Study of structure and orientation of mesentericin Y105, a bacteriocin from Gram-positive Leuconostoc mesenteroides, and its Trp-substituted analogues in phospholipid environments

AbstractMesentericin Y105 (Mes-Y105) is a bacteriocin secreted by Leuconostoc mesenteroides which is particularly active on Listeria. It is constituted by 37 residues and reticulated by one disulfide bridge. It has two W residues, W18 and W37, which can be studied by fluorescence. Two single substituted W/F analogues were synthesized (Mes-Y105/W18 and Mes-Y105/W37) to differentiate the local environment around each W and to study their changes in the presence of lipid vesicles.Fluorescence experiments show that, for the pure Trp-analogues, W18 and W37 are fully exposed to solvent whatever pH and buffer conditions. In the presence of lipid vesicles, both became buried. Lipid affinities were estimated: they are weak for zwitterionic phospholipids but an order of magnitude higher for negatively charged phosphatidylserine (PS) and phosphatidylglycerol (PG) lipids. On negatively charged PG lipids, Mes-Y105 and Mes-Y105/W37 display comparable lipid affinities. A decrease in lipid affinity is observed for Mes-Y105/W18 compared to Mes-Y105, which means that W37 would seem to be required for increased lipid selectivity. In the lipid-bound state W18 is strongly dehydrated, probably embedded into the acyl chains, while W37 stands more at the interface.Mes-Y105 was also studied by polarization modulation infrared reflection absorption spectroscopy (PMIRRAS), alone and in various phospholipid environments, to obtain structural information and to assess lipid perturbations. At nanomolar concentrations close to those required for anti-Listeria activity, Mes-Y105 forms films at the air/water interface and inserts into negatively charged lipid monolayers. In situ infrared data show that Mes-Y105 binding only affects the polar head group vibrations while the lipid order of the acyl chains remains unaffected. The PMIRRAS show that Mes-Y105 folds into an N-terminal antiparallel β-sheet followed by an α-helix, both structures being tilted (40°) compared to the normal at the interface, which is in agreement with the thickness estimated by Brewster angle microscopy (BAM). All these data support the proposal of a new model for Mes-Y105 at the membrane interface

Raman Spectroscopy of Synthetic Antimicrobial Frog Peptides Magainin 2a and PGLa

Magainin and PGLa are 23- and 21-residue peptides isolated from the skin of the African clawed frog Xenopus lueuis. They protect the frog from infection and exhibit a broad-spectrum antimicrobial activity in vitro. The mechanism of this activity involves the interaction of magainin with microbial membranes. We have measured the secondary structure and membrane-perturbing ability of these peptides to obtain information about this mechanism. Our results show that mgn2a forms a helix with an average length of less than 20 Å upon binding to liposomes. At high concentrations (50 mg/mL) mgn2a spontaneously solubilizes phosphatidylcholine liposomes at temperatures above the gel-liquid-crystalline phase transition. Mgn2a appears to bind to the surface of liposomes made of negatively charged lipids without spontaneously penetrating the bilayer. Finally, mgn2a and PGLa interact together with liposomes in a synergistic way that enhances the helix content of one or both of the peptides and allows the peptides to more easily penetrate the bilayer. PGLa mixed with a small nonperturbing amount of magainin 2 amide is 25-43 times as potent as PGLa alone at inducing the release of carboxyfluorescein from liposomes. The results suggest that the mechanism of antimicrobial activity does not involve a channel formed by transmembrane helical peptides

Molecular mechanism of action of tyrocidine antimicrobial peptides using NMR spectroscopy and computational techniques

Includes abstract.Includes bibliographical references.The need to come up with new and novel antibiotics that utilize unique mechanisms, to which bacteria cannot generate resistance, was the main motivation of this study. Tyrocidine peptides are non-selective antibiotics that have such properties. However, very limited information is available about their mechanism of action. The aim of this study was to determine the mechanism of action of tyrocidine peptides, tyrocidine A, tyrocidine B and tyrocidine C

Development of Amphipathic Beta-Strand Mimics as Potential Membrane Active Antibiotics

In recent years, there have been increasing numbers of bacterial strains emerging that are resistant to the currently available antibiotics. In the search for new antibiotics, attention has been focused on natural antimicrobial peptides that act by selectively disrupting the membranes of bacterial cells, a mechanism that is thought to be non- conducive to the development of resistance. It is desirable to mimic the structures and activities of these peptides, while introducing properties such as resistance to proteolytic degradation, which make molecules more ideal for development as drugs. Described here is the design and synthesis of P-strand mimetic oligomers based on alternating a-amino acids and azacyclohexenone units that segregate cationic lysine and hydrophobic valine side chains on opposite faces of the p-strand. *HNMR dilution studies demonstrated that despite the incorporation of alternating D- and L-amino acids in order to obtain facial amphiphilicity, these oligomers are capable of dimerizing to P-sheet mimics in a manner similar to the oligomers containing all L-amino acids. The ability of the molecules to disrupt phospholipid vesicles mimicking the membranes o f both bacterial and mammalian cells was investigated using a fluorescent dye leakage assay. Several of the oligomers were found to exhibit activity and selectivity for the bacterial over mammalian membranes. Overall, these studies demonstrate the promise of this class of molecules for the development of new potential antibiotics, and provide information on the structural features that are important for activity

