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Here the hypothesis that linearized esculentin 2EM (E2EM-lin) from Glandirana emeljanovi possesses pH dependent activity is investigated. The peptide showed weak activity against Gram-negative bacteria (MLCs ≥ 75.0 μM) but potent efficacy towards Gram-positive bacteria (MLCs ≤ 6.25 μM). E2EM-lin adopted an α-helical structure in the presence of bacterial membranes that increased as pH was increased from 6 to 8 (↑ 15.5–26.9%), whilst similar increases in pH enhanced the ability of the peptide to penetrate (↑ 2.3–5.1 mN m−1) and lyse (↑ 15.1–32.5%) these membranes. Theoretical analysis predicted that this membranolytic mechanism involved a tilted segment, that increased along the α-helical long axis of E2EM-lin (1–23) in the N → C direction, with − µH > increasing overall from circa − 0.8 to − 0.3. In combination, these data showed that E2EM-lin killed bacteria via novel mechanisms that were enhanced by alkaline conditions and involved the formation of tilted and membranolytic, α-helical structure. The preference of E2EM-lin for Gram-positive bacteria over Gram-negative organisms was primarily driven by the superior ability of phosphatidylglycerol to induce α-helical structure in the peptide as compared to phosphatidylethanolamine. These data were used to generate a novel pore-forming model for the membranolytic activity of E2EM-lin, which would appear to be the first, major reported instance of pH dependent AMPs with alkaline optima using tilted structure to drive a pore-forming process. It is proposed that E2EM-lin has the potential for development to serve purposes ranging from therapeutic usage, such as chronic wound disinfection, to food preservation by killing food spoilage organisms.

Aurein 2.1, aurein 2.6 and aurein 3.1 are amphibian host defence peptides that kill bacteria via the use of lytic amphiphilic α-helical structures. The C-terminal PEGylation of these peptides led to decreased antibacterial activity (Minimum Lethal Concentration (MLCs) ↓ circa one and a half to threefold), reduced levels of amphiphilic α-helical structure in solvents (α-helicity ↓ circa 15.0%) and lower surface activity (Δπ ↓ > 1.5 mN m ). This PEGylation of aureins also led to decreased levels of amphiphilic α-helical structure in the presence of anionic membranes and zwitterionic membranes (α-helicity↓ > 10.0%) as well as reduced levels of penetration (Δπ ↓ > 3.0 mN m ) and lysis (lysis ↓ > 10.0%) of these membranes. Based on these data, it was proposed that the antibacterial action of PEGylated aureins involved the adoption of α-helical structures that promote the lysis of bacterial membranes, but with lower efficacy than their native counterparts. However, PEGylation also reduced the haemolytic activity of native aureins to negligible levels (haemolysis ↓ from circa 10% to 3% or less) and improved their relative therapeutic indices (RTIs ↑ circa three to sixfold). Based on these data, it is proposed that PEGylated aureins possess the potential for therapeutic development; for example, to combat infections due to multi-drug resistant strains of S. aureus, designated as high priority by the World Health Organization. [Abstract copyright: Copyright © 2021 Elsevier B.V. All rights reserved.]

A number of disorders and diseases are associated with conditions of high pH, and many conventional antibiotics lose their efficacy under these pH conditions, generating a need for novel antimicrobials. A potential solution to fulfill this need is Antimicrobial Peptides (AMPs) with high pH optima. This review shows that a variety of anionic and cationic AMPs with this pH dependency are produced by creatures across the eukaryotic kingdom, including rabbits, cattle, sheep, fish, crabs and frog. These AMPs exhibit activity against viruses, bacteria, and fungi that involve membrane interactions and appear to be facilitated by a variety of mechanisms that generally promote passage across membranes to attack intracellular targets, such as DNA or protein synthesisand/or membrane lysis. Some of these mechanisms are unknown, but those elucidated include the use of bacterial pores and transporters, the self-promoted uptake pathway, and established models of membrane interaction, such as the carpet mechanism, toroidal pore formation, the adoption of tilted peptide, and the SHM model. A variety of potential roles have been proposed for these AMPs, including use as antivirals, antibacterials, antifungals, adjuvants to antimicrobial therapy, biomarkers of disease, and probes for pathogenic microbes. In this review, these properties are described and discussed, emphasizing the antimicrobial mechanisms used by these AMPs and the pH dependency of these mechanisms.

