Design and synthesis of novel antimicrobial pseudopeptides with selective membrane-perturbation activity

By incorporating carbamate bond(s) into a cytolytic peptide, novel pseudopeptides with potent antibacterial activity and low hemolytic activity were synthesized. Circular dichroism spectra suggested that the incorporation of carbamate bond(s) decrease the alpha helical conformation of the peptide in lipid membrane circumstances, which must be regarded as a major factor for the separation of antibacterial activity from cytotoxic activity for mammalian cell. Experiments in which dye was released from vesicles indicated that the potent antibacterial activity and low hemolytic activity of the pseudopeptides must be due to their great lipid membrane selectivity. The present result suggest that backbone modifications can be a great tool for developing pseudopeptides with improved biological activity and bioavailability from cytolytic peptides.     

Structure and mode of action of the antimicrobial peptide arenicin

The solution structure and the mode of action of arenicin isoform 1, an antimicrobial peptide with a unique 18-residue loop structure, from the lugworm Arenicola marina were elucidated here. Arenicin folds into a two-stranded antiparallel beta-sheet. It exhibits high antibacterial activity at 37 and 4 degrees C against Gram-negative bacteria, including polymyxin B-resistant Proteus mirabilis. Bacterial killing occurs within minutes and is accompanied by membrane permeabilization, membrane detachment and release of cytoplasm. Interaction of arenicin with reconstituted membranes that mimic the lipopolysaccharide-containing outer membrane or the phospholipid-containing plasma membrane of Gram-negative bacteria exhibited no pronounced lipid specificity. Arenicin-induced current fluctuations in planar lipid bilayers correspond to the formation of short-lived heterogeneously structured lesions. Our results strongly suggest that membrane interaction plays a pivotal role in the antibacterial activity of arenicin.     

Effects of tryptophan residues of porcine myeloid antibacterial peptide PMAP-23 on antibiotic activity.

PMAP-23 is a 23-residue antimicrobial peptide from porcine myeloid cells. In order to determine the effects of two Trp residues in positions 7 and 21 of PMAP-23 on antibacterial activity and phospholipid vesicle interacting property, two analogues in which Ala is substituted for Trp residue in position 7 or 21 were synthesized. A(21)-PMAP-23 exhibited reduced antibacterial activity and phospholipid vesicle disrupting activity when compared to those of PMAP-23 and A(7)-PMAP-23. PMAP-23 readily interacted with model lipid membrane and induced membrane destabilization. Therefore antibacterial activity induced by PMAP-23 is due to the interaction of cell membrane with peptide followed by membrane perturbation. A significant structural change on the SDS micelle was not found by Ala substitution of the Trp residue of PMAP-23. Also, there is a good correlation between hydrophobic interaction on RP-HPLC, expressed as retention time on RP-HPLC, and antibacterial activity. The vesicle titration experiment indicated that Trp residues located at near C-terminus are accessible to hydrophobic tail of phospholipid vesicle. This result suggests that the C-terminal end of PMAP-23 penetrates into the lipid bilayer in the course of the interaction with phospholipid membranes and is important for its antibacterial activity.     

Effect of N-terminal acetylation on lytic activity and lipid-packing perturbation induced in model membranes by a mastoparan-like peptide

L1A (IDGLKAIWKKVADLLKNT-NH2) is a peptide that displays a selective antibacterial activity to Gram-negative bacteria without being hemolytic. Its lytic activity in anionic lipid vesicles was strongly enhanced when its N-terminus was acetylated (ac-L1A). This modification seems to favor the perturbation of the lipid core of the bilayer by the peptide, resulting in higher membrane lysis. In the present study, we used lipid monolayers and bilayers as membrane model systems to explore the impact of acetylation on the L1A lytic activity and its correlation with lipid-packing perturbation. The lytic activity investigated in giant unilamellar vesicles (GUVs) revealed that the acetylated peptide permeated the membrane at higher rates compared with L1A, and modified the membrane's mechanical properties, promoting shape changes. The peptide secondary structure and the changes in the environment of the tryptophan upon adsorption to large unilamellar vesicles (LUVs) were monitored by circular dichroism (CD) and red-edge excitation shift experiments (REES), respectively. These experiments showed that the N-terminus acetylation has an important effect on both, peptide secondary structure and peptide insertion into the bilayer. This was also confirmed by experiments of insertion into lipid monolayers. Compression isotherms for peptide/lipid mixed films revealed that ac-L1A dragged lipid molecules to the more disordered phase, generating a more favorable environment and preventing the lipid molecules from forming stiff films. Enthalpy changes in the main phase transition of the lipid membrane upon peptide insertion suggested that the acetylated peptide induced higher impact than the non-acetylated one on the thermotropic behavior of anionic vesicles.     

