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BBA - Biomembranes (v.1758, #9)

Editorial Board (pp. ii).

Preface by Ayyalusamy Ramamoorthy (pp. 1177-1177).

Membrane interactions of antimicrobial peptides from Australian tree frogs by Martin P. Boland; Frances Separovic (pp. 1178-1183).

The skin secretions of amphibians are rich in host defence peptides. The membrane interactions of the antimicrobial peptides, aurein 1.2, citropin 1.1 and maculatin 1.1, isolated from Australian tree frogs, are reviewed. Although all three peptides are amphipathic α-helices, the mode of action of these membrane-active peptides is not defined. The peptides have a net positive charge and range in length from 13 to 21 residues, with the longest, maculatin 1.1, having a proline at position 15. Interestingly, alanine substitution at Pro-15 leads to loss of activity. The effects of these peptides on phospholipid bilayers indicate different mechanisms for pore formation and lysis of model membranes, with the shorter peptides exhibiting a carpet-like mechanism and the longest peptide forming pores in phospholipid bilayer membranes.

Keywords: Peptide–lipid interactions; Model membranes; Antibacterial peptides; Solid-state NMR; Pore formation

Tryptophan- and arginine-rich antimicrobial peptides: Structures and mechanisms of action by David I. Chan; Elmar J. Prenner; Hans J. Vogel (pp. 1184-1202).

Antimicrobial peptides encompass a number of different classes, including those that are rich in a particular amino acid. An important subset are peptides rich in Arg and Trp residues, such as indolicidin and tritrpticin, that have broad and potent antimicrobial activity. The importance of these two amino acids for antimicrobial activity was highlighted through the screening of a complete combinatorial library of hexapeptides. These residues possess some crucial chemical properties that make them suitable components of antimicrobial peptides. Trp has a distinct preference for the interfacial region of lipid bilayers, while Arg residues endow the peptides with cationic charges and hydrogen bonding properties necessary for interaction with the abundant anionic components of bacterial membranes. In combination, these two residues are capable of participating in cation–π interactions, thereby facilitating enhanced peptide–membrane interactions. Trp sidechains are also implicated in peptide and protein folding in aqueous solution, where they contribute by maintaining native and nonnative hydrophobic contacts. This has been observed for the antimicrobial peptide from human lactoferrin, possibly restraining the peptide structure in a suitable conformation to interact with the bacterial membrane. These unique properties make the Arg- and Trp-rich antimicrobial peptides highly active even at very short peptide lengths. Moreover, they lead to structures for membrane-mimetic bound peptides that go far beyond regular α-helices and β-sheet structures. In this review, the structures of a number of different Trp- and Arg-rich antimicrobial peptides are examined and some of the major mechanistic studies are presented.

Keywords: Abbreviations; BLM; black lipid membranes; CD; circular dichroism; cLZ; human c-type lysozyme; combi-1 (or-2); combinatorial peptide-1 (or-2); CPP; cell-permeable peptide; DiPoPE; dipalmitoleoyl PE; DOPC (or DOPE or DOPG); 1,2-dioleoyl-; sn; -glycero-3-PC (or PE, PG); DPC; dodecylphosphocholine; DPPC (or DPPE or DPPG); 1,2-dipalmitoyl-; sn; -glycero-3-PC (or PE or PG); DSC; differential scanning calorimetry; HEWL; hen egg white lysozyme; hLZ; human lysozyme; IC; ₅₀; inhibitory concentration at which 50% inhibition is achieved; ITC; isothermal titration calorimetry; Lfcin; lactoferricin; LfcinB; bovine Lfcin; LPS; lipopolysaccharide; LUV; large unilamellar vesicle; LysC; HEWL peptide, residues 98–112 in HEWL; LysH; residues 107–113 in hLZ; MD; molecular dynamics; MIC; minimal inhibitory concentration; MIP-3α; macrophage inflammatory protein-3α; MRSA; methicillin resistant; Staphylococcus aureus; NMR; nuclear magnetic resonance; NOE; nuclear Overhauser effect; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PG; phosphatidylglycerol; PDB; Protein Data Bank; PIN-a (or-b); puroindoline A (or B); puroA (or B); puroindoline A (or B) peptide; SDS; sodium dodecyl sulfate; SMH; Shai–Matsuzaki–Huang; VRE; vancomycin resistant enterococciAntimicrobial peptide; Structure; Tryptophan; Arginine; Cation–π interaction

NMR studies of aurein 1.2 analogs by Xia Li; Yifeng Li; Alan Peterkofsky; Guangshun Wang (pp. 1203-1214).

Aurein 1.2 is an antimicrobial and anticancer peptide isolated from an Australian frog. To improve our understanding of the mechanism of action, two series of peptides were designed. The first series includes the N-terminal membrane anchor of bacterial glucose-specific enzyme IIA, aurein 1.2, and a newly identified aurein 1.2 analog from human LL-37 (LLAA). The order of antibacterial activity is LLAA>aurein 1.2>>the membrane anchor (inactive). The structure of LLAA in detergent micelles was determined by¹H NMR spectroscopy, including structural refinement by natural abundance¹³C^α,¹³C^β, and¹⁵N chemical shifts. The hydrophobic surface area of the 3D structure is related to the retention time of the peptide on a reverse-phase HPLC column. The higher activity of LLAA compared to aurein 1.2 was attributed to additional cationic residues that enhance the membrane perturbation potential. The second peptide series was created by changing the C-terminal phenylalanine (F13) of aurein 1.2 to either phenylglycine or tryptophan. A closer or further location of the aromatic rings to the peptide backbone in the mutants relative to F13 is proposed to cause a drop in activity. Phenylglycine with unique chemical shifts may be a useful NMR probe for structure–activity relationship studies of antimicrobial peptides. To facilitate potential future use for NMR studies, random-coil chemical shifts for phenylglycine (X) were measured using the synthetic peptide GGXGG. Aromatic rings of phenylalanines in all the peptides penetrated 2–5 Å below the lipid head group and are essential for membrane targeting as illustrated by intermolecular peptide–lipid NOE patterns.

Keywords: Abbreviations; NMR; nuclear magnetic resonance; APD; the antimicrobial peptide database; DHPG; dihexanoylphosphatidylglycerol; D8PG; dioctanoyl phosphatidylglycerol; D10PG; didecanoyl phosphatidylglycerol; DQF-COSY; double-quantum filtered correlation spectroscopy; DSS; 2,2-dimethyl-silapentane-5-sulfonate sodium salt; HSQC; heteronuclear single quantum coherence; LLAA; the LL-37-derived aurein 1.2 analog; MPP; membrane perturbation potential; NOE; nuclear Overhauser effect; NOESY; nuclear Overhauser enhancement spectroscopy; PGs; phosphatidylglycerols; rmsd; root mean square deviation; SDS; sodium dodecyl sulfate; TOCSY; total correlation spectroscopyAntimicrobial peptides; Aurein 1.2; LL-37; Peptide–lipid interaction; Phenylglycine; Random-coil chemical shifts

Mode of action of the new antibiotic for Gram-positive pathogens daptomycin: Comparison with cationic antimicrobial peptides and lipopeptides by Suzana K. Straus; Robert E.W. Hancock (pp. 1215-1223).