Linearized esculentin 2 EM (E2EM-lin) from the frog, Glandirana emeljanovi was highly active against Gram-positive bacteria (minimum lethal concentration ≤ 5.0 μM) and strongly α-helical in the presence of lipid mimics of their membranes (>55.0%). The N-terminal α-helical structure adopted by E2EM-lin showed the potential to form a membrane interactive, tilted peptide with an hydrophobicity gradient over residues 9 to 23. E2EM-lin inserted strongly into lipid mimics of membranes from Gram-positive bacteria (maximal surface pressure changes ≥5.5 mN m ), inducing increased rigidity (C ↑), thermodynamic instability (ΔG 0) and high levels of lysis (>50.0%). These effects appeared to be driven by the high anionic lipid content of membranes from Gram-positive bacteria; namely phosphatidylglycerol (PG) and cardiolipin (CL) species. The high levels of α-helicity (60.0%), interaction (maximal surface pressure change = 6.7 mN m ) and lysis (66.0%) shown by E2EM-lin with PG species was a major driver in the ability of the peptide to lyse and kill Gram-positive bacteria. E2EM-lin also showed high levels of α-helicity (62.0%) with CL species but only low levels of interaction (maximal surface pressure change = 2.9 mN m ) and lysis (21.0%) with the lipid. These combined data suggest that E2EM-lin has a specificity for killing Gram-positive bacteria that involves the formation of tilted structure and appears to be primarily driven by PG-mediated membranolysis. These structure/function relationships are used to help explain the pore forming process proposed to describe the membranolytic, antibacterial action of E2EM-lin. [Abstract copyright: Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.]

Modelin-5-CONH2 (M5-NH2) is a synthetic antimicrobial peptide, which was found to show potent activity against Bacillus subtilis (Minimum lethal concentration = 8.47 μM) and to bind strongly to membranes of the organism (Kd = 10.44 μM). The peptide adopted high levels of amphiphilic α-helical structure in the presence of these membranes (> 50 %), which led to high levels of insertion (Δπ ≥ 8.0 mN m-1). M5-NH2 showed high affinity for anionic lipid (Kd = 7.46 µM) and zwitterionic lipid (Kd = 14.7 µM), which drove insertion into membranes formed from these lipids (Δπ = 11.5 and 3.5 mN m-1, respectively) and interactions with their head group and hydrophobic core regions. Neutron diffraction studies showed that M5-NH2 inserted into B. subtilis membranes with its N-terminal residue, L16, located 5.5 Å from the membrane centre, in the acyl chain region of these membranes, and promoted a reduction in membrane thickness of circa 1.8 Å or 5 % of membrane width. Insertion into B. subtilis membranes by the peptide also promoted other effects associated with membrane thinning, including increases in membrane surface area (Cs-1 decrease) and fluidity (ΔGmix > 0 to < 0). Membrane insertion and thinning by M5-NH2 induced high levels of lysis (> 55 %) and, taken with MD simulation data, it is speculated that the antibacterial action of the peptide may involve the toroidal pore, carpet or tilted-type mechanism of membrane permeabilization.

Here we report the first major example of anionic amphibian host defence peptides (HDPs) with anticancer activity. Maximin H5 is a C-terminally amidated, anionic host defence peptide (MH5N) from toads of the Bombina genus, which was shown to possess activity against the glioma cell line, T98G (EC50 = 125 μM). The peptide adopted high levels of α-helical structure (57.3%) in the presence of model cancer membranes (DMPC:DMPS in a molar ratio of 10:1). MH5N also showed a strong ability to penetrate these model membranes (Π = 10.5 mN m-1), which correlated with levels of DMPS (R2 > 0.98). Taken with the high ability of the peptide to lyse these membranes (65.7%), it is proposed that maximin H5 kills cancer cells via membranolytic mechanisms that are promoted by anionic lipid. It was also found that C-terminally deaminated maximin H5 (MH5C) exhibited lower levels of α-helical structure in the presence of cancer membrane mimics (44.8%) along with a reduced ability to penetrate these membranes (Π = 8.1 mN m-1) and induce their lysis (56.6%). These data suggested that the two terminal amide groups of native maximin H5 are required for its optimal membranolytic and anticancer activity.

Globally, death due to cancers is likely to rise to over 20 million by 2030, which has created an urgent need for novel approaches to anticancer therapies such as the development of host defence peptides. Cn-AMP2 (TESYFVFSVGM), an anionic host defence peptide from green coconut water of the plant Cocos nucifera, showed anti-proliferative activity against the 1321N1 and U87MG human glioma cell lines with IC50 values of 1.25 and 1.85 mM, respectively. The membrane interactive form of the peptide was found to be an extended conformation, which primarily included β-type structures (levels > 45%) and random coil architecture (levels > 45%). On the basis of these and other data, it is suggested that the short anionic N-terminal sequence (TES) of Cn-AMP2 interacts with positively charged moieties in the cancer cell membrane. Concomitantly, the long hydrophobic C-terminal sequence (YFVFSVGM) of the peptide penetrates the membrane core region, thereby driving the translocation of Cn-AMP2 across the cancer cell membrane to attack intracellular targets and induce anti-proliferative mechanisms. This work is the first to demonstrate that anionic host defence peptides have activity against human glioblastoma, which potentially provides an untapped source of lead compounds for development as novel agents in the treatment of these and other cancers.

