Effective lipid‐detergent system for study of membrane active peptides in fluid liposomes

The structure of peptide antibiotic gramicidin A (gA) was studied in phosphatidylcholin liposomes modified by nonionic detergent Triton X‐100. First, the detergent : lipid ratio at which the saturation of lipid membrane by Triton X‐100 occurs (R_e^sat), was determined by light scattering. Measurements of steady‐state fluorescence anisotropy of 1,6‐diphenyl‐1,3,5‐hexatriene at sublytic concentrations of detergent showed that after saturation of the membrane by Triton X‐100 microviscosity of lipid bilayer is reduced by 20%. The equilibrium conformational state of gA in phosphatidylcholine liposomes at R_e^sat was studied by CD spectroscopy. It was found that the conformational state of this channel‐forming peptide changed crucially when Triton X‐100 induced transition to more fluid membranes. The gA single‐channel measurements were made with Triton X‐100 containing bilayers. Tentative assignment of the channel type and gA structures was made by correlation of CD data with conductance histograms. Lipid‐detergent system with variable viscosity developed in this work can be used to study the structure and folding of other membrane‐active peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Kinetic and equilibrium studies of bile salt–liposome interactions

Abstract

Research has suggested that exposure to sub-micellar concentrations of bile salts (BS) increases the permeability of lipid bilayers in a time-dependent manner. In this study, incubation of soy phosphatidylcholine small unilamellar vesicles (liposomes) with sub-micellar concentrations of cholate (C), deoxycholate (DC), 12-monoketocholate (MKC) or taurocholate (TC) in pH 7.2 buffer increased membrane fluidity and negative zeta potential in the order of increasing BS liposome-pH 7.2 buffer distribution coefficients (MKC?<?C?≈?TC?<?DC). In liposomes labeled with the dithionite-sensitive fluorescent lipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (NBD-PE) in both leaflets and equilibrated with sub-micellar concentrations of BS, fluorescence decline during continuous exposure to dithionite was biphasic involving a rapid initial phase followed by a slower second phase. Membrane permeability to dithionite as measured by the rate of the second phase increased in the order control?<?MKC?<?TC?～?C?<?DC. In liposomes labeled with NBD-PE in the inner leaflet only and incubated with the same concentrations of C, DC and MKC, membrane permeability to dithionite initially increased very rapidly in the order MKC?<?C?<?DC before impermeability to dithionite was restored after which fluorescence decline was consistent with NBD-PE flip-flop. For liposomes incubated with TC, membrane permeability to dithionite was only slightly increased and the decline in fluorescence was mainly the result of NBD-PE flip-flop. These results provide evidence that BS interact with lipid bilayers in a time-dependent manner that is different for conjugated and unconjugated BS. MKC appears to cause least disturbance to liposomal membranes but, when the actual MKC concentration in liposomes is taken into account, MKC is actually the most disruptive.     

Ca<Superscript>2+</Superscript>-Induced Phase Separation in the Membrane of Palmitate-Containing Liposomes and Its Possible Relation to Membrane Permeabilization

A Ca(2+)-induced phase separation of palmitic acid (PA) in the membrane of azolectin unilamellar liposomes has been demonstrated with the fluorescent membrane probe nonyl acridine orange (NAO). It has been shown that NAO, whose fluorescence in liposomal membranes is quenched in a concentration-dependent way, can be used to monitor changes in the volume of lipid phase. The incorporation of PA into NAO-labeled liposomes increased fluorescence corresponding to the expansion of membrane. After subsequent addition of Ca(2+), fluorescence decreased, which indicated separation of PA/Ca(2+) complexes into distinct membrane domains. The Ca(2+)-induced phase separation of PA was further studied in relation to membrane permeabilization caused by Ca(2+) in the PA-containing liposomes. A supposition was made that the mechanism of PA/Ca(2+)-induced membrane permeabilization relates to the initial stage of Ca(2+)-induced phase separation of PA and can be considered as formation of fast-tightening lipid pores due to chemotropic phase transition in the lipid bilayer.     