Interaction of 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes

We investigated the interaction of six 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes. The net charge of the peptides at neutral pH varies from -1 to +6. Circular dichroism spectra indicate that peptides with a high net positive charge tend to fold into a helical conformation in the presence of negatively charged lipid vesicles. In helical conformation, their average hydrophobic moment and hydrophobicity would render them surface-active. The composition of amino acids on the polar face of the helix in the peptides is considerably different. The peptides show variations in their ability to permeabilise zwitterionic and anionic lipid vesicles. Whereas increased net positive charge favours greater permeabilisation, the distribution of charged residues in the polar face also plays a role in determining membrane activity. The distribution of amino acids in the polar face of the helix in the peptides that were investigated do not fall into the canonical classes described. Amphipathic helices, which are part of proteins, with a pattern of amino acid distribution different from those observed in class L, A and others, could help in providing newer insights into peptide-membrane interactions

Small Changes in the Primary Structure of Transportan 10 Alter the Thermodynamics and Kinetics of its Interaction with Phospholipid Vesicles

ABSTRACT: The kinetics and thermodynamics of binding of transportan 10 (tp10) and four of its variants to phospholipid vesicles, and the kinetics of peptide-induced dye efflux, were compared. Tp10 is a 21-residue, amphipathic, cationic, cell-penetrating peptide similar to helical antimicrobial peptides. The tp10 variants examined include amidated and free peptides, and replacements of tyrosine by tryptophan. Carboxy-terminal amidation or substitution of tryptophan for tyrosine enhance binding and activity. The Gibbs energies of peptide binding to membranes determined experimentally and calculated from the interfacial hydrophobicity scale are in good agreement. The Gibbs energy for insertion into the bilayer core was calculated using hydrophobicity scales of residue transfer from water to octanol and to the membrane/ water interface. Peptide-induced efflux becomes faster as the Gibbs energies for binding and insertion of the tp10 variants decrease. If anionic lipids are included, binding and efflux rate increase, as expected because all tp10 variants are cationic and an electrostatic component is added. Whether the most important effect of peptide amidation is the change in charge or an enhancement of helical structure, however, still needs to be established. Nevertheless, it is clear that the changes in efflux rate reflect the differences in the thermodynamics of binding and insertion of the free and amidated peptide groups. We have recently reported a detailed investigation (1) o

Hydrophobic mismatch demonstrated for membranolytic peptides and their use as molecular rulers to measure bilayer thickness in native cells

Hydrophobic mismatch is a well-recognized principle in the interaction of transmembrane proteins with lipid bilayers. This concept was extended here to amphipathic membranolytic α-helices. Nine peptides with lengths between 14 and 28 amino acids were designed from repeated KIAGKIA motifs, and their helical nature was confirmed by circular dichroism spectroscopy. Biological assays for antimicrobial activity and hemolysis, as well as fluorescence vesicle leakage and solid-state NMR spectroscopy, were used to correlate peptide length with membranolytic activity. These data show that the formation of transmembrane pores is only possible under the condition of hydrophobic matching: the peptides have to be long enough to span the hydrophobic bilayer core to be able to induce vesicle leakage, kill bacteria, and cause hemolysis. By correlating the threshold lengths for biological activity with the biophysical results on model vesicles, the peptides could be utilized as molecular rulers to measure the membrane thickness in different cells

Mechanism of Magainin 2a Induced Permeabilization of Phospholipid Vesicles

The magainins, peptide antibiotics secreted by the frog Xenopus laevis, have previously been shown to permeabilize phospholipid vesicles. To elucidate the mechanism of permeabilization, we have conducted detailed kinetic studies of magainin 2 amide (mgn2a)hduced release of 6-carboxyfluorescein from vesicles of phosphatidylserine. The results show that dye release occurs in (at least) two stages-an initial rapid phase, with t1/2 ≈ 3 s, followed by a much slower phase that approaches zero leakage rate before all the dye is released. Light-scattering studies showed that mgn2a does not cause gross changes in vesicle structure. The peptide was found to rapidly equilibrate between vesicles; this was demonstrated by determining a binding isotherm for the peptidelipid interaction, and by showing that addition of unloaded vesicles rapidly quenches peptide-induced leakage from loaded vesicles. Transient dye release in the presence of an equilibrating peptide can be explained in two ways: (1) the peptide exists only transiently in an active form; (2) the vesicles are only transiently leaky. Preincubation of mgn2a at assay concentrations in buffer alone or with unloaded vesicles did not inactivate the peptide. Therefore, rapid leakage is probably due to transient destabilization of the vesicle upon addition of mgn2a