Aurein 2.5 (GLFDIVKKVVGAFGSL-NH2) is an antimicrobial peptide, which was seen to have activity against Stachybotris chartarum, Penicillium roseopurpureum and Aspergillus flavus with minimum fungicidal concentrations in the range 250-500 μM. S. chartarum showed enhanced susceptibility to lysis as compared to P. roseopurpureum and A. flavus, (44, 26 and 28 % respectively). Monolayers formed from lipid membrane extracts derived from S. chartarum, P. roseopurpureum and A. flavus showed maximal surface pressure changes of 13.5, 10.3 and 10.2 mN m(-1) respectively. However, aurein 2.5 adopted similar levels of α-helical structure (circa 45 %) in the presence of vesicles formed from membrane lipid extracts derived from all three fungi. These data imply that differential activity is not due to targeting and membrane association but linked to the ability of the bound peptide to lyse the cells. At sterol levels mimetic of eukaryotic systems, high levels of α-helical structure (circa 50 %) were also observed and hence similar binding. However, enhanced sterol levels (>0.6) led to a reduction in monolayer membrane interaction, suggesting that the sterols influence efficacy. Consistent with this suggestion, thermodynamic analysis showed that the peptide was able to destabilise model fungal monolayers, as indicated by negative values of ∆Gmix.

Host defense peptides show great potential for development as new antimicrobial agents with novel mechanisms of action. However, a small number of resistance mechanisms to their action are known, and here, we report a novel bacterial resistance mechanism mediated by a lipid receptor. Maximin H5 from Bombina maxima bound anionic and zwitterionic membranes with low affinity (Kd225 μM) while showing a strong ability to lyse (55%) and penetrate (π6.0 mN m(-1)) these membranes. However, the peptide bound Escherichia coli and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) membranes with higher affinity (Kd65 μM) and showed a very low ability for bilayer lysis (8%) and partitioning (π1.0 mN m(-1)). Increasing levels of membrane DMPE correlated with enhanced binding by the peptide (R(2) = 0.96) but inversely correlated with its lytic ability (R(2) = 0.98). Taken with molecular dynamic simulations, these results suggest that maximin H5 possesses membranolytic activity, primarily involving bilayer insertion of its strongly hydrophobic N-terminal region. However, this region was predicted to form multiple hydrogen bonds with phosphate and ammonium groups within PE head-groups, which in concert with charge-charge interactions anchor the peptide to the surface of E. coli membranes, inhibiting its membranolytic action.

Aurein 2.5 (GLFDIVKKVVGAFGSL-NH(2)) is an amphibian antimicrobial peptide. Here, characterisation studies showed the peptide to exhibit molecular areas at an air / water interface (1.77 - 3.1 nm(2)), which are in agreement with the adoption of alpha-helical structures. Lipid monolayer studies showed aurein 2.5 to induce maximal surface pressure changes of circa 7 mN m(-1) in monolayers formed from phosphatidylglycerol (PG) and circa 6 mN m(-1) in those formed from phosphatidylethanolamine (PE). These data indicate that the membrane interactions of the peptide are amphiphilicity driven with no apparent electrostatic requirement. Individually mutating the phenylalanine residues of aurein 2.5 to leucine had no major effect on the levels of PG and PE interactions, suggesting that these residues are not essential to the membrane interactions of the peptide, contrasting to other aureins where corresponding phenylalanine residues are required for efficient membrane interaction and antibacterial activity. This difference in the requirement is suggested to relate to the surface architecture as proposed by the concept of the molecular perturbation potential.

Aurein 2.5 (GLFDIVKKVVGAFGSL-NH(2)) is an uncharacterised antimicrobial peptide. At an air/water interface, it exhibited strong surface activity (maximal surface pressure 25mNm(-1)) and molecular areas consistent with the adoption of alpha-helical structure orientated either perpendicular (1.72nm(2)molecule(-1)) or parallel (3.6nm(2)molecule(-1)) to the interface. Aurein 2.5 was strongly antibacterial, exhibiting a minimum inhibitory concentration (MIC) of 30microM against Bacillus subtilis and Escherichia coli. The peptide induced maximal surface pressure changes of 9mNm(-1) and 5mNm(-1), respectively, in monolayers mimicking membranes of these organisms whilst compression isotherm analysis of these monolayers showed DeltaG(Mix)0, indicating destabilisation by Aurein 2.5. These combined data suggested that toxicity of the peptide to these organisms may involve membrane invasion via the use of oblique orientated alpha-helical structure. The peptide induced strong, comparable maximal surface changes in monolayers of DOPG (7.5mNm(-1)) and DOPE monolayers (6mNm(-1)) suggesting that the membrane interactions of Aurein 2.5 were driven by amphiphilicity rather than electrostatic interaction. Based on these data, it was suggested that the differing ability of Aurein 2.5 to insert into membranes of B. subtilis and E. coli was probably related to membrane-based factors such as differences in lipid packing characteristics. The peptide was active against both sessile E. coli and Staphylococcus aureus with an MIC of 125microM. The broad-spectrum antibacterial activity and non-specific modes of membrane action used by Aurein 2.5 suggested use as an anti-biofilm agent such as in the decontamination of medical devices.