SNARE protein analog‐mediated membrane fusion

Fusion of lipid membranes to form a single bilayer is an essential process for life and provides important biological functions including neurotransmitter release. Membrane fusion proteins facilitate approximation of interacting membranes to overcome the energy barrier. In case of synaptic transmission, proteins involved are known as soluble N‐ethylmaleimide‐sensitive‐factor attachment receptor (SNARE) proteins. The SNAREs from synaptic vesicles interact with the SNAREs from the target membrane to form a coiled‐coil bundle of four helices, thus pulling the membranes tightly together and initiating fusion. However, it remains unclear how these proteins function at molecular level. Natural systems are often too complex to obtain unambiguous results. Simple model systems mimicking natural proteins in synthetic lipid bilayers are powerful tools for obtaining insights into this essential biological process. An important advantage of such systems is their well‐defined composition, which can be systematically varied in order to fully understand events at molecular level. In this review, selected model systems are presented based upon specific interactions between recognition units embedded in separate lipid bilayers mimicking native SNARE protein‐mediated membrane fusion. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Lipid interactions of acylated tryptophan‐methylated lactoferricin peptides by solid‐state NMR

Lactoferricin (LfB) is a 25‐residue innate immunity peptide released by pepsin from the N‐terminal region of bovine lactoferrin. A smaller amidated peptide, LfB6 (RRWQWR‐NH₂) retains antimicrobial activity and is thought to constitute the “antimicrobial active‐site” (Tomita, Acta Paediatr Jpn. 1994; 36 : 585–91). Here we report on N‐acylation of 1‐Me‐Trp⁵‐LfB6, Cn‐RRWQ[1‐Me‐W]R‐NH₂, where Cn is an acyl chain having n = 0, 2, 4, 6 or 12 carbons. Tryptophan 5 (Trp⁵) was methylated to enhance membrane binding and to allow for selective deuteration at that position. Peptide/lipid interactions of Cn‐RRWQ[1‐Me‐W ]R‐NH₂ (deuterated 1‐Me‐Trp⁵ underlined), were monitored by solid state ³¹P NMR and ²H NMR. The samples consisted of macroscopically oriented bilayers of mixed neutral (dimyristoylphosphatidylcholine, DMPC) and anionic (dimyristoylphosphatidylglycerol, DMPG) lipids in a 3:1 ratio with Cn‐RRWQ[&1‐Me‐W ]R‐NH₂ peptides added at a 1:25 peptide to lipid ratio. ²H‐NMR spectra reveal that the acylated peptides are well aligned in DMPC:DMPG bilayers. The ²H NMR quadrupolar splittings suggest that the 1‐Me‐Trp is located in a motionally restricted environment, indicating partial alignment at the membrane interface. ³¹P‐NMR spectra reveal that the lipids are predominantly in a bilayer configuration, with little perturbation by the peptides. Methylation alone, in C0‐RRWQ[1‐Me‐W ]R‐NH₂, resulted in a 3–4 fold increase in antimicrobial activity against E. coli. N‐acylation with a C12 fatty acid enhanced activity almost 90 fold. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Nucleoterpenes of Thymidine and 2′‐Deoxyinosine: Synthons for a Biomimetic Lipophilization of Oligonucleotides

2′‐Deoxyinosine ( 1 ) and thymidine ( 7 ) were N‐alkylated with geranyl and farnesyl moieties. These hydrophobic derivatives, 3a and 3b , and 9a and 9b , respectively, represent the first synthetic biomimetic nucleoterpenes and were subsequently 5′‐protected and converted into the corresponding 3′‐O‐phosphoramidites, 5a and 5b and 11a and 11b , respectively. The latter were used to prepare a series of lipophilized oligonucleotide dodecamers, a part of which were additionally labelled with indocarbocyanine fluorescent dyes (Cy3 or Cy5), 18 – 23 . The insertion of the lipooligonucleotides into, as well as duplex formation at artificial lipid bilayers was studied by single‐molecule fluorescence spectroscopy and fluorescence microscopy.     

Production of functional bacteriorhodopsin by an Escherichia coli cell‐free protein synthesis system supplemented with steroid detergent and lipid

Cell‐free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis‐based Escherichia coli cell‐free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light‐driven proton pump bacteriorhodopsin, consisting of seven transmembrane α‐helices. The cell‐free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3–0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent‐lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.     

