Effect of lipid composition on the topography of membrane-associated hydrophobic helices: stabilization of transmembrane topography by anionic lipids

To investigate the effect of lipid structure upon the membrane topography of hydrophobic helices, the behavior of hydrophobic peptides was studied in model membrane vesicles. To define topography, fluorescence and fluorescence quenching methods were used to determine the location of a Trp at the center of the hydrophobic sequence. For peptides with cationic residues flanking the hydrophobic sequence, the stability of the transmembrane (TM) configuration (relative to a membrane-bound non-TM state) increased as a function of lipid composition on the order: 1:1 (mol:mol) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine ∼ 6:4 POPC:cholesterol < POPC ∼ dioleoylphosphatidylcholine (DOPC) < 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DOPG) ≤ 1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] sodium salt (DOPS), indicating that the anionic lipids DOPG and DOPS most strongly stabilized the TM configuration. TM stabilization was near maximal at 20-30 mol% anionic lipid, which are physiologically relevant values. TM stabilization by anionic lipid was observed for hydrophobic sequences with a diverse set of sequences (including polyAla), diverse lengths (from 12 to 22 residues), and various cationic flanking residues (H, R, or K), but not when the flanking residues were uncharged. TM stabilization by anionic lipid was also dependent on the number of cationic residues flanking the hydrophobic sequence, but was still significant with only one cationic residue flanking each end of the peptide. These observations are consistent with TM-stabilizing effects being electrostatic in origin. However, Trp located more deeply in DOPS vesicles relative to DOPG vesicles, and peptides in DOPS vesicles showed increased helix formation relative to DOPG and all other lipid compositions. These observations fit a model in which DOPS anchors flanking residues near the membrane surface more strongly than does DOPG and/or increases the stability of the TM state to a greater degree than DOPG. We conclude that anionic lipids can have significant and headgroup structure-specific effects upon membrane protein topography.     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from + 4 to + 5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an α-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

Heterologous expression and solution structure of defensin from lentil Lens culinaris

A new defensin Lc-def, isolated from germinated seeds of the lentil Lens culinaris, has molecular mass 5440.4 Da and consists of 47 amino acid residues. Lc-def and its ¹⁵N-labeled analog were overexpressed in Escherichia coli. Antimicrobial activity of the recombinant protein was examined, and its spatial structure, dynamics, and interaction with lipid vesicles were studied by NMR spectroscopy. It was shown that Lc-def is active against fungi, but does not inhibit the growth of Gram-positive and Gram-negative bacteria. The peptide is monomeric in aqueous solution and contains one α-helix and triple-stranded β-sheet, which form cysteine-stabilized αβ motif (CSαβ) previously found in other plant defensins. The sterically neighboring loop1 and loop3 protrude from the defensin core and demonstrate significant mobility on the μs–ms timescale. Lc-def does not bind to the zwitterionic lipid (POPC) vesicles but interacts with the partially anionic (POPC/DOPG, 7:3) membranes under low-salt conditions. The Lc-def antifungal activity might be mediated through electrostatic interaction with anionic lipid components of fungal membranes.     

Interaction of gramicidin S and its aromatic amino-acid analog with phospholipid membranes

To investigate the mechanism of interaction of gramicidin S-like antimicrobial peptides with biological membranes, a series of five decameric cyclic cationic β-sheet-β-turn peptides with all possible combinations of aromatic D-amino acids, Cyclo(Val-Lys-Leu-D-Ar1-Pro-Val-Lys-Leu-D-Ar2-Pro) (Ar ≡ Phe, Tyr, Trp), were synthesized. Conformations of these cyclic peptides were comparable in aqueous solutions and lipid vesicles. Isothermal titration calorimetry measurements revealed entropy-driven binding of cyclic peptides to POPC and POPE/POPG lipid vesicles. Binding of peptides to both vesicle systems was endothermic—exceptions were peptides containing the Trp-Trp and Tyr-Trp pairs with exothermic binding to POPC vesicles. Application of one- and two-site binding (partitioning) models to binding isotherms of exothermic and endothermic binding processes, respectively, resulted in determination of peptide-lipid membrane binding constants (K_b). The K_b1 and K_b2 values for endothermic two-step binding processes corresponded to high and low binding affinities (K_b1 ≥ 100 K_b2). Conformational change of cyclic peptides in transferring from buffer to lipid bilayer surfaces was estimated using fluorescence resonance energy transfer between the Tyr-Trp pair in one of the peptide constructs. The cyclic peptide conformation expands upon adsorption on lipid bilayer surface and interacts more deeply with the outer monolayer causing bilayer deformation, which may lead to formation of nonspecific transient peptide-lipid porelike zones causing membrane lysis.     