With the steady rise in the number of antibiotic-resistant Gram-positive pathogens, it has become increasingly important to find new antibacterial agents which are highly active and have novel and diversified mechanisms of action. Two classes will be discussed here: the cationic antimicrobial peptides, which are amphiphilic in nature, targeting membranes and increasing their permeability; and lipopeptides, which consist of linear or cyclic peptides with an N-terminus that is acylated with a fatty acid side chain. One member of the cyclic lipopeptide family, the anionic molecule daptomycin, has been extensively studied and is the major focus of this review. Models will be presented on its mode of action and comparisons will be made to the known modes of action of cationic antimicrobial peptides and other lipopeptides.

Keywords: Daptomycin; Lipopeptide; Cationic peptide; Gram-positive pathogen; Mechanism of action

Driving engineering of novel antimicrobial peptides from simulations of peptide–micelle interactions by Himanshu Khandelia; Allison A. Langham; Yiannis N. Kaznessis (pp. 1224-1234).

Simulations of antimicrobial peptides in membrane mimics can provide the high resolution, atomistic picture that is necessary to decipher which sequence and structure components are responsible for activity and toxicity. With such detailed insight, engineering new sequences that are active but non-toxic can, in principle, be rationalized. Armed with supercomputers and accurate force fields for biomolecular interactions, we can now investigate phenomena that span hundreds of nanoseconds. Although the phenomena involved in antimicrobial activity, (i.e., diffusion of peptides, interaction with lipid layers, secondary structure attainment, possible surface aggregation, possible formation of pores, and destruction of the lipid layer integrity) collectively span time scales still prohibitively long for classical mechanics simulations, it is now feasible to investigate the initial approach of single peptides and their interaction with membrane mimics. In this article, we discuss the promise and the challenges of widely used models and detail our recent work on peptide–micelle simulations as an attractive alternative to peptide–bilayer simulations. We detail our results with two large structural classes of peptides, helical and beta-sheet and demonstrate how simulations can assist in engineering of novel antimicrobials with therapeutic potential.

Keywords: Antimicrobial peptide; Peptide–micelle simulation; Molecular dynamics; Mechanism of action

Biophysical studies of the interactions between 14-mer and 21-mer model amphipathic peptides and membranes: Insights on their modes of action by Marise Ouellet; François Otis; Normand Voyer; Michèle Auger (pp. 1235-1244).

We have investigated the interactions between synthetic amphipathic peptides and zwitterionic model membranes. Peptides with 14 and 21 amino acids composed of leucines and phenylalanines modified by the addition of crown ethers have been synthesized. The 14-mer and 21-mer peptides both possess a helical amphipathic structure as revealed by circular dichroism. To shed light on their mechanism of membrane interaction, different complementary biophysical techniques have been used such as circular dichroism, fluorescence, membrane conductivity measurement and NMR spectroscopy. Results obtained by these different techniques show that the 14-mer peptide is a membrane perturbator that facilitate the leakage of species such as calcein and Na ions, while the 21-mer peptide acts as an ion channel.³¹P solid-state NMR experiments on multilamellar vesicles reveal that the dynamics and/or orientation of the polar headgroups are greatly affected by the presence of the peptides. Similar results have also been obtained in mechanically oriented DLPC and DMPC bilayers where different acyl chain lengths seem to play a role in the interaction. On the other hand,²H NMR experiments on multilamellar vesicles demonstrate that the acyl chain order is affected differently by the two peptides. Based on these studies, mechanisms of action are proposed for the 14-mer and 21-mer peptides with zwitterionic membranes.

Keywords: Synthetic amphipathic peptide; Model membrane; NMR spectroscopy; Circular dichroism; Single-channel conductivity; Fluorescence

Peptide–membrane interactions and mechanisms of membrane destruction by amphipathic α-helical antimicrobial peptides by Hiromi Sato; Jimmy B. Feix (pp. 1245-1256).

Antimicrobial peptides (AMPs) have received considerable interest as a source of new antibiotics with the potential for treatment of multiple-drug resistant infections. An important class of AMPs is composed of linear, cationic peptides that form amphipathic α-helices. Among the most potent of these are the cecropins and synthetic peptides that are hybrids of cecropin and the bee venom peptide, mellitin. Both cecropins and cecropin-mellitin hybrids exist in solution as unstructured monomers, folding into predominantly α-helical structures upon membrane binding with their long helical axis parallel to the bilayer surface. Studies using model membranes have shown that these peptides intercalate into the lipid bilayer just below the level of the phospholipid glycerol backbone in a location that requires expansion of the outer leaflet of the bilayer, and evidence from a variety of experimental approaches indicates that expansion and thinning of the bilayer are common characteristics during the early stages of antimicrobial peptide–membrane interactions. Subsequent disruption of the membrane permeability barrier may occur by a variety of mechanisms, leading ultimately to loss of cytoplasmic membrane integrity and cell death.

Keywords: Antimicrobial peptide; Cecropin; Mellitin; Magainin; Spin label; EPR

SFG studies on interactions between antimicrobial peptides and supported lipid bilayers by Xiaoyun Chen; Zhan Chen (pp. 1257-1273).

The mode of action of antimicrobial peptides (AMPs) in disrupting cell membrane bilayers is of fundamental importance in understanding the efficiency of different AMPs, which is crucial to design antibiotics with improved properties. Recent developments in the field of sum frequency generation (SFG) vibrational spectroscopy have made it a powerful and unique biophysical technique in investigating the interactions between AMPs and a single substrate supported planar lipid bilayer. We will review some of the recent progress in applying SFG to study membrane lipid bilayers and discuss how SFG can provide novel information such as real-time bilayer structure change and AMP orientation during AMP–lipid bilayer interactions in a very biologically relevant manner. Several examples of applying SFG to monitor such interactions between AMPs and a dipalmitoyl phosphatidylglycerol (DPPG) bilayer are presented. Different modes of actions are observed for melittin, tachyplesin I,d-magainin 2, MSI-843, and a synthetic antibacterial oligomer, demonstrating that SFG is very effective in the study of AMPs and AMP–lipid bilayer interactions.

Keywords: SFG; Lipid bilayer; Antimicrobial peptide; Melittin; Magainin; Tachyplesin

Effect of salt on the interactions of antimicrobial peptides with zwitterionic lipid bilayers by Senthil K. Kandasamy; Ronald G. Larson (pp. 1274-1284).

The effect of salt on the binding of the antimicrobial peptide magainin to POPC lipid bilayers is studied by 40–50 ns molecular dynamics simulations of a POPC bilayer in the presence of different concentrations of Na+ and Cl− ions, corresponding to effective concentrations of 0, 100, 150, 200, 250 and 300 millimolar NaCl, with and without a single molecule of antimicrobial peptide magainin. Simulations without magainin showed that increasing salt concentration leads to the decrease in the area per lipid, a decrease in the head group tilt of the lipids, as well as increased order of lipid tails, in agreement with other recent simulations. Simulations with magainin show that peptide binding to the lipids is stronger at lower concentrations of salt. The peptides disorder the lipids in their immediate vicinity, but this effect is diminished as the salt concentration increases. Our studies indicate that while 50 ns simulations give information on peptide hydrogen bonding and lipid tail ordering that is insensitive to the initial peptide orientation, this run time is not sufficient to equilibrate the peptide position and orientation within the bilayer.