Correlation of three-dimensional structures with the antibacterial activity of a group of peptides designed based on a nontoxic bacterial membrane anchor

To understand the functional differences between a nontoxic membrane anchor corresponding to the N-terminal sequence of the Escherichia coli enzyme IIA(Glc) and a toxic antimicrobial peptide aurein 1.2 of similar sequence, a series of peptides was designed to bridge the gap between them. An alteration of a single residue of the membrane anchor converted it into an antibacterial peptide. Circular dichroism spectra indicate that all peptides are disordered in water but helical in micelles. Structures of the peptides were determined in membrane-mimetic micelles by solution NMR spectroscopy. The quality of the distance-based structures was improved by including backbone angle restraints derived from a set of chemical shifts ((1)H(alpha), (15)N, (13)C(alpha), and (13)C(beta)) from natural abundance two-dimensional heteronuclear correlated spectroscopy. Different from the membrane anchor, antibacterial peptides possess a broader and longer hydrophobic surface, allowing a deeper penetration into the membrane, as supported by intermolecular nuclear Overhauser effect cross-peaks between the peptide and short chain dioctanoyl phosphatidylglycerol. An attempt was made to correlate the NMR structures of these peptides with their antibacterial activity. The activity of this group of peptides does not correlate exactly with helicity, amphipathicity, charge, the number of charges, the size of the hydrophobic surface, or hydrophobic transfer free energy. However, a correlation is established between the peptide activity and membrane perturbation potential, which is defined by interfacial hydrophobic patches and basic residues in the case of cationic peptides. Indeed, (31)P solid state NMR spectroscopy of lipid bilayers showed that the extent of lipid vesicle disruption by these peptides is proportional to their membrane perturbation potential.     

Membrane active antimicrobial activity and molecular dynamics study of a novel cationic antimicrobial peptide polybia-MPI,from the venom of Polybia paulista

As the frequent emergence of the resistant bacteria, the development of new agents with a new action mode attracts a great deal of interest. It is now widely accepted that antimicrobial peptides (AMPs) are promising alternatives to conventional antibiotics. In this study, antimicrobial peptide polybia-MPI and its analogs were synthesized and their antibacterial activity was studied. Our results revealed that polybia-MPI has potent antibacterial activity against both Gram-positive and Gram-negative bacteria. Its ability to make PI permeate into bacteria and lead to the leakage of calcein from model membrane LUVs, suggests a killing mechanism involving membrane perturbation. SEM and TEM microscopy experiments verified that the morphology of bacteria was changed greatly under the treatment of polybia-MPI. Compared with the conventional chemotherapy, polybia-MPI targets the cell membrane rather than entering into the cell to exert its antibacterial activity. Furthermore, molecular dynamics (MD) simulations were employed to investigate the mechanism of membrane perturbation. The results indicated that the α-helical conformation in the membrane is required for the exhibition of antibacterial activity and the membrane disturbance by polybia-MPI is a cooperative process. In conclusion, with the increasing resistance to conventional antibiotics, there is no doubt that polybia-MPI could offer a new strategy to defend the resistant bacteria.     

Addition and omission analogs of the 13-residue antibacterial and hemolytic peptide PKLLKTFLSKWIG: structural preferences, model membrane binding and biological activities.