Membrane-active properties of α-MSH analogs: aggregation and fusion of liposomes triggered by surface-conjugated peptides

Reaction of the melanotropin hormone analogs [Nle⁴,D-Phe⁷]-α-MSH and [Nle⁴,D-Phe⁷]-α-MSH(4-10), which were extended at their N-terminus by a thiol-functionalized spacer arm, with preformed liposomes containing thiol-reactive (phospho)lipid derivatives resulted in the aggregation of the vesicles and in a partial leakage of their inner contents. This aggregation/leakage effect, which was only observed when the peptides were covalently conjugated to the surface of the liposomes, was correlated with the fusion of the vesicles as demonstrated by the observed decrease in resonance energy transfer between probes in a membrane lipid mixing assay. A limited fusion was confirmed by monitoring the mixing of the liposome inner contents (formation of 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylene bis(pyridinium bromide) complex). The membrane-active properties of the peptides could be correlated with changes in the fluorescence emission spectra of their tryptophan residue, which suggested that after their covalent binding to the outer surface of the liposomes they can partition within the core of the bilayers. A blue shift of 10 nm was observed for [Nle⁴,D-Phe⁷]-α-MSH which was correlated with an increase in fluorescence anisotropy and with changes in the accessibility of the coupled peptide as assessed by the quenching of fluorescence of its tryptophan residue by iodide (Stern-Volmer plots). These results should be related to the previously described capacity of α-MSH, and analogs, to interact with membranes and with the favored conformation of these peptides which, via a β-turn, segregate their central hydrophobic residues into a domain that could insert into membranes and, as shown here, trigger their destabilization.     

Reversible Liposome Association Induced by LAH4: A Peptide with Potent Antimicrobial and Nucleic Acid Transfection Activities

We report on the reversible association of anionic liposomes induced by an antimicrobial peptide (LAH4). The process has been characterized for mixed membranes of POPC and POPS at molar ratios of 1:1, 3:1, and 9:1. Although the vesicles remain in suspension in the presence of excess amounts of peptide, the addition of more lipids results in surface charge neutralization, aggregation of the liposomes, and formation of micrometer-sized structures that coexist in equilibrium with vesicles in suspension. At low ratios of anionic lipids, vesicle aggregation is a reversible process, and vesicle disassembly is observed upon inversion of the surface charge by further supplementation with anionic vesicles. In contrast, a different process, membrane fusion, occurs in the presence of high phosphatidylserine concentrations. Upon binding to membranes containing low POPS concentrations, the peptide adopts an in-plane α-helical structure, a secondary structure that is conserved during vesicle association and dissociation. Our finding that peptides are essential for vesicle aggregation contributes to a better understanding of the activity of antimicrobial peptides, and suggests an additional layer of complexity in membrane-protein lipid interactions.     

Cell selectivity correlates with membrane-specific interactions: A case study on the antimicrobial peptide G15 derived from granulysin