Keywords: Magainin; Molecular dynamics simulation; Salt; POPC; Antimicrobial peptide

Peptide–lipid interactions of the β-hairpin antimicrobial peptide tachyplesin and its linear derivatives from solid-state NMR by Timothy Doherty; Alan J. Waring; Mei Hong (pp. 1285-1291).

The peptide–lipid interaction of a β-hairpin antimicrobial peptide tachyplesin-1 (TP-1) and its linear derivatives are investigated to gain insight into the mechanism of antimicrobial activity.³¹P and²H NMR spectra of uniaxially aligned lipid bilayers of varying compositions and peptide concentrations are measured to determine the peptide-induced orientational disorder and the selectivity of membrane disruption by tachyplesin. The disulfide-linked TP-1 does not cause any disorder to the neutral POPC and POPC/cholesterol membranes but induces both micellization and random orientation distribution to the anionic POPE/POPG membranes above a peptide concentration of 2%. In comparison, the anionic POPC/POPG bilayer is completely unaffected by TP-1 binding, suggesting that TP-1 induces negative curvature strain to the membrane as a mechanism of its action. Removal of the disulfide bonds by substitution of Cys residues with Tyr and Ala abolishes the micellization of POPE/POPG bilayers but retains the orientation randomization of both POPC/POPG and POPE/POPG bilayers. Thus, linear tachyplesin derivatives have membrane disruptive abilities but use different mechanisms from the wild-type peptide. The different lipid–peptide interactions between TP-1 and other β-hairpin antimicrobial peptides are discussed in terms of their molecular structure.

Keywords: Abbreviations; TP-1; tachyplesin; PG-1; protegrin-1; POPE; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylethanolamine; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycerol-3-phosphatidylcholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycerol-3-phosphatidylglycerol; PA; phosphatidic acidAntimicrobial peptide; Tachyplesin; β-hairpin; Membrane disruption; Lipid bilayer; Solid-state NMR

Molecular mechanism of antimicrobial peptides: The origin of cooperativity by Huey W. Huang (pp. 1292-1302).

Based on very extensive studies on four peptides (alamethicin, melittin, magainin and protegrin), we propose a mechanism to explain the cooperativity exhibited by the activities of antimicrobial peptides, namely, a non-linear concentration dependence characterized by a threshold and a rapid rise to saturation as the concentration exceeds the threshold. We first review the structural basis of the mechanism. Experiments showed that peptide binding to lipid bilayers creates two distinct states depending on the bound-peptide to lipid ratio P/L. For P/L below a threshold P/L*, all of the peptide molecules are in the S state that has the following characteristics: (1) there are no pores in the membrane, (2) the axes of helical peptides are oriented parallel to the plane of membrane, and (3) the peptide causes membrane thinning in proportion to P/L. As P/L increases above P/L*, essentially all of the excessive peptide molecules occupy the I state that has the following characteristics: (1) transmembrane pores are detected in the membrane, (2) the axes of helical peptides are perpendicular to the plane of membrane, (3) the membrane thickness remains constant for P/L≥ P/L*. The free energy based on these two states agrees with the data quantitatively. The free energy also explains why lipids of positive curvature (lysoPC) facilitate and lipids of negative curvature (PE) inhibit pore formation.

Keywords: Threshold peptide concentration; Cooperative concentration dependence; Membrane thinning effect; Neutron in-plane scattering; Oriented circular dichroism; Two-state model

Exploring membrane selectivity of the antimicrobial peptide KIGAKI using solid-state NMR spectroscopy by Jun-xia Lu; Jack Blazyk; Gary A. Lorigan (pp. 1303-1313).

The designed antimicrobial peptide KIGAKIKIGAKIKIGAKI possesses enhanced membrane selectivity for bacterial lipids, such as phosphatidylethanolamine and phosphatidylglycerol. The perturbation of the bilayer by the peptide was first monitored using oriented bilayer samples on glass plates. The alignment of POPE/POPG model membranes with respect to the bilayer normal was severely altered at 4 mol% KIGAKI while the alignment of POPC bilayers was retained. The interaction mechanism between the peptide and POPE/POPG bilayers was investigated by carefully comparing three bilayer MLV samples (POPE bilayers, POPG bilayers, and POPE/POPG 4/1 bilayers). KIGAKI induces the formation of an isotropic phase for POPE/POPG bilayers, but only a slight change in the³¹P NMR CSA line shape for both POPE and POPG bilayers, indicating the synergistic roles of POPE and POPG lipids in the disruption of the membrane structure by KIGAKI.²H NMR powder spectra show no reduction of the lipid chain order for both POPG and POPE/POPG bilayers upon peptide incorporation, supporting the evidence that the peptide acts as a surface peptide.³¹P longitudinal relaxation studies confirmed that different dynamic changes occurred upon interaction of the peptide with the three different lipid bilayers, indicating that the strong electrostatic interaction between the cationic peptide KIGAKI and anionic POPG lipids is not the only factor in determining the antimicrobial activity. Furthermore,³¹P and²H NMR powder spectra demonstrated a change in membrane characteristics upon mixing of POPE and POPG lipids. The interaction between different lipids, such as POPE and POPG, in the mixed bilayers may provide the molecular basis for the KIGAKI carpet mechanism in the permeation of the membrane.

Keywords: Abbreviations; KIGAKI; KIGAKIKIGAKIKIGAKI-NH; ₂; POPC; 1-Palmitoyl-2-oleoyl-3-; sn; -glycero-3-phosphocholine; POPG; 1-Palmitoyl-2-oleoyl-3-; sn; -glycero-3-[phospho-rac-1-glycerol]; POPE; 1-Palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoethanolamine; POPC-d; ₃₁; 1-Palmitoyl-d; ₃₁; -2-oleoyl-3-; sn; -glycero-3-phosphocholine; POPG-d; ₃₁; 1-Palmitoyl-d; ₃₁; -2-oleoyl-3-; sn; -glycero-3-[phospho-rac-1-glycerol]; HEPES; N-[2-hydroxyethyl] piperazine-; N′; -[2-ethanesulfonic acid]; TFE; 2,2,2, trifluoroethanol; EDTA; ethylenediamine tetraacetic acid; Fmoc; N-(9-fluorenyl)methoxycarbonyl; HPLC; high performance liquid chromatography; CP; cross-polarization; MAS; magic angle spinning; MLVs; multilamellar vesicles; LUVs; large unilamellar vesicles; T; ₁; longitudinal relaxation timeKIGAKI; Antimicrobial activity; Membrane selectivity; Electrostatic interaction; Lipid/lipid interaction; Carpet mechanism

Peptide antibiotics in action: Investigation of polypeptide chains in insoluble environments by rotational-echo double resonance by Orsolya Toke; Lynette Cegelski; Jacob Schaefer (pp. 1314-1329).

Rotational-echo double resonance (REDOR) is a solid-state NMR technique that has the capability of providing intra- and intermolecular distance and orientational restraints in non-crystallizable, poorly soluble heterogeneous molecular systems such as cell membranes and cell walls. In this review, we will present two applications of REDOR: the investigation of a magainin-related antimicrobial peptide in lipid bilayers and the study of a vancomycin-like glycopeptide in the cell walls of Staphylococcus aureus.