VP1, a putative alpha-helical antimicrobial peptide (alpha-AMP) inhibited growth of Bacillus subtilis and Escherichia coli at 500microM. The peptide induced stable surface pressure changes in monolayers formed from B. subtilis native lipid extract (circa 4.5mNm(-1)) but transient pressure changes in corresponding E. coli monolayers (circa 1.0mNm(-1)), which led to monolayer disintegration. Synthetic lipid monolayers mimetic of the extracts were used to generate compression isotherms. Thermodynamic analysis of B. subtilis isotherms indicated membrane stabilisation by VP1 (DeltaG(Mix)0), via a mechanism dependent upon the phosphatidylglycerol to cardiolipin ratio. Corresponding analysis of E. coli isotherms indicated membrane destabilisation by the peptide (DeltaG(Mix)0). Destabilisation correlated with PE levels present and appeared to involve a mechanism resembling those used by tilted peptides. These data emphasise that structure/function analysis of alpha-AMPs must consider not only their structural characteristics but also the lipid make-up of the target microbial membrane.

The barley yellow dwarf virus movement protein (BYDV-MP) requires its N-terminal sequence to promote the transport of viral RNA into the nuclear compartment of host plant cells. Here, graphical analysis predicts that this sequence would form a membrane interactive amphiphilic α-helix. Confirming this prediction, NT1, a peptide homologue of the BYDV-MP N-terminal sequence, was found to be α-helical (65%) in the presence of vesicles mimics of the nuclear membrane. The peptide increased the fluidity of these nuclear membrane mimics (rise in wavenumber of circa 0.5–1.0 cm −1 ) and induced surface pressure changes of 2 mN m −1 in lipid monolayers with corresponding compositions. Taken with isotherm analysis these results suggest that BYDV-MP forms an N-terminal amphiphilic α-helix, which partitions into the nuclear membrane primarily through thermodynamically stable associations with the membrane lipid headgroup region. We speculate that these associations may play a role in targeting of the nuclear membrane by BYDM-MP.

Maximin H5 is an anionic antimicrobial peptide from amphibians, which carries a C-terminal amide moiety, and was found to be moderately haemolytic (20%). The α-helicity of the peptide was 42% in the presence of lipid mimics of erythrocyte membranes and was found able to penetrate (10.8 mN m(-1)) and lyse these model membranes (64 %). In contrast, the deaminated peptide exhibited lower levels of haemolysis (12%) and α-helicity (16%) along with a reduced ability to penetrate (7.8 m Nm(-1)) and lyse (55%) lipid mimics of erythrocyte membranes. Taken with molecular dynamic simulations and theoretical analysis, these data suggest that native maximin H5 primarily exerts its haemolytic action via the formation of an oblique orientated α-helical structure and tilted membrane insertion. However, the C-terminal deamination of maximin H5 induces a loss of tilted α-helical structure, which abolishes the ability of the peptide's N-terminal and C-terminal regions to H-bond and leads to a loss in haemolytic ability. Taken in combination, these observations strongly suggest that the C-terminal amide moiety carried by maximin H5 is required to stabilise the adoption of membrane interactive tilted structure by the peptide. Consistent with previous reports, these data show that the efficacy of interaction and specificity of maximin H5 for membranes can be attenuated by sequence modification and may assist in the development of variants of the peptide with the potential to serve as anti-infectives.

Oblique orientated α-helices possess hydrophobicity gradients, which allow the parent α-helices to penetrate the membrane at a shallow angle, thereby destabilising membrane lipid organisation and promoting a range of biological processes. These α-helices occur in a variety of membrane interactive proteins and a number of techniques have been developed to guide their identification using sequence data alone. Hydrophobicity profiling, which provides a onedimensional analysis of sequence data, identified only 30% of known tilted peptides in a control dataset and was thus of limited predictive use. In contrast, extended hydrophobic moment plot methodology and amphipilic profiling which take residue distribution into account and provide two-dimensional analysis of primary structural data, were found to be good indicators of tilted peptide structure. Amphiphilic profiling identified 67% of tilted peptides in the control dataset and showed that potentially, approximately 40% of transmembrane a-helices possess tilted peptide structure. However, it has been shown that extending these simple methods to take into account the three-dimensional spatial distribution of residues gives no clear additional benefit to identifying tilted peptides.