A 15-residue peptide dimer G15 derived from the cell lytic protein granulysin has been shown to exert potent activity against microbes, including E. coli, but not against human Jurkat cells [Z. Wang, E. Choice, A. Kaspar, D. Hanson, S. Okada, S.C. Lyu, A.M. Krensky, C. Clayberger, Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin. J. Immunol. 165 (2000) 1486-1490]. We investigated the target membrane selectivity of G15 using fluorescence, circular dichroism and ³¹P NMR methods. The ANS uptake assay shows that the extent of E. coli outer membrane disruption depends on G15 concentration. ³¹P NMR spectra obtained from E. coli total lipid bilayers incorporated with G15 show disruption of lipid bilayers. Fluorescence binding studies on the interaction of G15 with synthetic liposomes formed of E. coli lipids suggest a tight binding of the peptide at the membrane interface. The peptide also binds to negatively charged POPC/POPG (3:1) lipid vesicles but fails to insert deep into the membrane interior. These results are supported by the peptide-induced changes in the measured isotropic chemical shift and T1 values of POPG in 3:1 POPC:POPG multilamellar vesicles while neither a non-lamellar phase nor a fragmentation of bilayers was observed from NMR studies. The circular dichroism studies reveal that the peptide exists as a random coil in solution but folds into a less ordered conformation upon binding to POPC/POPG (3:1) vesicles. However, G15 does not bind to lipid vesicles made of POPC/POPG/Chl (9:1:1) mixture, mimicking tumor cell membrane. These results explain the susceptibility of E. coli and the resistance of human Jurkat cells to G15, and may have implications in designing membrane-selective therapeutic agents.     

Bilayer interaction and localization of cell penetrating peptides with model membranes: A comparative study of a human calcitonin (hCT)-derived peptide with pVEC and pAntp(43-58)

Cell-penetrating peptides (CPPs) are able to translocate problematic therapeutic cargoes across cellular membranes. The exact mechanisms of translocation are still under investigation. However, evidence for endocytic uptake is increasing. We investigated the interactions of CPPs with phospholipid bilayers as first step of translocation. To this purpose, we employed four independent techniques, comprising (i) liposome buffer equilibrium dialysis, (ii) Trp fluorescence quenching, (iii) fluorescence polarization, and (iv) determination of ζ-potentials. Using unilamellar vesicles (LUVs) of different phospholipid composition, we compared weakly cationic human calcitonin (hCT)-derived peptides with the oligocationic CPPs pVEC and penetratin (pAntp). Apparent partition coefficients of hCT-derived peptides in neutral POPC LUVs were dependent on amino acid composition and secondary structure; partitioning in negatively charged POPC/POPG (80:20) LUVs was increased and mainly governed by electrostatic interactions. For hCT(9-32) and its derivatives, D values raised from about 100-200 in POPC to about 1000 to 1500 when negatively charged lipids were present. Localization profiles of CPPs obtained by Trp fluorescence quenching were dependent on the charge density of LUVs. In POPC/POPG, hCT-derived CPPs were located on the bilayer surface, whereas pVEC and pAntp resided deeper in the membrane. In POPG LUVs, an increase of fluorescence polarization was observed for pVEC and pAntp but not for hCT-derived peptides. Generally, we found strong peptide-phospholipid interactions, especially when negatively charged lipids were present.     

Effect of Membrane Composition on Antimicrobial Peptides Aurein 2.2 and 2.3 From Australian Southern Bell Frogs

The effects of hydrophobic thickness and the molar phosphatidylglycerol (PG) content of lipid bilayers on the structure and membrane interaction of three cationic antimicrobial peptides were examined: aurein 2.2, aurein 2.3 (almost identical to aurein 2.2, except for a point mutation at residue 13), and a carboxy C-terminal analog of aurein 2.3. Circular dichroism results indicated that all three peptides adopt an α-helical structure in the presence of a 3:1 molar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPC/DMPG), and 1:1 and 3:1 molar mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPC/POPG). Oriented circular dichroism data for three different lipid compositions showed that all three peptides were surface-adsorbed at low peptide concentrations, but were inserted into the membrane at higher peptide concentrations. The ³¹P solid-state NMR data of the three peptides in the DMPC/DMPG and POPC/POPG bilayers showed that all three peptides significantly perturbed lipid headgroups, in a peptide or lipid composition-dependent manner. Differential scanning calorimetry results demonstrated that both amidated aurein peptides perturbed the overall phase structure of DMPC/DMPG bilayers, but perturbed the POPC/POPG chains less. The nature of the perturbation of DMPC/DMPG bilayers was most likely micellization, and for the POPC/POPG bilayers, distorted toroidal pores or localized membrane aggregate formation. Calcein release assay results showed that aurein peptide-induced membrane leakage was more severe in DMPC/DMPG liposomes than in POPC/POPG liposomes, and that aurein 2.2 induced higher calcein release than aurein 2.3 and aurein 2.3-COOH from 1:1 and 3:1 POPC/POPG liposomes. Finally, DiSC₃5 assay data further delineated aurein 2.2 from the others by showing that it perturbed the lipid membranes of intact S. aureus C622 most efficiently, whereas aurein 2.3 had the same efficiency as gramicidin S, and aurein 2.3-COOH was the least efficient. Taken together, these data show that the membrane interactions of aurein peptides are affected by the hydrophobic thickness of the lipid bilayers and the PG content.     