Keywords: Abbreviations; antimicrobial peptide; AMP; dimyristoylphosphatidylglycerol; DMPG; dipalmitoylphosphatidylcholine; DPPC; dipalmitoylphosphatidylglycerol; DPPG; fluorophenylbenzyl-vancomycin; FPBV; KIAGKIAKIAGKIAKIAGKIA; K3; lipid-to-lipid molar ratio; L/P; magic angle spinning; MAS; minimal inhibitory concentration; MIC; nuclear magnetic resonance; NMR; polarization inversion spin exchange at the magic angle; PISEMA; radiofrequency; RF; rotational-echo double resonance; REDOR; separated local field; SLP; vancomycin-resistant enterococci; VRE; vancomycin-resistant; Staphylococcus aureus; VRSA; transferred echo double resonance; TEDORHost-defense peptides; Ion-channels; Membrane pores; Vancomycin; Oritavancin; Cell wall; Solid-state NMR; Dipolar recoupling

Conditions affecting the re-alignment of the antimicrobial peptide PGLa in membranes as monitored by solid state²H-NMR by Pierre Tremouilhac; Erik Strandberg; Parvesh Wadhwani; Anne S. Ulrich (pp. 1330-1342).

The cationic antimicrobial peptide PGLa is electrostatically attracted to bacterial membranes, binds as an amphiphilic α-helix, and is thus able to permeabilize the lipid bilayer. Using solid state²H-NMR of non-perturbing Ala-d₃ labels on the peptide, we have characterized the helix alignment under a range of different conditions. Even at a very high peptide-to-lipid ratio (1:20) and in the presence of negatively charged lipids, there was no indication of a toroidal wormhole structure. Instead, PGLa re-aligns from a surface-bound S-state to an obliquely tilted T-state, which is presumably dimeric. An intermediate structure half-way between the S- and T-state was observed in fully hydrated multilamellar DMPC vesicles at 1:50, suggesting a fast exchange between the two states on the time scale of >50 kHz. We demonstrate that this equilibrium is shifted from the S- towards the T-state either upon (i) increasing the peptide concentration, (ii) adding negatively charged DMPG, or (iii) decreasing the level of hydration. The threshold concentration for re-alignment in DMPC is found to be between 1:200 and 1:100 in oriented samples at 96% humidity. In fully hydrated multilamellar DMPC vesicles, it shifts to an effective peptide-to-lipid ratio of 1:50 as some peptides are able to escape into the bulk water phase.

Keywords: Abbreviations; Ala-d; ₃; 3,3,3-; ²; H; ₃; -; l; -alanine; CD; circular dichroism; DMPC; 1-,2-dimyristoyl-; sn; -glycero-3-phosphatidylcholine; DMPG; 1-,2-dimyristoyl-; sn; -glycero-3-phosphatidylglycerol; MALDI-TOF; matrix assisted laser desorption ionization-time of flight; MLV; multilamellar vesicles; NMR; nuclear magnetic resonance; OCD; oriented circular dichroism; OS; oriented sample; PC; phosphatidylcholine; PG; phosphatidylglycerol; PGLa; peptidyl–glycine–leucine–carboxyamide; P/L; peptide-to-lipid molar ratio; RMSD; root mean square deviation; S; _mol; molecular order parameter; Δν; _Q; quadrupole splitting; �?; peptide azimuthal rotation angle; τ; peptide tilt angleα-helical peptide; Magainin family antimicrobial peptide; Solid-state; ²; H-NMR; Alanine-d; ₃; isotope label; Oriented membrane sample; Dimerization equilibrium; Lipid hydration; DMPC and DMPG

Role of membrane lipids in the mechanism of bacterial species selective toxicity by two α/β-antimicrobial peptides by Raquel F. Epand; Margaret A. Schmitt; Samuel H. Gellman; Richard M. Epand (pp. 1343-1350).

We have previously shown that two synthetic antimicrobial peptides with alternating α- and β-amino acid residues, designated simply as α/β-peptide I and α/β-peptide II, had toxicity toward bacteria and affected the morphology of bacterial membranes in a manner that correlated with their effects on liposomes with lipid composition similar to those of the bacteria. In the present study we account for the weak effects of α/β-peptide I on liposomes or bacteria whose membranes are enriched in phosphatidylethanolamine (PE) and why such membranes are particularly susceptible to damage by α/β-peptide II. The α/β-peptide II has marked effects on unilamellar vesicles enriched in PE causing vesicle aggregation and loss of their internal aqueous contents. The molecular basis of these effects is the ability of α/β-peptide II to induce phase segregation of anionic and zwitterionic lipids as shown by fluorescence and differential scanning calorimetry. This phase separation could result in the formation of defects through which polar materials could pass across the membrane as well as form a PE-rich membrane domain that would not be a stable bilayer. α/β-Peptide II is more effective in this regard because, unlike α/β-peptide I, it has a string of two or three adjacent cationic residues that can interact with anionic lipids. Although α/β-peptide I can destroy membrane barriers by converting lamellar to non-lamellar structures, it does so only weakly with unilamellar vesicles or with bacteria because it is not as efficient in the aggregation of these membranes leading to the bilayer–bilayer contacts required for this phase conversion. This study provides further understanding of why α/β-peptide II is more toxic to micro-organisms with a high PE content in their membrane as well as for the lack of toxicity of α/β-peptide I with these cells, emphasizing the potential importance of the lipid composition of the cell surface in determining selective toxicity of anti-microbial agents.

Keywords: Abbreviations; LUV; large unilamellar vesicle; MLV; multilamellar vesicles; DO; dioleoyl; DP; dipalmitoyl; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PG; phosphatidylglycerol; CL; tetraoleoyl cardiolipin; L/P; lipid to α/β-peptide molar ratio; C6-NBD-PE; 1-Myristoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-; sn; -Glycero-3-Phosphoethanolamine; C6-NBD-PC; 1-myristoyl-2[6-(7-nitro-2,1,3-benzoxadiazol-4-yl) )amino)caproyl]; sn; -glycero-3-phosphocholine; C6-NBD-PG; 1-Myristoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-; sn; -Glycero-3-[Phospho-; rac; -(1-glycerol)]; Rh-PE; N-(lissamine Rhodamine B sulfonyl)phosphatidylethanolamine; FRET; fluorescence resonance energy transfer; DSC; differential scanning calorimetryAntimicrobial peptide; Species specificity; Phase separation; Phosphatidylethanolamine; Bacterial membrane lipid

Expression and purification of a recombinant LL-37 from Escherichia coli by Ja-Young Moon; Katherine A. Henzler-Wildman; A. Ramamoorthy (pp. 1351-1358).