The consequences of selective addition or deletion of polar amino acids in a 13-residue antibacterial peptide PKLLKTFLSKWIG on structure, membrane binding and biological activities have been investigated. The variants generated are (a) S and T residues replaced by K, (b) S and T residues deleted individually and together, (c) introduction of two additional K and (d) deletion of L and L with T. In the aqueous environment all the peptides were unordered. In trifluoroethanol, the spectra of peptides belonging to groups (a-c) suggest distorted helical conformation. Peptides in group (d) appear to adopt beta-sheet conformation. The peptides bind to zwitterionic and negatively charged lipid vesicles, although to different extents. With the exception of peptides in group (d), all the other peptides exhibited comparable antibacterial activity against Escherichia coli and Staphylococcus aureus. However, the changes made in the peptides in groups (a-c) resulted in reduction of hemolytic activity compared to the parent peptide. Extent of binding to lipid vesicles composed of phosphatidylcholine and cholesterol appears to correlate with hemolytic activity. It appears that polar and charged residues play a major role in modulating the biological activities of the 13-residue peptide PKLLKTFLSKWIG. The 11-residue peptide-like PKLLKFLKWIG has selective antibacterial activity. Thus, by judicious engineering it should be possible to generate short peptides with selective antibacterial activity.     

Interactions of mouse Paneth cell alpha-defensins and alpha-defensin precursors with membranes. Prosegment inhibition of peptide association with biomimetic membranes

The bactericidal activity of mouse alpha-defensins (cryptdins) requires proteolytic activation of inactive precursors by matrix metalloproteinase-7 (matrilysin, EC, MMP-7(a)). To investigate mechanisms of cryptdin-4 (Crp4) peptide interactions with membrane bilayers and to determine whether MMP-7-mediated proteolysis activates the membrane disruptive activity of Crp4, associations of Crp4 and melittin with biomimetic lipid/polydiacetylene chromatic vesicles were characterized. The peptides differ in their sensitivity to vesicle lipid composition and their depth of bilayer penetration. Crp4 undergoes strong interfacial binding onto lipid bilayers with disruption of the bilayer head group region, unlike melittin, which inserts more deeply into the hydrophobic core of the bilayer. Colorimetric and tryptophan fluorescence studies showed that Crp4 insertion is favored by negatively charged phospholipids and that zwitterionic and Escherichia coli phospholipids promote stronger interfacial binding; melittin-membrane interactions were independent of either variable. In contrast to the membrane disruptive activity of Crp4, pro-Crp4 did not perturb vesicular membranes, consistent with the lack of bactericidal activity of the precursor, and incubation of Crp4 with prosegment in trans blocked Crp4 and G1W-Crp4 membrane interactions at concentrations that inhibit Crp4 bactericidal activity. CD measurements showed that Crp4 has an expected beta-sheet structure that is not evident in the pro-Crp4 CD trace or when Crp4 is incubated with prosegment, indicating that the beta-sheet signal is attenuated by proregion interactions or possibly disrupted by the prosegment. Collectively, the results suggest that the prosegment inhibits Crp4 bactericidal activity by blocking peptide-mediated perturbation of target cell membranes, a constraint that is relieved when MMP-7 cleaves the prosegment.     

Design and synthesis of cyclic disulfide-bonded antibacterial peptides on the basis of the alpha helical domain of Tenecin 1, an insect defensin

We synthesized cyclic disulfide-bonded (i, i+4) peptides with various net positive charges (+2-+5) from linear peptides derived from the alpha helical domain of Tenecin 1, an insect defensin, and investigated the effect of the intradisulfide bridge (i, i+4) on hydrophobicity, secondary structure, leakage activity and binding activity for large unilamellar vesicles, antimicrobial activity, and hemolytic activity. Intradisulfide bridge formation of the peptides resulted in the increase of amphiphilicity and hydrophobicity. Cyclic forms of the peptides did not deeply penetrate into PG/PC (1:1, mole ratio) large unilamellar vesicles and had a decreased lipid membrane perturbation activity for PG/PC LUVs. When the peptides interacted with PG/CL (2:1, mole ratio) LUVs, cyclic peptides with a high net positive charge (+4-+5) showed similar binding affinities and leakage activities for vesicles to those of linear forms, whereas cyclic peptides with a low net positive charge (+2-+3) exhibited lower leakage activity than their linear forms. CD spectra indicate that the intradisulfide bridge (i, i+4) provided little conformational constraint to linear peptides in buffer solution but resulted in the decrease of alpha helicity of the peptides in lipid membrane mimic conditions. The cyclic peptide with the highest net positive charge had a similar antibacterial activity to that of the linear peptide, whereas the cyclic peptides with a low net positive charge (+3-+4) exhibited lower antibacterial activity than their linear forms. The cyclic peptides of an appropriate net charge showed more potent activities against some bacteria than those of linear forms under high salt conditions.     