Interaction of rifampicin and isoniazid with large unilamellar liposomes: spectroscopic location studies

The location of isoniazid and rifampicin, two tuberculostatics commonly used for the treatment of Mycobacterium tuberculosis and Mycobacterium avium complex infectious diseases, in bilayers of dimyristoyl-L-alpha-phosphatidylcholine (DMPC) and dimyristoyl-L-a-phosphatidylglycerol (DMPG) have been studied by 1H NMR and fluorimetric methods. Steady-state fluorescence intensity and fluorescence energy transfer studies between rifampicin and a set of functionalized probes [n-(9-anthroyloxy)stearic acids, n=2, 12] reveal that, in both systems, isoniazid is located at the membrane surface whereas rifampicin is deeply buried inside the lipid bilayers. Steady-state fluorescence anisotropy studies performed with the probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-diphenylhexa-triene (TMA-DPH), not only corroborate the above results, but also show that no changes in membrane fluidity were detected in either liposome. The 1H NMR results, in DMPC liposomes, confirm the location of rifampicin near the methylene group of the acyl chains of the lipid bilayers.     

Interaction of synthetic peptide corresponding to signal sequence of glucitol permease of Escherichia Coli and its analogue with liposomes

The N-terminal signal sequence of glucitol pcrmease of Escherichia Coli (Gut22) and its analogue (Gut22Ana) were synthesized. The analogue had a Pro residue substituting for the His at the 7th position of Gut22 and a Val residue substituting for the Glu at the 10th position. The intrinsic fluorescence emission spectra indicated that the binding of Gut22 with lipid bilayer was much stronger than that of Gut22Ana. The leakage experiments with calcein-loaded liposomes showed that Gut22 strongly perturbed lipid bilayers while Gut22Ana did not. The apparent partition constant of Gut22 for partitioning into phosphatidylserine/phosphatidylcholine bilayers was measured; the effect of membrane potential on the interaction of Gut22 with lipid bilayers was studied and the conformation changes of Gut22 and Gut22Ana upon interacting with liposomes were studied by the method of circular dichroism analysis.     

G‐protein coupled receptor array technologies: Site directed immobilisation of liposomes containing the H1‐histamine or M2‐muscarinic receptors

This paper describes a novel strategy to create a microarray of G‐protein coupled receptors (GPCRs), an important group of membrane proteins both physiologically and pharmacologically. The H₁‐histamine receptor and the M₂‐muscarinic receptor were both used as model GPCRs in this study. The receptor proteins were embedded in liposomes created from the cellular membrane extracts of Spodoptera frugiperda (Sf9) insect cell culture line with its accompanying baculovirus protein insert used for overexpression of the receptors. Once captured onto a surface these liposomes provide a favourable lipidic environment for the integral membrane proteins. Site directed immobilisation of these liposomes was achieved by introduction of cholesterol‐modified oligonucleotides (oligos). These oligo/cholesterol conjugates incorporate within the lipid bilayer and were captured by the complementary oligo strand exposed on the surface. Sequence specific immobilisation was demonstrated using a quartz crystal microbalance with dissipation (QCM‐D). Confirmatory results were also obtained by monitoring fluorescent ligand binding to GPCRs captured on a spotted oligo microarray using Confocal Laser Scanning Microscopy and the ZeptoREADER microarray imaging system. Sequence specific immobilisation of such biologically important membrane proteins could lead to the development of a heterogeneous self‐sorting liposome array of GPCRs which would underpin a variety of future novel applications.     

Virulence‐associated protein A from Rhodococcus equi is an intercompartmental pH‐neutralising virulence factor