Human cathelicidin-derived LL-37 is a 37-residue cationic, amphipathic α-helical peptide. It is an active component of mammalian innate immunity. LL-37 has several biological functions including a broad spectrum of antimicrobial activities and LPS-neutralizing activity. In order to determine the high-resolution three-dimensional structure of LL-37 using NMR spectroscopy, it is important to obtain the peptide with isotopic labels such as¹⁵N,¹³C and/or²H. Since it is less expensive to obtain such a peptide biologically, in this study, we report for the first time a method to express in E. coli and purify LL-37 using Glutathione S-transferase (GST) fusion system. LL-37 gene was inserted into vector pGEX-4T3 and expressed as a GST-LL-37 fusion protein in BL21(DE3) strain. The recombinant GST-LL-37 protein was purified with a yield of 8 mg/l by affinity chromatography and analyzed its biochemical and spectroscopic properties. Factor Xa was used to cleave a 4.5-kDa LL-37 from the GST-LL-37 fusion protein and the peptide was purified using a reverse-phase HPLC on a Vydac C₁₈ column with a final yield of 0.3 mg/l. The protein purified using reverse-phase HPLC was confirmed to be LL-37 by the analyses of Western blot and MALDI-TOF-Mass spectrometry. E. coli cells harboring the expression vector pGEX-4T3-LL-37 were grown in the presence of the¹⁵N-labeled M9 minimal medium and culture conditions were optimized to obtain uniform¹⁵N enrichment in the constitutively expressed LL-37 peptide. These results suggest that our production method will be useful in obtaining a large quantity of recombinant LL-37 peptide for NMR studies.

Keywords: Cathelicidin; Antimicrobial peptide; LL37; NMR; Expression; Antibiotics

Investigating molecular recognition and biological function at interfaces using piscidins, antimicrobial peptides from fish by Eduard Y. Chekmenev; Breanna S. Vollmar; Kristen T. Forseth; McKenna N. Manion; Shiela M. Jones; Tim J. Wagner; RaeLynn M. Endicott; Brandon P. Kyriss; Lorraine M. Homem; Michelle Pate; Jing He; Joshua Raines; Peter L. Gor'kov; William W. Brey; Dan J. Mitchell; Ann J. Auman; Mary J. Ellard-Ivey; Jack Blazyk; Myriam Cotten (pp. 1359-1372).

We studied amidated and non-amidated piscidins 1 and 3, amphipathic cationic antimicrobial peptides from fish, to characterize functional and structural similarities and differences between these peptides and better understand the structural motifs involved in biological activity and functional diversity among amidated and non-amidated isoforms. Antimicrobial and hemolytic assays were carried out to assess their potency and toxicity, respectively. Site-specific high-resolution solid-state NMR orientational restraints were obtained from¹⁵N-labeled amidated and non-amidated piscidins 1 and 3 in the presence of hydrated oriented lipid bilayers. Solid-state NMR and circular dichroism results indicate that the peptides are α-helical and oriented parallel to the membrane surface. This orientation was expected since peptide–lipid interactions are enhanced at the water–bilayer interface for amphipathic cationic antimicrobial peptides.¹⁵N solid-state NMR performed on oriented samples demonstrate that piscidin experiences fast, large amplitude backbone motions around an axis parallel to the bilayer normal. Under the conditions tested here, piscidin 1 was confirmed to be more antimicrobially potent than piscidin 3 and antimicrobial activity was not affected by amidation. In light of functional and structural similarities between piscidins 1 and 3, we propose that their topology and fast dynamics are related to their mechanism of action.

Keywords: Abbreviations; ACAPs; antimicrobial, cationic, amphipathic peptides; CD; circular dichroism; DPG; diphosphatidylglycerate; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DMPG; 1,2-dimyristoyl-; sn; -glycero-3-phosphoglycerate; DTPC; 1,2-; O; -ditetradecyl-; sn; -glycero-3-phosphocholine; DTPG; 1,2-; O; -ditetradecyl-; sn; -glycero-3-phosphoglycerate; HPLC; high performance liquid chromatography; LPS:; lipopolysaccharides; LUVs; large unilamellar vesicles; MIC; minimal inhibitory concentration; NMR; nuclear magnetic resonance; p1(or 3)-COO; ⁻; non-amidated piscidin 1 (or 3); p1(or 3)-NH; ₂; amidated piscidin 1 (or 3); PC; phosphatidylcholine; PDB; protein data bank; PE; phosphatidylethanolamine; PISEMA; Polarization Inversion Spin Exchange at the Magic Angle; PG; phosphatidylglycerol; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylcholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylglycerate; REDOR; Rotational Echo DOuble Resonance; TFE; trifluoroethanol; UV; ultravioletPiscidin; Structure–function relationship; Solid-state NMR; Oriented lipid bilayer; Peptide dynamics; Water–bilayer interface

Structural congruence among membrane-active host defense polypeptides of diverse phylogeny by Nannette Y. Yount; Michael R. Yeaman (pp. 1373-1386).

A requisite for efficacious host defense against pathogens and predators has prioritized evolution of effector molecules thereof. A recent multidimensional analysis of physicochemical properties revealed a novel, unifying structural signature among virtually all classes of cysteine-containing antimicrobial peptides. This motif, termed the γ-core, is seen in host defense peptides from organisms spanning more than 2.6 billion years of evolution. Interestingly, many toxins possess the γ-core signature, consistent with discoveries of their direct antimicrobial activity. Many microbicidal chemokines (kinocidins) likewise contain iterations of the γ-core motif, reconciling their antimicrobial efficacy. Importantly, these polypeptide classes have evolved to target and modulate biomembranes in protecting respective hosts against unfavorable interactions with potential pathogens or predators. Extending on this concept, the current report addresses the hypothesis that antimicrobial peptides, kinocidins, and polypeptide toxins are structurally congruent and share a remarkably close phylogenetic relationship, paralleling their roles in host–pathogen relationships. Analyses of their mature amino acid sequences demonstrated that cysteine-stabilized antimicrobial peptides, kinocidins, and toxins share ancient evolutionary relatedness stemming from early precursors of the γ-core signature. Moreover, comparative 3-D structure analysis revealed recurring iterations of antimicrobial peptide γ-core motifs within kinocidins and toxins. However, despite such congruence in γ-core motifs, the kinocidins diverged in overall homology from microbicidal peptides or toxins. These findings are consistent with observations that chemokines are not toxic to mammalian cells, in contrast to many antimicrobial peptides and toxins. Thus, specific functions of these molecular effectors may be governed by specific configurations of structural modules associated with a common γ-core motif. These concepts are consistent with the hypothesis that the γ-core is an archetype determinant in polypeptides that target or regulate with biological membranes, with specific iterations optimized to unique or cognate host defense contexts. Quantitative and qualitative data suggest these protein families emerged through both parallel and divergent processes of modular evolution. Taken together, the current and prior findings imply that the γ-core motif contributes to conserved structures and functions of host defense polypeptides. The presence of this unifying molecular signature in otherwise diverse categories of membrane-active host defense peptides implies an ancient and essential role for such a motif in effector molecules governing host–pathogen relationships.

Keywords: Antimicrobial peptide; Toxin; Chemokine; Host defense; Structure; Evolution

Antimicrobial activity of an abiotic host defense peptide mimic by Gregory N. Tew; Dylan Clements; Haizong Tang; Lachelle Arnt; Richard W. Scott (pp. 1387-1392).