Consequences of introducing a disulfide bond into an antibacterial and hemolytic peptide.

The effect of introducing a disulfide bridge between the N- and C-terminal ends on the structure and biological activities of the 13-residue linear peptide PKLLKTFLSKWIG(SPFK), which has both antibacterial and hemolytic activity, have been investigated. The terminal amino acids P and G in SPFK were replaced by cysteines to form a disulfide bridge. The linear peptides C(Acm)KLLKTFLSKWIC(Acm) and C(Acm) KLLKTFLSKWIC(Acm)-amide, where Acm is acetamidomethyl group, showed antibacterial activity but did not possess hemolytic activity unlike SPFK. Introduction of an S-S bridge resulted in enhanced hemolytic activity compared with SPFK. The hemolytic activity was particularly pronounced in the cyclic peptide CKLLKTFLSKWIC-amide. Circular dichroism studies indicate that the cyclic peptides tend to adopt distorted helical structures. The cyclic peptides also have a greater affinity for lipid vesicles, which could be the reason for the effective perturbation of the erythrocyte membrane.     

Interaction studies of novel cell selective antimicrobial peptides with model membranes and E. coli ATCC 11775

Cationic antimicrobial peptides (CAMPs) are novel candidates for drug development. Here we describe design of six short and potent CAMPs (SA-1 to SA-6) based on a minimalist template of 12 residues H+HHG+HH+HH+NH2 (where H: hydrophobic amino acid and +: charged hydrophilic amino acid). Designed peptides exhibit good antibacterial activity in micro molar concentration range (1-32 μg/ml) and rapid clearance of Gram-positive and Gram-negative bacterial strains at concentrations higher than MIC. For elucidating mode of action of designed peptides various biophysical studies including CD and Trp fluorescence were performed using model membranes. Further based on activity, selectivity and membrane bound structure; modes of action of Trp rich peptide SA-3 and template based peptide SA-4 were compared. Calcein dye leakage and transmission electron microscopic studies with model membranes exhibited selective membrane active mode of action for peptide SA-3 and SA-4. Extending our work from model membranes to intact E. coli ATCC 11775 in scanning electron micrographs we could visualize different patterns of surface perturbation caused by peptide SA-3 and SA-4. Further at low concentration rapid translocation of FITC-tagged peptide SA-3 into the cytoplasm of E. coli cells without concomitant membrane perturbation indicates involvement of intracellular targeting mechanism as an alternate mode of action as was also evidenced in DNA retardation assay. For peptide SA-4 concentration dependent translocation into the bacterial cytoplasm along with membrane perturbation was observed. Establishment of a non specific membrane lytic mode of action of these peptides makes them suitable candidates for drug development.     

Fusogenic Alzheimer's peptide fragment Abeta (29-42) in interaction with lipid bilayers: secondary structure, dynamics, and specific interaction with phosphatidyl ethanolamine polar heads as revealed by solid-state NMR

The interaction of the native Alzheimer's peptide C-terminal fragment Abeta (29-42), and two mutants (G33A and G37A) with neutral lipid bilayers made of POPC and POPE in a 9:1 molar ratio was investigated by solid-state NMR. This fragment and the lipid composition were selected because they represent the minimum requirement for the fusogenic activity of the Alzheimer's peptide. The chemical shifts of alanine methyl isotropic carbon were determined by MAS NMR, and they clearly demonstrated that the major form of the peptide equilibrated in membrane is not in a helical conformation. (2)H NMR, performed with acyl chain deuterated POPC, demonstrated that there is no perturbation of the acyl chain's dynamics and of the lipid phase transition temperature. (2)H NMR, performed with alanine methyl-deuterated peptide demonstrated that the peptide itself has a limited mobility below and above the lipid phase transition temperature (molecular order parameter equal to 0.94). MAS (31)P NMR revealed a specific interaction with POPE polar head as seen by the enhancement of POPE phosphorus nuclei T(2) relaxation. All these results are in favor of a beta-sheet oligomeric association of the peptide at the bilayer interface, preferentially recruiting phosphatidyl ethanolamine polar heads.     