Cancer is a major cause of premature death and there is an urgent need for new anticancer agents with novel mechanisms of action. Here we review recent studies on a group of peptides that show much promise in this regard, exemplified by arthropod cecropins and amphibian magainins and aureins. These molecules are alpha-helical defence peptides, which show potent anticancer activity (alpha-ACPs) in addition to their established roles as antimicrobial factors and modulators of innate immune systems. Generally, alpha-ACPs exhibit selectivity for cancer and microbial cells primarily due to their elevated levels of negative membrane surface charge as compared to non-cancerous eukaryotic cells. The anticancer activity of alpha-ACPs normally occurs at micromolar levels but is not accompanied by significant levels of haemolysis or toxicity to other mammalian cells. Structure/function studies have established that architectural features of alpha-ACPs such as amphiphilicty levels and hydrophobic arc size are of major importance to the ability of these peptides to invade cancer cell membranes. In the vast majority of cases the mechanisms underlying such killing involves disruption of mitochondrial membrane integrity and/or that of the plasma membrane of the target tumour cells. Moreover, these mechanisms do not appear to proceed via receptor-mediated routes but are thought to be effected in most cases by the carpet/toroidal pore model and variants. Usually, these membrane interactions lead to loss of membrane integrity and cell death utilising apoptic and necrotic pathways. It is concluded that that alpha-ACPs are major contenders in the search for new anticancer drugs, underlined by the fact that a number of these peptides have been patented in this capacity.

Here, it is shown that amphiphilicity profiling based on the mean hydrophobic moment provides a simple visual guide for the identification of oblique orientated a-helices. The methodology has an efficiency of circa 70% and predicts that approximately 40% of transmembrane a-helices may possess these structures.

The replacement of hydrogen with deuterium is frequently used in conjunction with neutron diffraction to investigate peptide-membrane interaction. This isotopic substitution in an amino acid residue radically changes the neutron scatter pattern of the peptide, thereby allowing its localisation within the bilayer with the aid of derived Fourier maps. Nonetheless, this technique relies on the generally held assumption that normal and isotopically enriched protein species do not differ significantly in structure or biological activity. Recently, this assumption has been questioned and here, diffraction data from studies on a membrane interactive peptide clearly challenge the reliability of this assumption.

m-calpain is a calcium-dependent heterodimeric protease implicated in a number of pathological conditions. The activation of m-calpain appears to be modulated by membrane interaction, which has been predicted to involve oblique-orientated alpha-helix formation by a GTAMRILGGVI segment located in domain V of the protein's small subunit. Here, we have investigated this prediction. Fourier transform infrared conformational analysis showed that VP1, a peptide homolog of this segment, exhibited alpha-helicity of approximately 45% in the presence of dimyristoylphosphatidylcholine/dimyristoylphosphatidylserine (DMPS) vesicles. The level of helicity was unaffected over a 1- to 8-mM concentration range and did not alter when the anionic lipid composition of these vesicles was varied between 1% and 10% DMPS. Similar levels of alpha-helicity were observed in trifluoroethanol and the peptide appeared to adopt alpha-helical structure at an air/water interface with a molecular area of 164 A(2) at the monolayer collapse pressure. VP1 was found to penetrate dimyristoylphosphatidylcholine/DMPS monolayers, and at an initial surface pressure of 30 mN m(-1), the peptide induced surface pressure changes in these monolayers that correlated strongly with their anionic lipid content (maximal at 4 mN m(-1) in the presence of 10% DMPS). Neutron diffraction studies showed VP1 to be localized at the hydrophobic core of model palmitoyloleylphosphatidylcholine/palmitoyloleylphosphatidylserine (10:1 molar ratio) bilayer structures and, in combination, these results are consistent with the oblique membrane penetration predicted for the peptide. It would also appear that although not needed for structural stabilization anionic lipid was required for membrane penetration.

To facilitate microbial membrane invasion, amphiphilic α-helical antimicrobial peptides (α-AMPs) show a spatial segregation of hydrophobic and hydrophilic residues about the α-helical long axis. Here we discuss potential mechanisms by which these peptides are able to disrupt membrane structure and the structural characteristics, which are required for function.