Antimicrobial activity and membrane selective interactions of a synthetic lipopeptide MSI-843

Lipopeptide MSI-843 consisting of the nonstandard amino acid ornithine (Oct-OOLLOOLOOL-NH₂) was designed with an objective towards generating non-lytic short antimicrobial peptides, which can have significant pharmaceutical applications. Octanoic acid was coupled to the N-terminus of the peptide to increase the overall hydrophobicity of the peptide. MSI-843 shows activity against bacteria and fungi at micromolar concentrations. It permeabilizes the outer membrane of Gram-negative bacterium and a model membrane mimicking bacterial inner membrane. Circular dichroism investigations demonstrate that the peptide adopts α-helical conformation upon binding to lipid membranes. Isothermal titration calorimetry studies suggest that the peptide binding to membranes results in exothermic heat of reaction, which arises from helix formation and membrane insertion of the peptide. ²H NMR of deuterated-POPC multilamellar vesicles shows the peptide-induced disorder in the hydrophobic core of bilayers. ³¹P NMR data indicate changes in the lipid head group orientation of POPC, POPG and Escherichia colitotal lipid bilayers upon peptide binding. Results from ³¹P NMR and dye leakage experiments suggest that the peptide selectively interacts with anionic bilayers at low concentrations (up to 5 mol%). Differential scanning calorimetry experiments on DiPOPE bilayers and ³¹P NMR data from E.coli total lipid multilamellar vesicles indicate that MSI-843 increases the fluid lamellar to inverted hexagonal phase transition temperature of bilayers by inducing positive curvature strain. Combination of all these data suggests the formation of a lipid-peptide complex resulting in a transient pore as a plausible mechanism for the membrane permeabilization and antimicrobial activity of the lipopeptide MSI-843.     

Antimicrobial and Membrane Disrupting Activities of a Peptide Derived from the Human Cathelicidin Antimicrobial Peptide LL37

A 21-residue peptide segment, LL7-27 (RKSKEKIGKEFKRIVQRIKDF), corresponding to residues 7-27 of the only human cathelicidin antimicrobial peptide, LL37, is shown to exhibit potent activity against microbes (particularly Gram-positive bacteria) but not against erythrocytes. The structure, membrane orientation, and target membrane selectivity of LL7-27 are characterized by differential scanning calorimetry, fluorescence, circular dichroism, and NMR experiments. An anilinonaphthalene-8-sulfonic acid uptake assay reveals two distinct modes of Escherichia coli outer membrane perturbation elicited by LL37 and LL7-27. The circular dichroism results show that conformational transitions are mediated by lipid-specific interactions in the case of LL7-27, unlike LL37. It folds into an α-helical conformation upon binding to anionic (but not zwitterionic) vesicles, and also does not induce dye leakage from zwitterionic lipid vesicles. Differential scanning calorimetry thermograms show that LL7-27 is completely integrated with DMPC/DMPG (3:1) liposomes, but induces peptide-rich and peptide-poor domains in DMPC liposomes. ¹⁵N NMR experiments on mechanically aligned lipid bilayers suggest that, like the full-length peptide LL37, the peptide LL7-27 is oriented close to the bilayer surface, indicating a carpet-type mechanism of action for the peptide. ³¹P NMR spectra obtained from POPC/POPG (3:1) bilayers containing LL7-27 show substantial disruption of the lipid bilayer structure and agree with the peptide's ability to induce dye leakage from POPC/POPG (3:1) vesicles. Cholesterol is shown to suppress peptide-induced disorder in the lipid bilayer structure. These results explain the susceptibility of bacteria and the resistance of erythrocytes to LL7-27, and may have implications for the design of membrane-selective therapeutic agents.     