Multivalent presentation of the cell-penetrating peptide nona-arginine on a linear scaffold strongly increases its membrane-perturbing capacity

Arginine-rich cell-penetrating peptides (CPP) are widely employed as delivery vehicles for a large variety of macromolecular cargos. As a mechanism-of-action for induction of uptake cross-linking of heparan sulfates and interaction with lipid head groups have been proposed. Here, we employed a multivalent display of the CPP nona-arginine (R9) on a linear dextran scaffold to assess the impact of heparan sulfate and lipid interactions on uptake and membrane perturbation. Increased avidity through multivalency should potentiate molecular phenomena that may only play a minor role if only individual peptides are used. To this point, the impact of multivalency has only been explored for dendrimers, CPP-decorated proteins and nanoparticles. We reasoned that multivalency on a linear scaffold would more faithfully mimic the arrangement of peptides at the membrane at high local peptide concentrations. On average, five R9 were coupled to a linear dextran backbone. The conjugate displayed a direct cytoplasmic uptake similar to free R9 at concentrations higher than 10 μM. However, this uptake was accompanied by an increased membrane disturbance and cellular toxicity that was independent of the presence of heparan sulfates. In contrast, for erythrocytes, the multivalent conjugate induced aggregation, however, showed only limited membrane perturbation. Overall, the results demonstrate that multivalency of R9 on a linear scaffold strongly increases the capacity to interact with the plasma membrane. However, the induction of membrane perturbation is a function of the cellular response to peptide binding.     

Enhanced membrane pore formation through high-affinity targeted antimicrobial peptides

Many cationic antimicrobial peptides (AMPs) target the unique lipid composition of the prokaryotic cell membrane. However, the micromolar activities common for these peptides are considered weak in comparison to nisin, which follows a targeted, pore-forming mode of action. Here we show that AMPs can be modified with a high-affinity targeting module, which enables membrane permeabilization at low concentration. Magainin 2 and a truncated peptide analog were conjugated to vancomycin using click chemistry, and could be directed towards specific membrane embedded receptors both in model membrane systems and whole cells. Compared with untargeted vesicles, a gain in permeabilization efficacy of two orders of magnitude was reached with large unilamellar vesicles that included lipid II, the target of vancomycin. The truncated vancomycin-peptide conjugate showed an increased activity against vancomycin resistant Enterococci, whereas the full-length conjugate was more active against a targeted eukaryotic cell model: lipid II containing erythrocytes. This study highlights that AMPs can be made more selective and more potent against biological membranes that contain structures that can be targeted.     

Orientation of a beta-hairpin antimicrobial peptide in lipid bilayers from two-dimensional dipolar chemical-shift correlation NMR

The orientation of a beta-sheet membrane peptide in lipid bilayers is determined, for the first time, using two-dimensional (2D) (15)N solid-state NMR. Retrocyclin-2 is a disulfide-stabilized cyclic beta-hairpin peptide with antibacterial and antiviral activities. We used 2D separated local field spectroscopy correlating (15)N-(1)H dipolar coupling with (15)N chemical shift to determine the orientation of multiply (15)N-labeled retrocyclin-2 in uniaxially aligned phosphocholine bilayers. Calculated 2D spectra exhibit characteristic resonance patterns that are sensitive to both the tilt of the beta-strand axis and the rotation of the beta-sheet plane from the bilayer normal and that yield resonance assignment without the need for singly labeled samples. Retrocyclin-2 adopts a transmembrane orientation in dilauroylphosphatidylcholine bilayers, with the strand axis tilted at 20 degrees +/- 10 degrees from the bilayer normal, but changes to a more in-plane orientation in thicker 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-choline (POPC) bilayers with a tilt angle of 65 degrees +/- 15 degrees . These indicate that hydrophobic mismatch regulates the peptide orientation. The 2D spectra are sensitive not only to the peptide orientation but also to its backbone (phi, psi) angles. Neither a bent hairpin conformation, which is populated in solution, nor an ideal beta-hairpin with uniform (phi, psi) angles and coplanar strands, agrees with the experimental spectrum. Thus, membrane binding orders the retrocyclin conformation by reducing the beta-sheet curvature but does not make it ideal. (31)P NMR spectra of lipid bilayers with different compositions indicate that retrocyclin-2 selectively disrupts the orientational order of anionic membranes while leaving zwitteronic membranes intact. These structural results provide insights into the mechanism of action of this beta-hairpin antimicrobial peptide.     