A number of DD-peptidases have been reported to interact with the membrane via C-terminal amphiphilic alpha-helices, but experimental support for this rests with a few well-characterized cases. These show the C-terminal interactions of DD-carboxypeptidases to involve high levels of membrane penetration, DD-endopeptidases to involve membrane surface binding and class C penicillin-binding proteins to involve membrane binding with intermediate properties. Here, we have characterized C-terminal alpha-helices from each of these peptidase groups according to their amphiphilicity, as measured by meanmicroH, and the corresponding mean hydrophobicity,H. Regression and statistical analyses showed these properties to exhibit parallel negative linear relationships, which resulted from the spatial ordering of alpha-helix amino acid residues. Taken with the results of compositional and graphical analyses, our results suggest that the use of C-terminal alpha-helices may be a universal feature of the membrane anchoring for each of these groups of DD-peptidases. Moreover, to accommodate differences between these mechanisms, each group of C-terminal alpha-helices optimizes its structural amphiphilicity and hydrophobicity to fulfil its individual membrane-anchoring function. Our results also show that each anchor type analysed requires a similar overall balance between amphiphilicity for membrane interaction, which we propose is necessary to stabilize their initial membrane associations. In addition, we present a methodology for the prediction of C-terminal alpha-helical anchors from the classes of DD-peptidases analysed, based on a parallel linear model.

Escherichia coli low molecular mass penicillin-binding proteins (PBPs) include PBP4, PBP5, PBP6 and PBP6b. Evidence suggests that these proteins interact with the inner membrane via C-terminal amphiphilic α-helices. Nonetheless, the membrane interactive mechanisms utilized by the C-terminal anchors of PBP4 and PBP6b show differences to those utilized by PBP5 and PBP6. Here, hydrophobic moment-based analyses have predicted that, in contrast to the PBP4 and PBP6b C-termini, those of PBP5 and PBP6 are candidates to form oblique orientated α-helices. Consistent with these predictions, Fourier transform infrared spectroscopy (FTIR) has shown that peptide homologs of the PBP4 and PBP5 C-terminal regions, P4 and P5, respectively, both possessed the ability to adopt α-helical structure in the presence of lipid. However, whereas P4 appeared to show a preference for interaction with the surface regions of dimyristoylglycerophosphoethanolamine and dimyristoylglycerophosphoglycerol membranes, P5 appeared to show deep penetration of both these latter membranes and dimyristoylglycerophosphocholine membranes. Based on these results, we have suggested that in contrast to the membrane anchoring of the PBP4 and PBP6b C-terminal α-helices, the PBP5 and PBP6 C-terminal α-helices may possess hydrophobicity gradients and penetrate membranes in an oblique orientation.

The activity of m-calpain, a heterodimeric, Ca2+-dependent cysteine protease appears to be modulated by membrane interactions involving oblique-orientated α-helix formation by a segment, GTAMRILGGVI, in the protein's smaller subunit. Here, graphical and hydrophobic moment-based analyses predicted that this segment may form an α-helix with strong structural resemblance to the influenza virus peptide, HA2, a known oblique-orientated α-helix former. Fourier transform infrared spectroscopy showed that a peptide homologue of the GTAMRILGGVI segment, VP1, adopted low levels of α-helical structure (≈ 20%) in the presence of zwitterionic lipid and induced a minor decrease (3 °C) in the gel to liquid-crystalline phase transition temperature, TC, of the hydrocarbon chains of zwitterionic membranes, suggesting interaction with the lipid headgroup region. In contrast, VP1 adopted high levels of α-helical structure (65%) in the presence of anionic lipid, induced a large increase (10 °C) in the TC of anionic membranes, and showed high levels of anionic lipid monolayer penetration (ΔSP = 5.5 mN·m−1), suggesting deep levels of membrane penetration. VP1 showed strong haemolytic ability (LD50 = 1.45 mm), but in the presence of ionic agents, this ability, and that of VP1 to penetrate anionic lipid monolayers, was greatly reduced. In combination, our results suggest that m-calpain domain V may penetrate membranes via the adoption of an oblique-orientated α-helix and electrostatic interactions. We speculate that these interactions may involve snorkelling by an arginine residue located in the polar face of this α-helix.

Amphiphilic alpha-helices play a major role in membrane dependent processes and are manifested in the primary structure of a protein by the periodic appearance of hydrophobic residues. Based on these periodic sequences, the hydrophobic moment was introduced,microH, which essentially treats the hydrophobicity of amino acid residues as a two-dimensional vector sum and provides a measure of amphiphilicity within regular repeat structures. To identify putative amphiphilic alpha-helix forming sequences, hydrophobic moment analysis assumes an amino acid residue periodicity of 100 and scans protein primary structures to find the 11-residue window with maximalmicroH. Taken with the window's mean hydrophobicity,H0, hydrophobic moment plot analysis uses the coordinate pair, [microH,H0] to classify alpha-helices as either surface active, globular or transmembrane. More recently, this latter analysis has been extended to recognize candidate oblique orientated alpha-helices. Here, the hydrophobic moment is reviewed and data to query the logic of using a fixed window length and a fixed residue angular periodicity in hydrophobic moment analysis are provided. In addition, problems associated with the use of such analysis to predict alpha-helix structure/function relationships are considered.