Bacterial drug resistance is emerging as one of the most significant challenges to human health. Antimicrobial peptides (AMPs), which are produced by many tissues and cell types of invertebrates, insects, and humans, as part of their innate immune system, have attracted considerable interest as alternative antibiotics. Interest in novel mimics of AMPs has increased greatly over the last few years. This report details a new AMP mimic, based on phenylene ethynylene, with improved antimicrobial activity and selectivity. Screening against a large set of bacterial and other organisms demonstrates broad spectrum antimicrobial activity including activity against antibiotic resistant bacterial like methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) as well as activity against yeast ( Candida albicans) and fungus ( Stachybotrys chartarum). Bacterial resistance development studies using Staphylococcus aureus show a rapid increase in MIC for conventional antibiotics, ciprofloxacin and norfloxacin. In sharp contrast, no change in MIC was observed for the AMP mimic. Cytotoxicity experiments show that the AMP mimic acts preferentially on microbes as opposed to mammalian red blood cells, 3T3 fibroblasts, and HEPG2 cells. In vivo experiments determined the maximum tolerated dose (MTD) to be 10 mg/kg suggesting a therapeutic window is available. These studies indicate that nonpeptidic amphiphilic AMP mimics could be developed as potential new treatments for antibiotic-resistant bacterial infections.

Keywords: Magainin; Phenylene ethynylene; Foldamer; Antimicrobial; Antibiotic-resistant

Investigations of antimicrobial peptides in planar film systems by Roman Volinsky; Sofiya Kolusheva; Amir Berman; Raz Jelinek (pp. 1393-1407).

Planar systems – monolayers and films – constitute a useful platform for studying membrane-active peptides. Here, we summarize varied approaches for studying peptide organization and peptide–lipid interactions at the air/water interface, and focus on three representative antimicrobial membrane-associated peptides—alamethicin, gramicidin, and valinomycin. Experimental data, specifically surface pressure/area isotherms and Brewster angle microscopy images, provided information on peptide association and the effects of the lipid monolayers on peptide surface organization. In general, film analysis emphasized the effects of lipid layers in promoting peptide association and aggregation at the air/water interface. Importantly, the data demonstrated that in many cases peptide domains are phase-separated within the phospholipid monolayers, suggesting that this behavior contributes to the biological actions of membrane-active antimicrobial peptides.

Keywords: Langmuir monolayer; Brewster angle microscopy; Peptide–membrane interaction; Peptide film

LL-37, the only human member of the cathelicidin family of antimicrobial peptides by Ulrich H.N. Dürr; U.S. Sudheendra; Ayyalusamy Ramamoorthy (pp. 1408-1425).

Antimicrobial peptides and their precursor molecules form a central part of human and mammalian innate immunity. The underlying genes have been thoroughly investigated and compared for a considerable number of species, allowing for phylogenetic characterization. On the phenotypical side, an ever-increasing number of very varied and distinctive influences of antimicrobial peptides on the innate immune system are reported. The basic biophysical understanding of mammalian antimicrobial peptides, however, is still very limited. This is especially unsatisfactory since knowledge of structural properties will greatly help in the understanding of their immunomodulatory functions. The focus of this review article will be on LL-37, the only cathelicidin-derived antimicrobial peptide found in humans. LL-37 is a 37-residue, amphipathic, helical peptide found throughout the body and has been shown to exhibit a broad spectrum of antimicrobial activity. It is expressed in epithelial cells of the testis, skin, the gastrointestinal tract, and the respiratory tract, and in leukocytes such as monocytes, neutrophils, T cells, NK cells, and B cells. It has been found to have additional defensive roles such as regulating the inflammatory response and chemo-attracting cells of the adaptive immune system to wound or infection sites, binding and neutralizing LPS, and promoting re-epthelialization and wound closure. The article aims to report the known biophysical facts, with an emphasis on structural evidence, and to set them into relation with insights gained on phylogenetically related antimicrobial peptides in other species. The multitude of immuno-functional roles is only outlined. We believe that this review will aid the future work on the biophysical, biochemical and immunological investigations of this highly intriguing molecule.

Keywords: Antimicrobial peptide; LL-37; Cathelicidin; Solid-state NMR; Antibiotic; Membrane

Influence of N-acylation of a peptide derived from human lactoferricin on membrane selectivity by Dagmar Zweytick; Georg Pabst; Peter M. Abuja; Alexander Jilek; Sylvie E. Blondelle; Jörg Andrä; Roman Jerala; Daniel Monreal; Guillermo Martinez de Tejada; Karl Lohner (pp. 1426-1435).

Increasing numbers of bacterial strains being resistant to conventional antibiotics emphasize the urgent need for new antimicrobial agents. One strategy is based on host defence peptides that can be found in every organism including humans. We have studied the antimicrobial peptide LF11, derived from the pepsin cleavage product of human lactoferrin, known for its antimicrobial and lipid A-binding activity, and peptide C12LF11, the N-lauryl-derivative of LF11, which has owing to the attached hydrocarbon chain an additional hydrophobic segment. The influence of this hydrocarbon chain on membrane selectivity was studied using model membranes composed of dipalmitoylphosphatidylglycerol (DPPG), mimicking bacterial plasma membranes, and of dipalmitoylphosphatidylcholine (DPPC), a model system for mammalian membranes. A variety of biophysical techniques was applied. Thereby, we found that LF11 did not affect DPPC bilayers and showed only moderate effects on DPPG membranes in accordance with its non-hemolytic and weak antimicrobial activity. In contrast, the introduction of the N-lauryl group caused significant changes in the phase behaviour and lipid chain packing in both model membrane systems. These findings correlate with the in vitro tests on methicillin resistant S. aureus, E. coli, P. aeruginosa and human red blood cells, showing increased biological activity of C12LF11 towards these test organisms. This provides evidence that both electrostatic and hydrophobic interactions are crucial for biological activity of antimicrobial peptides, whereas a certain balance between the two components has to be kept, in order not to loose the specificity for bacterial membranes.

Keywords: Acylation and antimicrobial peptide; Lactoferrin; Antimicrobial and hemolytic activity; Model membrane

Alpha-helical antimicrobial peptides—Using a sequence template to guide structure–activity relationship studies by Igor Zelezetsky; Alessandro Tossi (pp. 1436-1449).

An important class of cytolytic antimicrobial peptides (AMPs) assumes an amphipathic, α-helical conformation that permits efficient interaction with biological membranes. Host defence peptides of this type are widespread in nature, and numerous synthetic model AMPs have been derived from these or designed de novo based on their characteristics. In this review we provide an overview of the ‘sequence template’ approach which we have used to design potent artificial helical AMPs, to guide structure–activity relationship studies aimed at their optimization, and to help identify novel natural AMP sequences. Combining this approach with the rational use of natural and non-proteinogenic amino acid building blocks has allowed us to probe the individual effects on the peptides' activity of structural and physico-chemical parameters such as the size, propensity for helical structuring, amphipathic hydrophobicity, cationicity, and hydrophobic or polar sector characteristics. These studies furthermore provided useful insights into alternative modes of action for natural membrane-active helical peptides.

Keywords: Abbreviations; Abu; 2-aminobutyric acid; Acp; aminocylcopentanecarboxylic acid; Aib; 2-aminoisobutyric acid; AMP; antimicrobial peptide; Dab; 2,4-diaminobutyric acid; Dap; 2,3-diaminopropionic acid; Deg; diethylglycine; Dpg; dipropylglycine; FS; forward scattering; HDP; host defence peptide; Hse; homoserine; MIC; minimum inhibitory concentration; Nle; norleucine; Nva; norvaline; Orn; ornithine; SAR; structure–activity relationship; SEM; scanning electron microscopy; SS; side scattering; TFE; trifluoroethanolAntimicrobial peptide; Sequence template; Hydrophobicity; Amphipathicity; Structure–activity relationship; α-Helical

Insertion selectivity of antimicrobial peptide protegrin-1 into lipid monolayers: Effect of head group electrostatics and tail group packing by Yuji Ishitsuka; Duy S. Pham; Alan J. Waring; Robert I. Lehrer; Ka Yee C. Lee (pp. 1450-1460).

