resonance energy transfer experiments supported the clustering of GM1, but not phosphatidylglycerol, by MG. Quenching data suggested that MG is bound to the sugar region of GM1. The bound peptide assumed a helical structure and induced the leakage of calcein and the coupled flip-flop of lipids, indicating the peptide also forms a toroidal pore in GM1-containing vesicles. However, the membrane permeabilizing activity was weaker against GM1-containing membranes than phosphatidylglycerol-doped liposomes in accordance with the trapping of the peptide in the sugar region. These results shed light on AMP-human cell interaction.

Membrane-active peptides exhibit a wide variety of biological functions including pore formation, signaling and antimicrobial activities. They are also capable of transporting large cargo such as proteins or nucleic acids across cell membranes. This review summarizes biophysical and structural investigations on hydrophobic, amphipathic and heavily charged peptides that reveal a very dynamic view on their membrane interactions. Individual peptides are able to adopt a variety of different conformations and topology and at the same time exhibit multimodal functionalities. Examples discussed in this paper include peptaibols, magainins, cell penetrating peptides and designed histidine-rich sequences with potent antimicrobial and nucleic acid transfection activities.

We previously proposed three hypotheses relating the mechanism of antimicrobial and cytolytic peptides in model membranes to the Gibbs free energies of binding and insertion into the membrane [Almeida, P. F., and Pokorny, A. (2009) Biochemistry 48, 8083-8093]. Two sets of peptides were designed to test those hypotheses, by mutating of the sequences of δ-lysin, cecropin A, and magainin 2. Peptide binding and activity were measured on phosphatidylcholine membranes. In the first set, the peptide charge was changed by mutating basic to acidic residues or vice versa, but the amino acid sequence was not altered much otherwise. The type of dye release changed from graded to all-or-none according to prediction. However, location of charged residues in the sequence with the correct spacing to form salt bridges failed to improve binding. In the second set, the charged and other key residues were kept in the same positions, whereas most of the sequence was significantly but conservatively simplified, maintaining the same hydrophobicity and amphipathicity. This set behaved completely different from predicted. The type of release, which was expected to be maintained, changed dramatically from all-or-none to graded in the mutants of cecropin and magainin. Finally, contrary to the hypotheses, the results indicate that the Gibbs energy of binding to the membrane, not the Gibbs energy of insertion, is the primary determinant of peptide activity., (© 2011 American Chemical Society)

To gain further insight into the antimicrobial activities of cationic linear peptides, we investigated the topology of each of two peptides, PGLa and magainin 2, in oriented phospholipid bilayers in the presence and absence of the other peptide and as a function of the membrane lipid composition. Whereas proton-decoupled (15)N solid-state NMR spectroscopy indicates that magainin 2 exhibits stable in-plane alignments under all conditions investigated, PGLa adopts a number of different membrane topologies with considerable variations in tilt angle. Hydrophobic thickness is an important parameter that modulates the alignment of PGLa. In equimolar mixtures of PGLa and magainin 2, the former adopts transmembrane orientations in dimyristoyl-, but not 1-palmitoyl-2-oleoyl-, phospholipid bilayers, whereas magainin 2 remains associated with the surface in all cases. These results have important consequences for the mechanistic models explaining synergistic activities of the peptide mixtures and will be discussed. The ensemble of data suggests that the thinning of the dimyristoyl membranes caused by magainin 2 tips the topological equilibrium of PGLa toward a membrane-inserted configuration. Therefore, lipid-mediated interactions play a fundamental role in determining the topology of membrane peptides and proteins and thereby, possibly, in regulating their activities as well., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We combined molecular dynamics based free energy calculations with sum frequency generation (SFG) spectroscopy to study the orientational distribution of solvated peptides near hydrophobic surfaces. Using a simplified atomistic model of the polystyrene (PS) surface, molecular dynamics simulations have been applied to compute the orientational probability of an α-helical peptide, magainin 2, with respect to the PS/water interface. Free energy calculations revealed that the preferred (horizontal) peptide orientation was driven by the favorable interactions between the hydrophobic PS surface and the hydrophobic residues on the helix, and additional simulations examined the importance of small aggregate formation. Concentration-dependent measurements obtained via SFG vibrational spectroscopy suggest that, at very low peptide concentrations, magainin molecules tend to lie down at the PS/solution interface, which correlates well with the simulation results. When the concentration is increased, peptides exhibit behavior not captured by MD simulations using single helical peptides. A combination of simulations and experiments was shown to yield more reliable results with molecular-level insights into interaction between peptides and polymer surfaces.

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 alpha-helix, buforin II amide adopts an extended or polyproline II conformation when bound to DNA. These results show that an alpha-helix structure is not required for the DNA binding and condensation activity of buforin II amide., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize bacterial membranes. Understanding their mechanism of action might help design better antibiotics. Using an implicit membrane model, modified to include pores of different shapes, we show that four AMPs (alamethicin, melittin, a magainin analogue, MG-H2, and piscidin 1) bind more strongly to membrane pores, consistent with the idea that they stabilize them. The effective energy of alamethicin in cylindrical pores is similar to that in toroidal pores, whereas the effective energy of the other three peptides is lower in toroidal pores. Only alamethicin intercalates into the membrane core; MG-H2, melittin and piscidin are located exclusively at the hydrophobic/hydrophilic interface. In toroidal pores, the latter three peptides often bind at the edge of the pore, and are in an oblique orientation. The calculated binding energies of the peptides are correlated with their hemolytic activities. We hypothesize that one distinguishing feature of AMPs may be the fact that they are imperfectly amphipathic which allows them to bind more strongly to toroidal pores. An initial test on a melittin-based mutant seems to support this hypothesis., (Copyright 2010 Elsevier B.V. All rights reserved.)