The ability of α-helical antimicrobial peptides to kill microorganisms depends upon their ability to interact with the membranes of these organisms. To be effective, such peptides require the ability to exhibit considerable discrimination between target and host cells. Key factors in selectivity and efficiency of action include differences between the phospholipid and sterol composition of the target membrane and host cell membrane. In this chapter, we present an overview of the Langmuir–Blodgett monolayers technique, which provides a versatile system for studying the interfacial properties of peptides and peptide–membrane interactions under controlled conditions. This system can provide information on the surface activity of peptides such as antimicrobial peptides and provide insights into their biological function at a membrane interface.

Oblique orientated α-helices are highly specialised protein structural elements that penetrate membranes at a shallow angle and are used to promote membrane destabilisation by a number of protein classes. Here, the use of extended hydrophobic moment methodology shows that the amphibian extrudates, aurein 1.2 and citropin 1.1, may use oblique orientated α-helices in their antimicrobial action and that such use may be shared by other antimicrobial peptides. This appears to be the first systematic analysis of these peptides for the possession of oblique orientated a-helical structure.

Theoretical analysis indicates that peptide VP1 forms a membrane interactive amphiphilic alpha-helix with antibacterial properties. Fourier transform infra-red based analyses showed VP1 to be alpha-helical (45%) in the presence of vesicle mimics of membranes from Staphylococcus aureus and to induce increases in the fluidity of these vesicles, as indicated by a rise in wavenumber of circa 0.5 to 1.0 cm(-1). The peptide induced surface pressure increases of 5 mN m(-1) in monolayer mimics of S. aureus membranes confirm the formation of a membrane interactive alpha-helix. These interactions appeared to involve significant hydrophobic and electrostatic contributions as VP1 induced comparable surface pressure changes in anionic (5.5 mN m(-1)) and zwitterionic (4 mN m(-1)) lipid monolayers. It is suggested that whilst efficacy requires further sequence specific information, the peptides generic structure provides the basis for its broad antimicrobial activity.

Here, the interactions of aurein 1.2, a defence peptide, with T98G glioblastoma cell membranes are studied. The peptide induced maximal surface pressure changes of circa 9 mN m(-1) in monolayers of endogenous T98G membrane lipid. Reducing monolayer anionic lipid showed a positive correlation (R(2)>0.91) with decreases in maximal surface pressure changes induced by aurein 1.2 (circa 3 mN m(-1) in the absence of this lipid). Cancer cell membrane invasion by the peptide therefore appears not to be mediated by lipid receptors or specific lipid requirements but rather a general requirement for anionic lipid and/or other negatively charged membrane components.

AP1 (GEQGALAQFGEWL) was shown by theoretical analysis to be an anionic oblique-orientated alpha-helix former. The peptide exhibited a monolayer surface area of 1.42 nm(2), implying possession of alpha-helical structure at an air/water interface, and Fourier transform infrared spectroscopy (FTIR) showed the peptide to be alpha-helical (100%) in the presence of vesicle mimics of Escherichia coli membranes. FTIR lipid-phase transition analysis showed the peptide to induce large decreases in the fluidity of these E. coli membrane mimics, and Langmuir-Blodgett trough analysis found the peptide to induce large surface pressure changes in monolayer mimics of E. coli membranes (4.6 mN.m(-1)). Analysis of compression isotherms based on mixing enthalpy (DeltaH) and the Gibbs free energy of mixing (DeltaG(Mix)) predicted that these monolayers were thermodynamically stable (DeltaH and DeltaG(Mix) each negative) but were destabilized by the presence of the peptide (DeltaH and DeltaG(Mix) each positive). The peptide was found to have a minimum lethal concentration of 3 mm against E. coli and was seen to cause lysis of erythrocytes at 5 mm. In combination, these data clearly show that AP1 functions as an anionic alpha-helical antimicrobial peptide and suggest that both its tilted peptide characteristics and the composition of its target membrane are important determinants of its efficacy of action.

The antimicrobial activity of the anionic peptide, AP1 (GEQGALAQFGEWL), was investigated. AP1 was found to kill Staphylococcus aureus with an MLC of 3mM and to induce maximal surface pressure changes of 3.8 mN m(-1) over 1200s in monolayers formed from lipid extract of S. aureus membranes. FTIR spectroscopy showed the peptide to be alpha-helical (100%) in the presence of vesicles formed from this lipid extract and to induce increases in their fluidity (Deltanu circa 0.5 cm(-1)). These combined data show that AP1 is able to function as an alpha-helical antimicrobial peptide against Gram-positive bacteria and suggest that the killing mechanism used by the peptide involves interactions with the membrane lipid headgroup region. Moreover, this killing mechanism differs strongly from that previously reported for AP1 against Gram-negative bacteria, indicating the importance of considering the effects of membrane lipid composition when investigating the structure/function relationships of antimicrobial peptides.