The ability to selectively target the harmful microbial membrane over that of the host cell is one of the most important characteristics of the antimicrobial peptides (AMPs). This selectivity strongly depends on the chemical and structural properties of the lipids that make up the cell membrane. A systematic study of the initial membrane selectivity of protegrin-1 (PG-1), a β-sheet AMP, was performed using Langmuir monolayers. Constant pressure insertion assay was used to quantify the amount of PG-1 insertion and fluorescence microscopy was employed to observe the effect of PG-1 on lipid ordering. Charge and packing properties of the monolayer were altered by using lipids with different head groups, substituting saturated with unsaturated lipid tail group(s) and incorporating spacer molecules. PG-1 inserted most readily into anionic films composed of phosphatidylglycerol (PG) and lipid A, consistent with its high selectivity for microbial membranes. It also discriminated between zwitteranionic phospholipids, inserting more readily into phosphatidylcholine (PC) monolayers than those composed of phosphatidylethanolamine, potentially explaining why PG-1 is hemolytic for PC-rich human erythrocytes and not for the PE-rich erythrocytes of ruminants. Increased packing density of the monolayer by increased surface pressure, increased tail group saturation or incorporation of dihydrocholesterol diminishes the insertion of PG-1. Fluorescence microscopy shows that lipid packing is disordered upon PG-1 insertion. However, the presence of PG-1 can still affect lipid morphology even with no observed PG-1 insertion. These results show the important role that lipid composition of the cell membrane plays in the activity of AMPs.

Keywords: Antimicrobial peptide; Protegrin-1; Langmuir monolayer; Fluorescence microscopy; Peptide–membrane interaction; Model cell membrane; Selectivity; Disordering; Phospholipid; DPPC; DPPE; DPPG; POPC; POPE; POPG; Lipid A; Dihydrocholesterol; Ganglioside

Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: Implications for a novel mechanism of action by Hongxia Zhao; Rohit Sood; Arimatti Jutila; Shambhunath Bose; Gunnar Fimland; Jon Nissen-Meyer; Paavo K.J. Kinnunen (pp. 1461-1474).

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl- sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl- sn-glycero-3-phospho- rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl- sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein–lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.

Keywords: Abbreviations; BrainPS; brain phosphatidylserine; CF; carboxyfluorescein; DPH; diphenylhexatriene; LUVs; large unilamellar vesicles; NBD-PG; 1-palmitoyl-2-(N-4-nitrobenz-2-oxa-1,3-diazol)amino-caproyl-; sn; -glycero-3-phospho-rac-glycerol; PBS; phosphate buffer saline; PC; phosphatidylcholine; PG; phosphatidylglycerol; plA; Plantaricin A; POPE; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoethanolamine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phospho-rac-glycerol; PPDPC; 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-; sn; -glycero-3-phosphocholine; PPDPG; 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-; sn; -glycero-3-phospho-rac-glycerol; PS; phosphatidylserine; r; fluorescence anisotropy; RFI; _m; relative pyrene monomer fluorescence intensity; R; (; I; _e; /; I; _m; ); relative excimer to monomer ratio; SOPC; 1-stearoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; π; surface pressure; π; _c; critical surface pressure abolishing the intercalation of the peptides into the lipid monolayers; π; ₀; initial surface pressure; Δ; π; increment in surface pressureAntimicrobial peptide; Bacteriocin; Liposome; Acidic phospholipid; Protein fibril

The effect of binding of spider-derived antimicrobial peptides, oxyopinins, on lipid membranes by Kaoru Nomura; Gerardo Corzo (pp. 1475-1482).

Oxyopinins (Oxki1 and Oxki2) are antimicrobial peptides isolated from the crude venom of the wolf spider Oxyopes kitabensis. The effect of oxyopinins on lipid bilayers was investigated using high-sensitivity titration calorimetry and³¹P solid-state NMR spectroscopy. High-sensitivity titration calorimetry experiments showed that the binding of oxyopinins was exothermic, and the binding enthalpies (Δ H) to 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphatidylcholine (POPC) small unilamellar vesicles (SUVs) were −18.1 kcal/mol and −15.0 kcal/mol for Oxki1 and Oxki2, respectively, and peptide partition coefficient ( K_p) was found to be 3.9×10³ M−¹.³¹P NMR spectra of 1,2-dielaidoyl- sn-glycero-3-phosphoethanolamine (DEPE) membranes in the presence of oxyopinins indicated that they induced a positive curvature in lipid bilayers. The induced positive curvature was stronger in the presence of Oxki2 than in the presence of Oxki1.³¹P NMR spectra of phosphaditylcholine (PC) membranes in the presence of Oxki2 showed that Oxki2 produced micellization of membranes at low peptide concentrations, but unsaturated PC membranes or acidic phospholipids prevented micellization from occurring. Furthermore,³¹P NMR spectra using membrane lipids from E. coli suggested that Oxki1 was more disruptive to bacterial membranes than Oxki2. These results strongly correlate to the known biological activity of the oxyopinins.

Keywords: Abbreviations; CD; circular dichroism; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphatidylcholine; DMPG; 1,2-dimyristoyl-; sn; -glycero-3-phosphatidylglycerol; DEPE; 1,2-dielaidoyl-; sn; -glycero-3-phosphoethanolamine; H; _II; inverted hexagonal phase; HPLC; high-performance liquid chromatography; ITC; isothermal titration calorimetry; L; _α; lamellar phase; MALDI-TOF; matrix-assisted laser-desorption ionization-time-of-flight; MLV; multi-lamellar vesicles; NMR; nuclear magnetic resonance; ODS; octa decyl silica; PC; phosphaditylcholine; PE; phosphaditylethanolamine; PG; phosphatidylglycerol; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylcholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylglycerol; SUVs; small unilamellar vesiclesSpider-derived antimicrobial peptide; Membrane binding; Lipid bilayer; ³¹; P solid-state NMR; ITC; Curvature strain

Structure of antimicrobial peptides and lipid membranes probed by interface-sensitive X-ray scattering by Tim Salditt; Chenghao Li; Alexander Spaar (pp. 1483-1498).

The conformation and correlations of amphiphilic and antimicrobial peptides and the associated changes of lipid bilayers can be studied in oriented lipid membranes deposited on solid substrates. Here we review recent work on these systems, as studied by modern interface-sensitive X-ray and neutron scattering methods. Density profile, short range order of acyl chains and molecular conformations of peptides and lipids are probed in the fluid state of the bilayer. With an emphasis on technical aspects, we review recent work illustrating the potential of the methods and discuss its potential in the field.