Professional phagocytic cells such as macrophages are a central part of innate immune defence. They ingest microorganisms into membrane‐bound compartments (phagosomes), which acidify and eventually fuse with lysosomes, exposing their contents to a microbicidal environment. Gram‐positive Rhodococcus equi can cause pneumonia in young foals and in immunocompromised humans. The possession of a virulence plasmid allows them to subvert host defence mechanisms and to multiply in macrophages. Here, we show that the plasmid‐encoded and secreted virulence‐associated protein A (VapA) participates in exclusion of the proton‐pumping vacuolar‐ATPase complex from phagosomes and causes membrane permeabilisation, thus contributing to a pH‐neutral phagosome lumen. Using fluorescence and electron microscopy, we show that VapA is also transferred from phagosomes to lysosomes where it permeabilises the limiting membranes for small ions such as protons. This permeabilisation process is different from that of known membrane pore formers as revealed by experiments with artificial lipid bilayers. We demonstrate that, at 24 hr of infection, virulent R. equi is contained in a vacuole, which is enriched in lysosome material, yet possesses a pH of 7.2 whereas phagosomes containing a vapA deletion mutant have a pH of 5.8 and those with virulence plasmid‐less sister strains have a pH of 5.2. Experimentally neutralising the macrophage endocytic system allows avirulent R. equi to multiply. This observation is mirrored in the fact that virulent and avirulent R. equi multiply well in extracts of purified lysosomes at pH 7.2 but not at pH 5.1. Together these data indicate that the major function of VapA is to generate a pH‐neutral and hence growth‐promoting intracellular niche. VapA represents a new type of Gram‐positive virulence factor by trafficking from one subcellular compartment to another, affecting membrane permeability, excluding proton‐pumping ATPase, and consequently disarming host defences.     

Use of liposomes as membrane models to evaluate the contribution of drug–membrane interactions to antioxidant properties of etodolac

Abstract

This work stresses the need to combine antioxidant assays and drug–membrane interaction studies to describe more accurately the antioxidant profile of non-steroidal anti-inflammatory drugs (NSAIDs). Different experiments performed in liposomes and aqueous solution were compared and used to evaluate the protective effect of etodolac in lipid peroxidation. Lipid peroxidation was induced by the peroxyl radical (ROO?) derived from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and hydroxyl radical (HO?) generated by the Fenton reaction and was assessed by the fluorescence intensity decay of three fluorescence probes with distinct lipophilic properties – fluorescein; hexadecanoyl aminofluorescein (HDAF) and diphenylhexatriene propionic acid (DPHPA). Membrane fluidity changes due to lipid peroxidation were also evaluated by steady-state anisotropy measurements. Interactions of etodolac with lipid bilayers were evaluated by membrane zeta-potential measurements. Results indicate a drug location near the membrane surface and show that etodolac can scavenge the radicals studied but to a variable extent, depending on the assayed media and reactive species. The use of different probes and liposomes as membrane mimetic systems allowed us to conclude that membrane lipoperoxidation is not only related to the scavenging characteristics of the antioxidants, but also to their ability to interact with lipid bilayers.     

A facile,sensitive and quantitative membrane‐binding assay for proteins

Soluble proteins that bind membranes function in numerous cellular pathways yet facile, sensitive and quantitative methods that complement and improve sensitivity of widely used liposomes‐based assays remain unavailable. Here, we describe the utility of a photoactivable fluorescent lipid as a generic reporter of protein‐membrane interactions. When incorporated into liposomes and exposed to ultraviolet (UV), proteins bound to liposomes become crosslinked with the fluorescent lipid and can be readily detected and quantitated by in‐gel fluorescence analysis. This modification obviates the requirement for high‐speed centrifugation spins common to most liposome‐binding assays. We refer to this assay as Proximity‐based Labeling of Membrane‐Associated Proteins (PLiMAP).     

Which physical and structural factors of liposome carriers control their drug-loading efficiency?

The correlation between structural and physical properties of lipid membrane and its drug-loading efficiency were studied. The properties of bilayer were altered by incorporation of several lipidic modifiers: cholesterol, oleic acid, methyl oleate, and pegylated lipid. By using the molecular probe technique it was demonstrated that the membrane properties, such as micropolarity, microviscosity and free volume were considerably changed by incorporation of the modifiers. The partitioning of two different porphyrins between the bulk aqueous phase and the modified liposomes was studied using the fluorescence methods, and liposome-binding constants were determined. It was found that cholesterol reduced the partitioning of both porphyrins into liposomal bilayer. On the contrary, the incorporation of methyl oleate and pegylated lipid causes a pronounced increase in the value of the binding constants of both porphyrins. It was concluded that the free volume rather than hydrophobicity of bilayer is a governing factor in the solute partitioning into lipid bilayers.     