Molecular characterization of the interaction of crotamine-derived nucleolar targeting peptides with lipid membranes

A novel class of cell-penetrating, nucleolar-targeting peptides (NrTPs), was recently developed from the rattlesnake venom toxin crotamine. Based on the intrinsic fluorescence of tyrosine or tryptophan residues, the partition of NrTPs and crotamine to membranes with variable lipid compositions was studied. Partition coefficient values (in the 10(2)-10(5) range) followed essentially the compositional trend POPC:POPG≤POPG相似文献   

Biophysical studies of the membrane location of the voltage-gated sensors in the HsapBK and KvAP K channels

The membrane location of two fragments in two different K⁺-channels, the KvAP (from Aeropyrum pernix) and the HsapBK (human) corresponding to the putative “paddle” domains, has been investigated by CD, fluorescence and NMR spectroscopy. Both domains interact with q = 0.5 phospholipid bicelles, DHPC micelles and with POPC vesicles. CD spectra demonstrate that both peptides become largely helical in the presence of phospholipid bicelles. Fluorescence quenching studies using soluble acrylamide or lipid-attached doxyl-groups show that the arginine-rich domains are located within the bilayered region in phospholipid bicelles. Nuclear magnetic relaxation parameters, T₁ and ¹³C-¹H NOE, for DMPC in DMPC/DHPC bicelles and for DHPC in micelles showed that the lipid acyl chains in the bicelles become less flexible in the presence of either of the fragments. An even more pronounced effect is seen on the glycerol carbons. ²H NMR spectra of magnetically aligned bicelles showed that the peptide derived from KvAP had no or little effect on bilayer order, while the peptide derived from HsapBK had the effect of lowering the order of the bilayer. The present study demonstrates that the fragments derived from the full-length proteins interact with the bilayered interior of model membranes, and that they affect both the local mobility and lipid order of model membrane systems.     

Hemolytic Activity of Membrane-Active Peptides Correlates with the Thermodynamics of Binding to 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine Bilayers

Understanding the mechanisms of antimicrobial, cytolytic and cell-penetrating peptides is important for the design of new peptides to be used as cargo-delivery systems or antimicrobials. But these peptides should not be hemolytic. Recently, we designed a series of such membrane-active peptides and tested several hypotheses about their mechanisms on model membranes. To that end, the Gibbs free energy of binding to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles was determined experimentally. Because the main lipid components of the outermost monolayer of erythrocyte membranes are zwitterionic, like POPC, we hypothesized that the Gibbs free energy of binding of these peptides to POPC would also be a good indicator of their hemolytic activity. Now, the hemolytic activity of those synthetic peptides was examined by measuring the lysis of sheep erythrocyte suspensions after peptide addition. Indeed, the Gibbs free energy of binding was in good correlation with the hemolytic activity, which was represented by the concentration of peptide in solution that produced 50 % hemolysis. Furthermore, with two exceptions, those peptides that caused graded dye release from POPC vesicles were also hemolytic, while most of those that caused all-or-none release were not.     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from +4 to +5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an alpha-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

Membrane fusion activity of the influenza virus hemagglutinin: interaction of HA2 N-terminal peptides with phospholipid vesicles