The polar region consecutive to the HIV fusion peptide participates in membrane fusion

The fusion peptide of HIV-1 gp41 is formed by the 16 N-terminal residues of the protein. This 16-amino acid peptide, in common with several other viral fusion peptides, caused a reduction in the bilayer to hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine (T(H)), suggesting its ability to promote negative curvature in membranes. Surprisingly, an elongated peptide corresponding to the 33 N-terminal amino acids raised T(H), although it was more potent than the 16-amino acid fusion peptide in inducing lipid mixing with large unilamellar liposomes of 1:1:1 dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine/choleste rol. The 17-amino acid C-terminal fragment of the peptide can induce membrane fusion by itself, if it is anchored to a membrane by palmitoylation of the amino terminus, indicating that the additional 17 hydrophilic amino acids contribute to the fusogenic potency of the peptide. This is not solely a consequence of the palmitoylation, as a random peptide with the same amino acid composition with a palmitoyl anchor was less potent in promoting membrane fusion and palmitic acid itself had no fusogenic activity. The 16-amino acid N-terminal fusion peptide and the longer 33-amino acid peptide were labeled with NBD. Fluorescence binding studies indicate that both peptides bind to the membrane with similar affinities, indicating that the increased fusogenic activity of the longer peptide was not a consequence of a greater extent of membrane partitioning. We also determined the secondary structure of the peptides using FTIR spectroscopy. We find that the amino-terminal fusion peptide is inserted into the membrane as a beta-sheet and the 17 C-terminal amino acids lie on the surface of the membrane, adopting an alpha-helical conformation. It was further demonstrated with the use of rhodamine-labeled peptides that the 33-amino acid peptide self-associated in the membrane while the 16-amino acid N-terminal peptide did not. Thus, the 16-amino acid N-terminal fusion peptide of HIV inserts into the membrane and, like other viral fusion peptides, lowers T(H). In addition, the 17 consecutive amino acids enhance the fusogenic activity of the fusion peptide presumably by promoting its self-association.     

Antibacterial activity of bactenecin 5 fragments and their interaction with phospholipid membranes.

Bactenecin 5 (Bac 5) is an antibacterial 43mer peptide isolated from bovine neutrophils. It consists of an Arg-rich N-terminal region and successive repeats of Arg-Pro-Pro-Ile (or Phe). We synthesized Bac 5(1-23) and several related peptides to clarify the roles these regions play in antibacterial activity. An assay of antibacterial activity revealed that such activity requires the presence of Arg residues at or near the N-terminus, as well as a chain length exceeding 15 residues. None of the peptides exhibited haemolytic activity. Polyproline II-like CD curves were observed for most of the peptides. Measurements of the membrane perturbation and fusion indicated that the perturbation and fusogenic activities of the peptides were, generally, parallel to their antibacterial activities. Amino acid substitution in the repeating region had some effect on antibacterial activity.     

The anti-angiogenic peptide anginex disrupts the cell membrane

Anginex is a synthetic beta-sheet peptide with anti-angiogenic and anti-tumor activity. When added to cultured endothelial cells at concentrations ranging from 2.5 microM to 25 microM, anginex induced cell death, which was reflected by a strong increase of subdiploid cells and fragments, loss of cellular ATP, and LDH release. Cytotoxicity remained the same whether cells were treated with anginex at 4 degrees C or at 37 degrees C. At low temperatures, fluorescein-conjugated anginex accumulated on the endothelial surface, but did not reach into the cytoplasm, indicating that the cell membrane is the primary target for the peptide. Within minutes of treatment, anginex caused endothelial cells to take up propidium iodide and undergo depolarization, both parameters characteristic for permeabilization of the cell membrane. This process was amplified when cells were activated with hydrogen peroxide. Red blood cell membranes were essentially unaffected by anginex. Anginex bound lipid bilayers with high affinity and with a clear preference for anionic over zwitterionic phospholipids. Structural studies by circular dichroism and solid-state nuclear magnetic resonance showed that anginex forms a beta-sheet and adopts a unique and highly ordered conformation upon binding to lipid membranes. This is consistent with lipid micellization or the formation of pore-forming beta-barrels. The data suggest that the cytotoxicity of anginex stems from its ability to target and disrupt the endothelial cell membrane, providing a possible explanation for the angiostatic activity of the peptide.     