4‐Cyano‐α‐methyl‐l‐phenylalanine as a Spectroscopic Marker for the Investigation of PeptaibioticMembrane Interactions

Two analogs of the ten‐amino acid residue, membrane‐active lipopeptaibiotic trichogin GA IV, mono‐labeled with 4‐cyano‐α‐methyl‐L ‐phenylalanine, a potentially useful fluorescence and IR absorption probe of the local microenvironment, were synthesized by the solid‐phase methodology and conformationally characterized. The single modification was incorporated either at the N‐terminus (position 1) or near the C‐terminus (position 8) of the peptide main chain. In both cases, the replaced amino acid was the equally helicogenic α‐aminoisobutyric acid (Aib) residue. We performed a solution conformational analysis by use of FT‐IR absorption, CD, and 2D‐NMR spectroscopies. The results indicate that both labeled analogs essentially maintain the overall helical propensity of the naturally occurring lipopeptaibiotic. Peptide? membrane interactions were assessed by fluorescence and ATR‐IR absorption techniques. Analogies and differences between the two peptides were highlighted. Taken together, our data confirm literature results that some of the spectroscopic parameters of the 4‐cyanobenzyl chromophore are sensitive markers of the local microenvironment.     

Antioxidant Activity of Vitamin E and Trolox: Understanding of the Factors that Govern Lipid Peroxidation Studies In Vitro

Peroxidation of lipids is of significant interest owing to the evidence that peroxyl radicals and products of lipid peroxidation may be involved in the toxicity of compounds initiating a deteriorative reaction in the processing and storage of lipid-containing foods. In view of the significance of the antioxidant role of the dietary compound vitamin E and its water-soluble analogue Trolox in research of lipid-containing foods, it is desirable to determine more specifically how and where they operate its antioxidant activity in lipid membranes. In this study, unilamellar liposomes of phosphatidylcholine were used as membrane mimetic systems to estimate the antioxidant properties of vitamin E and Trolox and establish a relationship between their interactions with the membrane and their consequent antioxidant activity. Lipid peroxidation was initiated by the peroxyl radical (ROO^•) in lipid and aqueous media by the thermal decomposition of azocompounds and was assessed by the fluorescence intensity decay of the fluorescent probe diphenylhexatriene propionic acid. Results obtained showed that membrane lipoperoxidation is related not only to the scavenging characteristics of the compounds studied but also to their ability to interact with the lipid bilayers, and consequently liposomes provide additional information to that obtained currently from assays performed in aqueous buffer media.     

Cationic carbosilane dendrimers-lipid membrane interactions

The aim of this work was to study interactions between cationic carbosilane dendrimers (CBS) and lipid bilayers or monolayers. Two kinds of second generation carbosilane dendrimers were used: NN16 with Si-O bonds and BDBR0011 with Si-C bonds. The results show that cationic carbosilane dendrimers interact both with liposomes and lipid monolayers. Interactions were stronger for negatively charged membranes and high concentration of dendrimers. In liposomes interactions were studied by measuring fluorescence anisotropy changes of fluorescent labels incorporated into the bilayer. An increase in fluorescence anisotropy was observed for both fluorescent probes when dendrimers were added to lipids that means the decreased membrane fluidity. Both the hydrophobic and hydrophilic parts of liposome bilayers became more rigid. This may be due to dendrimers' incorporation into liposome bilayer. For higher concentrations of both dendrimers precipitation occurred in negatively charged liposomes. NN16 dendrimer interacted stronger with hydrophilic part of bilayers whereas BDBR0011 greatly modified the hydrophobic area. Monolayers method brought similar results. Both dendrimers influenced lipid monolayers and changed surface pressure. For negatively charged lipids the monitored parameter changed stronger than for uncharged DMPC lipids. Moreover, NN16 dendrimer interacted stronger than the BDBR0011.     

Surface-linked liposomal antigen induces ige-selective unresponsiveness regardless of the lipid components of liposomes