We have investigated the interaction of a number of synthetic 20-residue peptides, corresponding to the HA2 N-terminus of the influenza virus hemagglutinin (X31 strain), with phospholipid vesicles and monolayers. Besides the wild-type sequence, two peptides were studied with mutations corresponding to those previously studied in entire HA's expressed in transfected cells [Gething et al., (1986) J. Cell. Biol. 102, 11-23]. These mutations comprised a single Glu replacement for Gly at the N-terminus ("El" mutant) or at position 4 ("E4") of the HA2 subunit and were shown to produce striking alterations in virus-induced hemolysis and syncytia formation, especially for E1. The X31 "wild-type" (wt) peptide and its E4 variant are shown here to have the capacity to insert into phosphatidylcholine (POPC) large unilamellar vesicle (LUV) membranes in a strictly pH-dependent manner, penetration being marginal at pH 7.4 and significant at pH 5.0. Bilayer insertion was evident from a shift in the intrinsic Trp fluorescence of the wt and E4 peptides and from the induction of calcein leakage from POPC LUV and correlated well with the peptides' ability at pH 5.0 to penetrate into POPC monolayers at initial surface pressures higher than 30 mN/m. By contrast, the E1 peptide was found, at pH 5.0, to bind less tightly to vesicles (assessed by a physical separation method) and to cause much less leakage of POPC LUV than the wt, even under conditions where the peptides were bound to approximately the same extent. Consistent with the correlation between leakage and penetration observed for the wt peptide at pH 5 versus 7, the E1 peptide, even at low pH, showed much less lipid-vesicle-induced shift of its Trp fluorescence than wt, caused a much slower rate of leakage of vesicle contents, and did not insert into POPC monolayers at surface pressures beyond 28.5 mN/m. Circular dichroism spectroscopy measurements of peptides in POPC SUV showed that the conformations of all three peptides are sensitive to pH, but only the wt and E4 peptides became predominantly alpha-helical at acid pH.     

Biophysical characterization and antitumor activity of synthetic Pantinin peptides from scorpion's venom

Antimicrobial peptides have been extensively described as bioactive agents, mainly considering their selective toxicity towards bacteria but not to healthy mammalian cells. In past years, this class of compounds has been classified as an attractive and novel family of anticancer agents. Pantinin peptides isolated from scorpion Pandinus imperator presented antimicrobial activity. In this study, we have explored the in vitro antitumor activity of antimicrobial pantinin peptides against the tumor cell lines MDA-MB-231 (breast adenocarcinoma) and DU − 145 (prostate adenocarcinoma) and healthy fibroblasts HGF − 1. To further improve our mechanistic understanding for this class of compounds, we have also performed a biophysical characterization of these peptides in lipid model membranes. Cell viability assays revealed that all peptides were more effective on tumor cells when compared to fibroblasts, indicating selectivity towards cancer cells. Furthermore, flow cytometry analysis revealed that all peptides induced apoptosis in cancer cells in a different way from fibroblasts. Circular dichroism spectroscopy showed that all peptides adopted an α-helical structure and an evaluation of the binding constant indicates a higher affinity of the peptides to negatively charged phospholipids. Additionally, permeabilization assays showed that POPG and POPS anionic vesicles were more susceptible to peptide-induced lysis than POPC:Chol and POPC:POPE vesicles. Moreover, we have observed that increasing concentrations of cholesterol inhibits peptide binding process. Therefore, our findings suggest that Pantinin peptides may have chemotherapeutic potential for cancer treatment.     

Surface behaviour and peptide-lipid interactions of the antibiotic peptides, Maculatin and Citropin

Surface behaviour of Maculatin 1.1 and Citropin 1.1 antibiotic peptides have been studied using the Langmuir monolayer technique in order to understand the peptide-membrane interaction proposed as critical for cellular lysis. Both peptides have a spontaneous adsorption at the air-water interface, reaching surface potentials similar to those obtained by direct spreading. Collapse pressures (Π_c, stability to lateral compression), molecular areas at maximal packing and surface potentials (ΔV) obtained from compression isotherms of both pure peptide monolayers are characteristic of peptides adopting mainly α-helical structure at the interface. The stability of Maculatin monolayers depended on the subphase and increased when pH was raised. In an alkaline environment, Maculatin exhibits a molecular reorganization showing a reproducible discontinuity in the Π-A compression isotherm. Both peptides in lipid films with the zwitterionic palmitoyl-oleoyl-phosphatidylcholine (POPC) showed an immiscible behaviour at all lipid-peptide proportions studied. By contrast, in films with the anionic palmitoyl-oleoyl-phosphatidylglycerol (POPG), the peptides showed miscible behaviour when the peptides represented less than 50% of total surface area. Additional penetration experiments also demonstrated that both peptides better interact with POPG compared with POPC monolayers. This lipid preference is discussed as a possible explanation of their antibiotic properties.     