Many antimicrobial peptides (AMPs) function by forming various oligomeric structures and/or pores upon binding to bacterial membranes. Because such peptide aggregates are capable of inducing membrane thinning and membrane permeabilization, we expected that AMP binding would also affect the diffusivity or mobility of the lipid molecules in the membrane. Herein, we show that measurements of the diffusion times of individual lipids through a confocal volume via fluorescence correlation spectroscopy (FCS) provide a sensitive means of probing the underlying AMP-membrane interactions. In particular, results obtained with two well-studied AMPs, magainin 2 and mastoparan X, and two model membranes indicate that this method is capable of revealing structural information, especially the heterogeneity of the peptide-membrane system, that is otherwise difficult to obtain using common ensemble methods. Moreover, because of the high sensitivity of FCS, this method allows examination of the effect of AMPs on the membrane structure at very low peptide/lipid ratios.

We have designed and synthesized a gene encoding a fusion protein comprising Magainin2 and a carrier protein with the aim of screening for a suitable carrier protein expressing antibacterial peptides in the mammalian expression system. The antibacterial peptide Magainin2 was used as a model. Our results on mammalian cell expression showed that there was no exceptional splicing in the transfected CHO-s cells. Analysis of the transgenic mouse model revealed that the expression level of this fusion protein in one transgenic positive mouse was up to 10 g/l, which is close to the level of beta-casein in goat milk. The bioactivity analysis showed that the digested fusion protein had antibacterial activity. These results demonstrate that the synthetic gene of the carrier protein is suitable for expressing an antibacterial peptide in a mammalian cell system at high productivity and efficiency. Moreover, they demonstrate the potential for producing antibacterial peptides in a transgenic animal bioreactor on a large scale and inexpensively.

A major component of green tea extracts, catechin (-)-Epigallocatechin gallate (EGCg), has been reported to be biologically active and interacting with membranes. A recent study reported drastic effects of EGCg on giant unilamellar vesicles (GUVs). In particular, EGCg above 30 microM caused GUVs to burst. Here we investigated the effect of EGCg on single GUVs at lower concentrations, believing that its molecular mechanism would be more clearly revealed. We used the micropipette aspiration method, by which the changes of surface area and volume of a GUV could be measured as a result of interaction with EGCg. We also used x-ray diffraction to measure the membrane thinning effect by EGCg. To understand the property of EGCg, we compared its effect with other membrane-active molecules, including pore-forming peptide magainin, the turmeric (curry) extract curcumin, and detergent Triton X100. We found the effect of EGCg somewhat unique. Although EGCg readily binds to lipid bilayers, its membrane area expansion effect is one order of magnitude smaller than curcumin. EGCg also solubilizes lipid molecules from lipid bilayers without forming pores, but its effect is different from that of Triton X100.

Sexually transmitted infections (STIs) and human immunodeficiency virus (HIV), the causative agents of acquired immunodeficiency syndrome (AIDS), are two great concerns in the reproductive health of women. Thus, the challenge is to find products with a double activity, on the one hand having antimicrobial/antiviral properties with a role in the reduction of STI, and on the other hand having spermicidal action to be used as a contraceptive. In the absence of an effective microbicide along with the disadvantages of the most commonly used spermicidal contraceptive worldwide, nonoxynol-9, new emphasis has been focused on the development of more potential intravaginal microbicidal agents. Topical microbicides spermicides would ideally provide a female-controlled method of self-protection against HIV as well as preventing pregnancies. Nonoxynol-9, the only recommended microbicide spermicide, damages cervicovaginal epithelium because of its membrane-disruptive properties. Clearly, there is an urgent need to identify new compounds with dual potential microbicidal properties; antimicrobial peptides should be candidates for such investigations. Dermaseptins and magainins are two classes of cationic, amphipathic alpha-helical peptides that have been identified in the skin extracts of frogs Phyllomedusa sauvagei and Xenopus laevis. Regarding their contraceptive activities and their effect against various STI-causing pathogens, we believe that these two peptides are appropriate candidates in the evaluation of newer and safer microbicides spermicides in the future.

Interactions of cationic antimicrobial peptides with living bacterial and mammalian cells are little understood, although model membranes have been used extensively to elucidate how peptides permeabilize membranes. In this study, the interaction of F5W-magainin 2 (GIGKWLHSAKKFGKAFVGEIMNS), an equipotent analogue of magainin 2 isolated from the African clawed frog Xenopus laevis, with unfixed Bacillus megaterium and Chinese hamster ovary (CHO)-K1 cells was investigated, using confocal laser scanning microscopy. A small amount of tetramethylrhodamine-labeled F5W-magainin 2 was incorporated into the unlabeled peptide for imaging. The influx of fluorescent markers of various sizes into the cytosol revealed that magainin 2 permeabilized bacterial and mammalian membranes in significantly different ways. The peptide formed pores with a diameter of approximately 2.8 nm (< 6.6 nm) in B. megaterium, and translocated into the cytosol. In contrast, the peptide significantly perturbed the membrane of CHO-K1 cells, permitting the entry of a large molecule (diameter, >23 nm) into the cytosol, accompanied by membrane budding and lipid flip-flop, mainly accumulating in mitochondria and nuclei. Adenosine triphosphate and negatively charged glycosaminoglycans were little involved in the magainin-induced permeabilization of membranes in CHO-K1 cells. Furthermore, the susceptibility of CHO-K1 cells to magainin was found to be similar to that of erythrocytes. Thus, the distinct membrane-permeabilizing processes of magainin 2 in bacterial and mammalian cells were, to the best of our knowledge, visualized and characterized in detail for the first time.