Keywords: Abbreviations; POPC; palmitoyl-oleoyl-sn-glycero-phosphocholine; DMPC; dimyristoyl-sn-glycero-phosphocholine; DLPC; dilauroyl-sn-glycero-phosphocholine; DMPG; Dimyristoyl-sn-Glycero-Phospho-rac-glycerol; DPhPC; diphytanoyl phosphatidylcholine; SM; sphingomyelin; PS; phosphatidyl-serine; PG; phospho-rac-glycerol; PE; phosphatidyl-ethanolamine; MD; Molecular Dynamics; RSM; Reciprocal Space Mapping; GID; Grazing Incidence Diffraction; 2D; Two-Dimensional; P/L; molar Peptide-to-Lipid ratio; DIC; Differential Phase Contrast

The human beta-defensin-3, an antibacterial peptide with multiple biological functions by Vishnu Dhople; Amy Krukemeyer; Ayyalusamy Ramamoorthy (pp. 1499-1512).

A group of interesting molecules called defensins exhibit multiple functions but have been primarily recognized to possess a broad spectrum of antimicrobial activities. Studies have reported two different types of defensins (α and β) from human and animals, a cyclic θ defensin from rhesus, and several defensin-like peptides from plants. There is no amino acid sequence homology between these peptides, but they all contain three Cys–Cys disulfide linkages while the connectivities are different. Human β-defensin-3 (HβD-3) is the most recently discovered member of the host-defense peptide family that has attracted much attention. This molecule is expressed either constitutively or induced upon a challenge, and a growing evidence indicates the involvement of such molecules in adaptive immunity as well. It has been shown to exhibit antibacterial activities towards Gram-negative and Gram-positive bacteria as well as an ability to act as a chemo-attractant. Analysis of NMR structural data suggested a symmetrical dimeric form of this peptide in solution, which consists of three β strands and a short helix in the N-terminal region. While the disulfide linkages are known to provide the structural stability and stability against proteases, the biological relevance of this dimeric form was contradicted by another biological study. Since there is considerable current interest in developing HβD-3 for possible pharmaceutical applications, studies to further our understanding on the determinants of antibacterial activities and immunomodulatory function of HβD-3 are considered to be highly significant. The knowledge of its biosynthetic regulation will also help in understanding the role of HβD-3 in immunity. This article presents an overview of the expression and regulation of HβD-3 in humans, and the structure–function correlations among HβD-3 and its modified peptides are discussed emphasizing the functional importance. The future scope for studies on HβD-3 and design of short potent antimicrobial peptides, based on the native HβD-3 molecule, that do not interfere in the immunomodulatory function is also outlined.

Keywords: Antibacterial peptide; Defensin; HβD-3; Structure; Innate and adaptive immunity; Membrane-disruption

Lipopolysaccharide (Endotoxin)-host defense antibacterial peptides interactions: Role in bacterial resistance and prevention of sepsis by Yosef Rosenfeld; Yechiel Shai (pp. 1513-1522).

Lipopolysaccharide (LPS) is the major molecular component of the outer membrane of Gram-negative bacteria and serves as a physical barrier providing the bacteria protection from its surroundings. LPS is also recognized by the immune system as a marker for the detection of bacterial pathogen invasion, responsible for the development of inflammatory response, and in extreme cases to endotoxic shock. Because of these functions, the interaction of LPS with LPS binding molecules attracts great attention. One example of such molecules are antimicrobial peptides (AMPs). These are large repertoire of gene-encoded peptides produced by living organisms of all types, which serve as part of the innate immunity protecting them from pathogen invasion. AMPs are known to interact with LPS with high affinities. The biophysical properties of AMPs and their mode of interaction with LPS determine their biological function, susceptibility of bacteria to them, as well as the ability of LPS to activate the immune system. This review will discuss recent studies on the molecular mechanisms underlying these interactions, their effects on the resistance of the bacteria to AMPs, as well as their potential to neutralize LPS-induced endotoxic shock.

Keywords: Antimicrobial peptide; Lipopolisaccharide; Peptide-membrane interaction; Endotoxin; Bacterial resistrance

Interactions of bovine lactoferricin with acidic phospholipid bilayers and its antimicrobial activity as studied by solid-state NMR by Masako Umeyama; Atsushi Kira; Katsuyuki Nishimura; Akira Naito (pp. 1523-1528).

Bovine lactoferricin (LfcinB) is an antimicrobial peptide released by pepsin cleavage of lactoferrin. In this work, the interaction between LfcinB and acidic phospholipid bilayers with the weight percentage of 65% dimyristoylphosphatidylglycerol (DMPG), 10% cardiolipin (CL) and 25% dimyristoylphosphatidylcholine (DMPC) was investigated as a mimic of cell membrane of Staphylococcus aureus by means of quartz crystal microbalance (QCM) and solid-state³¹P and¹H NMR spectroscopy. Moreover, we elucidated a molecular mechanism of the antimicrobial activity of LfcinB by means of potassium ion selective electrode (ISE). It turned out that affinity of LfcinB for acidic phospholipid bilayers was higher than that for neutral phospholipid bilayers. It was also revealed that the association constant of LfcinB was larger than that of lactoferrin as a result of QCM measurements.³¹P DD-static NMR spectra indicated that LfcinB interacted with acidic phospholipid bilayers and bilayer defects were observed in the bilayer systems because isotropic peaks were clearly appeared. Gel-to-liquid crystalline phase transition temperatures (Tc) in the mixed bilayer systems were determined by measuring the temperature variation of relative intensities of acyl chains in¹H MAS NMR spectra. Tc values of the acidic phospholipid and LfcinB-acidic phospholipid bilayer systems were 21.5 °C and 24.0 °C, respectively. To characterize the bilayer defects, potassium ion permeation across the membrane was observed by ISE measurements. The experimental results suggest that LfcinB caused pores in the acidic phospholipid bilayers. Because these pores lead the permeability across the membrane, the molecular mechanism of the antimicrobial activity could be attributed to the pore formation in the bacterial membrane induced by LfcinB.

Keywords: Lactoferricin; Antimicrobial peptide; Acidic phospholipid bilayers; Pore formation; Solid-state; ³¹; P and; ¹; H NMR; Gel-to-liquid crystalline phase transition temperature

Detergent-like actions of linear amphipathic cationic antimicrobial peptides by Burkhard Bechinger; Karl Lohner (pp. 1529-1539).

Antimicrobial peptides have raised much interest as pathogens become resistant against conventional antibiotics. We review biophysical studies that have been performed to better understand the interactions of linear amphipathic cationic peptides such as magainins, cecropins, dermaseptin, δ-lysin or melittin. The amphipathic character of these peptides and their interactions with membranes resemble the properties of detergent molecules and analogies between membrane-active peptide and detergents are presented. Several models have been suggested to explain the pore-forming, membrane-lytic and antibiotic activities of these peptides. Here we suggest that these might be ‘special cases’ within complicated phase diagrams describing the morphological plasticity of peptide/lipid supramolecular assemblies.

Keywords: Abbreviations; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DOPA; 1,2-dioleoyl-; sn; -glycero-3-phosphatidic acid; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; NMR; nuclear magnetic resonance; PC; 1-2-diacyl-; sn; -glycero-3-phosphocholine; PE; 1,2-diacyl-; sn; -glycerol-3-phosphoethanolamine; PG; 1,2-diacyl-; sn; -glycerol-3-phosphoglycerol; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoglycerolPolypeptide lipid interaction; Peptide pore formation; Phospholipid membrane; Bilayer; Regulation; Selectivity

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BBA - Biomembranes (v.1758, #9)

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BBA - Biomembranes (v.1758, #9)