Membrane interactions of the anuran antimicrobial peptide HSP1-NH2: Different aspects of the association to anionic and zwitterionic biomimetic systems

Studies have suggested that antimicrobial peptides act by different mechanisms, such as micellisation, self-assembly of nanostructures and pore formation on the membrane surface. This work presents an extensive investigation of the membrane interactions of the 14 amino-acid antimicrobial peptide hylaseptin P1-NH₂ (HSP1-NH₂), derived from the tree-frog Hyla punctata, which has stronger antifungal than antibacterial potential. Biophysical and structural analyses were performed and the correlated results were used to describe in detail the interactions of HSP1-NH₂ with zwitterionic and anionic detergent micelles and phospholipid vesicles. HSP1-NH₂ presents similar well-defined helical conformations in both zwitterionic and anionic micelles, although NMR spectroscopy revealed important structural differences in the peptide N-terminus. ²H exchange experiments of HSP1-NH₂ indicated the insertion of the most N-terminal residues (1–3) in the DPC-d₃₈ micelles. A higher enthalpic contribution was verified for the interaction of the peptide with anionic vesicles in comparison with zwitterionic vesicles. The pore formation ability of HSP1-NH₂ (examined by dye release assays) and its effect on the size and surface charge as well as on the lipid acyl chain ordering (evaluated by Fourier-transform infrared spectroscopy) of anionic phospholipid vesicles showed membrane disruption even at low peptide-to-phospholipid ratios, and the effect increases proportionately to the peptide concentration. On the other hand, these biophysical investigations showed that a critical peptide-to-phospholipid ratio around 0.6 is essential for promoting disruption of zwitterionic membranes. In conclusion, this study demonstrates that the binding process of the antimicrobial HSP1-NH₂ peptide depends on the membrane composition and peptide concentration.     

Lysylated phospholipids stabilize models of bacterial lipid bilayers and protect against antimicrobial peptides

Aminoacylated phosphatidylglycerols are common lipids in bacterial cytoplasmic membranes. Their presence in Staphylococcus aureus has been linked to increased resistance to a number of antibacterial agents, including antimicrobial peptides. Most commonly, the phosphatidylglycerol headgroup is esterified to lysine, which converts anionic phosphatidylglycerol into a cationic lipid with a considerably increased headgroup size. In the present work, we investigated the interactions of two well-studied antimicrobial peptides, cecropin A and mastoparan X, with lipid vesicles composed of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), containing varying fractions of an aminoacylated phosphatidylethanolamine, a stable analog of the corresponding phosphatidylglycerol-derivative. To differentiate between the effects of headgroup size and charge on peptide–lipid interactions, we synthesized two different derivatives. In one, the headgroup was modified by the addition of lysine, and in the other, by glutamine. The modification by glutamine results in a phospholipid with a headgroup size comparable to that of the lysylated version. However, whereas lysyl-phosphatidylethanolamine (Lys-PE) is cationic, glutaminyl-phosphatidylethanolamine (Gln-PE) is zwitterionic. We found that binding of mastoparan X and cecropin A was not significantly altered if the content of aminoacylated phosphatidylethanolamines did not exceed 20 mol.%, which is the concentration found in bacterial membranes. However, a lysyl-phosphatidylethanolamine content of 20 mol% significantly inhibits dye release from lipid vesicles, to a degree that depends on the peptide. In the case of mastoparan X, dye release is essentially abolished at 20 mol.% lysyl-phosphatidylethanolamine, whereas cecropin A is less sensitive to the presence of lysyl-phosphatidylethanolamine. These observations are understood through the complex interplay between peptide binding and membrane stabilization as a function of the aminoacylated lipid content. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.