We have previously reported that antigen coupled with liposomes induced antigen-specific and IgE-selective unresponsiveness in mice. This antigen preparation was investigated for application in a novel vaccine protocol to induce minimal IgE synthesis. In this study, ovalbumin (OVA)-liposome conjugates were made using liposomes of four different lipid components, including unsaturated carrier lipid and three different saturated carrier lipids, after which the induction of anti-OVA antibody production was investigated in mice. All of the OVA-liposome conjugates induced IgE-selective unresponsiveness. The membrane fluidity of liposomes, as measured by detecting changes in the fluorescence polarization of a 1,6-diphenyl-1,3,5-hexatriene (DPH) probe located in the bilayers, was significantly higher in liposomes consisting of unsaturated carrier lipids than those of the other liposomes consisting of saturated carrier lipids. The highest titer of anti-OVA IgG was observed in mice immunized with OVA-liposomes made using liposomes consisting of unsaturated carrier lipids. In addition, among these OVA-liposomes, the one possessing the longest carbon chain induced the lowest IgG antibody production. These results suggest that the membrane fluidity of liposomes might affect the adjuvant effect of liposomes but not the induction of IgE-selective unresponsiveness in immunizations with surface-linked liposomal antigens.     

Fluorescent probes for asymmetric lipid bilayers: Synthesis and properties in phosphatidyl choline liposomes and erythrocyte membranes

Summary We have synthesized three sets of fluorescent probes which we believe will be useful in studies of asymmetric membranes and have studied their interactions with model lipid bilayers and erythrocyte membranes. The probes were designed to partition preferentially into one face of a lipid bilayer with asymmetrically disposed phospholipids and to report lipid transitions in that monolayer.We synthesized more than twenty probes containing anthroyl-, dansyl-, or pyrene rings with acidic, basic, and neutral functional groups and alkyl spacers of various lengths. The interactions of these probes with liposomes of phosphatidyl choline and with erythrocyte membranes were characterized to determine whether probe insertion was asymmetric, how deeply the probe penetrated the bilayer, and whether the probe reflected thermotropic phase transitions in model membranes. The set of variously charged anthroyl esters, analogs of local anaesthetics, appears to be promising for studies of asymmetric membranes.Fluorescent probes have been used extensively to provide information on the lipid regions of biological membranes. Membrane fluidity, a composite of molecular packing and motion of acyl chains in lipid bilayers, has been assessed with a variety of fluorescent probes, the fluorescence of which undergoes some measurable change at the temperature of the membrane's thermotropic phase transition. A large number of fluorescent probes have been used for this purpose. Bashford, Morgan and Radda (Bashford, C.L., Morgan, C.G., Radda, G.K. 1976;Biochim. Biophys. Acta 426: 157) and Thulborn and Sawyer (Thulborn, K.R., Sawyer, W.H. 1978;Biochim. Biophys. Acta 511: 125) synthesized several fatty acid derivatives in which an anthracene group is attached (in ester linkage) along the acyl chain at various positions, and have shown that this set of probes may be useful in probing membrane fluidity at differentdepths within the bilayer.This report describes the synthesis and properties of several sets of amphipathic fluorescent probes, which may partition unequally into the two faces of an asymmetric lipid bilayer, and may therefore provide information about membranes complementary to that obtainable with existing probes.     

Antimicrobial activity of peptides derived from human ß‐amyloid precursor protein

Antimicrobial peptides are important effector molecules of the innate immune system. Here, we describe that peptides derived from the heparin‐binding disulfide‐constrained loop region of human ß‐amyloid precursor protein are antimicrobial. The peptides investigated were linear and cyclic forms of NWCKRGRKQCKTHPH (NWC15) as well as the cyclic form comprising the C‐terminal hydrophobic amino acid extension FVIPY (NWCKRGRKQCKTHPHFVIPY; NWC20c). Compared with the benchmark antimicrobial peptide LL‐37, these peptides efficiently killed the Gram‐negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram‐positive Staphylococcus aureus and Bacillus subtilis, and the fungi Candida albicans and Candida parapsilosis. Correspondingly, fluorescence and electron microscopy demonstrated that the peptides caused defects in bacterial membranes. Analogously, the peptides permeabilised negatively charged liposomes. Despite their bactericidal effect, the peptides displayed very limited hemolytic activities within the concentration range investigated and exerted very small membrane permeabilising effects on human epithelial cells. The efficiency of the peptides with respect to bacterial killing and liposome membrane leakage was in the order NWC20c > NWC15c > NWC15l, which also correlated to the adsorption density for these peptides at the model lipid membrane. Thus, whereas the cationic sequence is a minimum determinant for antimicrobial action, a constrained loop‐structure as well as a hydrophobic extension further contributes to membrane permeabilising activity of this region of amyloid precursor protein. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.