The ability of phenothiazinium-based photosensitizers to induce photodamage to Escherichia coli membranes is investigated. Phenothiazinium-based photosensitizers were found to be somewhat lipophilic (log P>0.7) and to induce surface-pressure changes (3-12 mN m(-1)) in lipid monolayers mimetic of bacterial membranes, implying that these molecules are able to penetrate biological membranes. Under dark and light conditions (3.15 J cm(-1) for 30 min), phenothiazinium-based photosensitizers were incubated with E. coli cells. These cells showed levels of dark bacteriolysis that ranged between 6% and 13%, with light conditions leading to no significant increase in these levels. Gas chromatography-based analyses showed such incubations to produce no significant changes in the levels of C(16) and C(18) fatty acid chain saturation found in E. coli whole lipid-extracts. It is concluded that the phenothiazinium-based photosensitizers studied may not use E. coli membranes as their primary photodynamic target, but may inflict photodamage on cytoplasmic targets, possibly DNA.

A key question associated with topology predictions for membrane proteins is whether there is sufficient variation in the biophysical properties of residues at the membrane interface to enable identification of TM spans in a robust and efficient manner using relatively simple methods of analysis. Here, a test for the homogeneity of multinomial populations is used to identify statistical differences between the residue compositions of windows within datasets of aligned non-homologous TM alpha-helices. Using this approach, the accuracy and robustness of the predicted boundaries for datasets of uncleaved signal (US) sequences and stop transfer sequences (ST) is tested. The validity of the 21 residue length, which is generally assumed for TM spans in membrane protein topology prediction is also investigated and it is suggested that ST sequences may be better represented by a length of 22 residues.

Escherichia coli penicillin-binding protein 5 (PBP5) anchors to the inner membrane in a pH-dependent manner via a C-terminal amphiphilic alpha-helix. Low pH was found to enhance both levels of PBP5 membrane anchoring and levels of alpha-helicity in an aqueous PBP5 C-terminal homologue, which led to the suggestion that levels of PBP5 membrane anchoring are related to levels of PBP5 C-terminal alpha-helicity. Here we have used Fourier-transformed infrared spectroscopy (FTIR) and a peptide homologue of the PBP5 C-terminal sequence to investigate the effect of pH on the conformational behavior of this sequence at a lipid interface and on its ability to interact with lipid. Our results suggest that the membrane-anchoring mechanism of PBP5 is unlikely to involve conformational change in the protein's C-terminal region and may therefore involve conformational changes in the protein's ectomembranous domain.

The Escherichia coli low molecular mass penicillin-binding proteins PBP4, PBP5 and PBP6 are DD-peptidases involved in murein biosynthesis. It has been suggested that these proteins may be anchored to the periplasmic face of the inner membrane via their C termini. Here, peptide homologues (P4, P5 and P6) of the PBP4, PBP5 and PBP5 C-terminal regions have been used to investigate potential protein-lipid interactions involved in this anchoring mechanism. Surface pressure changes observed for the interactions of P5 and P6 with a range of monolayers indicated that the peptides are membrane interactive and that the interactions proceeded via predominantly hydrophobic forces with only minor requirements for anionic lipid. In contrast, P4 interactions with monolayers appeared to proceed via predominantly electrostatic forces with a major requirement for anionic lipid. The lipid interactions of all three peptides were generally enhanced by low pH and for P5 and P6 were in the range of 10–15 mN m −1 whereas for P4 interactions they were in the range of 3–7 mN m −1 . CD analysis implied the presence of α-helical structure in P5 and P6 and molecular area determinations implied that P4 may also possess helical architecture in the presence of dioleoylphosphatidylglycerol monolayers. Overall, our results support the view that C-terminal amphiphilic α-helices are involved in the membrane anchoring of PBP5 and PBP6 and suggest that a similar mechanism could contribute to PBP4-membrane anchoring. Furthermore, we have speculated that the presence of cationic residues in the hydrophilic face of these α-helices may help facilitate membrane interaction.

Escherichia coli penicillin-binding protein 5 (PBP5) is anchored to the periplasmic face of the inner membrane via a C-terminal amphiphilic alpha-helix. The results of washing experiments have suggested an electrostatic contribution to the anchoring mechanism which may involve the cationic region of the C-terminal alpha-helix. Similarities between this anchor domain and some surface active agents, such as melittin, suggest that the cationic region of the PBP5 anchor may require the presence of anionic phospholipids for membrane interaction. Washing experiments performed on membranes of HDL11, an E. coli mutant in which the expression of the major anionic phospholipids is under lac control, found no such requirement. The results are discussed in relation to the hypothesis that the cationic region may interact with other sources of negative charge, possibly arising from a PBP complex.