Solid-state nuclear magnetic resonance relaxation studies of the interaction mechanism of antimicrobial peptides with phospholipid bilayer membranes

An 18-residue peptide, KWGAKIKIGAKIKIGAKI-NH(2) was designed to form amphiphilic beta-sheet structures when bound to lipid bilayers. The peptide possesses high antimicrobial activity when compared to naturally occurring linear antimicrobial peptides, most of which adopt an amphipathic alpha-helical conformation upon binding to the lipids. The perturbation of the bilayer by the peptide was studied by static (31)P and (2)H solid-state NMR spectroscopy using POPC and POPG/POPC (3/1) bilayer membranes with sn-1 chain perdeuterated POPC and POPG as the isotopic labels. (31)P NMR powder spectra exhibited two components for POPG/POPC bilayers upon addition of the peptide but only a slight change in the line shape for POPC bilayers, indicating that the peptide selectively disrupted the membrane structure consisting of POPG lipids. (2)H NMR powder spectra indicated a reduction in the lipid chain order for POPC bilayers and no significant change in the ordering for POPG/POPC bilayers upon association of the peptide with the bilayers, suggesting that the peptide acts as a surface peptide in POPG/POPC bilayers. Relaxation rates are more sensitive to the motions of the membranes over a large range of time scales. Longer (31)P longitudinal relaxation times for both POPG and POPC in the presence of the peptide indicated a direct interaction between the peptide and the POPG/POPC bilayer membranes. (31)P longitudinal relaxation studies also suggested that the peptide prefers to interact with the POPG phospholipids. However, inversion-recovery (2)H NMR spectroscopic experiments demonstrated a change in the relaxation rate of the lipid acyl chains for both the POPC membranes and the POPG/POPC membranes upon interaction with the peptide. Transverse relaxation studies indicated an increase in the spectral density of the collective membrane motion caused by the interaction between the peptide and the POPG/POPC membrane. The experimental results demonstrate significant dynamic changes in the membrane in the presence of the antimicrobial peptide and support a carpet mechanism for the disruption of the membranes by the antimicrobial peptide.     

Influence of tryptophan on lipid binding of linear amphipathic cationic antimicrobial peptides

We recently demonstrated that a linear 18-residue peptide, (KIGAKI)(3)-NH(2), designed to form amphipathic beta-sheet structure when bound to lipid bilayers, possessed potent antimicrobial activity and low hemolytic activity. The ability of (KIGAKI)(3)-NH(2) to induce leakage from lipid vesicles was compared to that of the amphipathic alpha-helical peptide, (KIAGKIA)(3)-NH(2), which had equivalent antimicrobial activity. Significantly, the lytic properties of (KIGAKI)(3)-NH(2) were enhanced for mixed acidic-neutral lipid vesicles containing phosphatidylethanolamine instead of phosphatidylcholine as the neutral component, while the potency of (KIAGKIA)(3)-NH(2) was significantly reduced [Blazyk, J., et al. (2001) J. Biol. Chem. 276, 27899-27906]. In this paper, we measured the lytic properties of these peptides, as well as several fluorescent analogues containing a single tryptophan residue, by monitoring permeability changes in large unilamellar vesicles with varying lipid compositions and in Escherichia coli cells. The binding of these peptides to lipid bilayers with defined compositions was compared using surface plasmon resonance, circular dichroism, and fluorescence spectroscopy. Surprisingly large differences were observed in membrane binding properties, particularly in the case of KIGAKIKWGAKIKIGAKI-NH(2). Since all of these peptides possess the same charge and very similar mean hydrophobicities, the binding data cannot be explained merely in terms of electrostatic and/or hydrophobic interactions. In light of their equivalent antimicrobial and hemolytic potencies, some of these peptides may employ mechanisms beyond simply increasing plasma membrane permeability to exert their lethal effects.