Wasp Mastoparans Follow the Same Mechanism as the Cell-Penetrating Peptide Transportan 10

Abstract

We have been examining the mechanism and kinetics of the interactions of a selected set of peptides with phospholipid membranes, in a quantitative manner. This set was chosen to cover a broad range of physical-chemical properties and cell specificities. Mastoparan (masL) and mastoparan X (masX) are two similar peptides from the venoms of the wasps Vespula lewisii and Vespa xanthoptera, respectively, and were chosen to complete the set. The rate constants for masX association with, and dissociation from membranes are reported here for the first time. The kinetics of dye efflux induced by both mastoparans from phospholipid vesicles were also examined and quantitatively analyzed. We find that masL and masX follow the same graded kinetic model that we previously proposed for the cell-penetrating peptide transportan 10 (tp10), but with different parameters. This comparison is relevant because tp10 is derived from masL by addition of a mostly nonpolar segment of 7 residues at the N-terminus. Tp10 is more active than the mastoparans toward phosphatidylcholine vesicles, but the mastoparans are more sensitive to the effect of anionic lipids. Furthermore, the Gibbs free energies of binding and insertion of the peptides calculated using the Wimley-White transfer scales are in good agreement with the values derived from our experimental data, and are useful for understanding peptide behavior.During the past several years, we have been examining the mechanisms of a set of antimicrobial, cytolytic, and cell-penetrating peptides using a unified approach that includes a quantitative analysis of their function on phospholipid large unilamellar vesicles (LUVs) 1 . In this manner, all peptides can be directly compared, and the origin of the differences and similarities in their mechanisms better rationalized. This set of peptides includes δ-lysin (1, 2), cecropin A (3), magainin 2 (4), and the cell-penetrating peptide transportan 10 (tp10) (5, 6), all of which form amphipathic α-helices when bound to membrane surfaces. These peptides vary in length from 21-37 residues, which is enough to span the lipid bilayer if inserted perpendicularly to the membrane surface, and their net charges vary from 0 to +7 at pH 7.5. Thus, they cover a broad range of physical-chemical properties. Tp10 is a man-made, 21-residue, chimeric construct obtained by linking the 6 residues of the neuropeptide galanin, through an extra lysine residue, to the 14 residues of mastoparan from Vespula lewisii, which becomes the C-terminal segment of tp10 (7,8). Previously, we investigated the kinetics of tp10 binding to phospholipid membranes, the kinetics of tp10-induced dye efflux from vesicles, and its mechanism of dye release (5). Tp10 causes graded release of lipid vesicle contents, and its interaction with membranes is consistent with peptide translocation across the bilayer (5), which is essential for its function (8). † This work was supported by National Institutes of Health Grant GM072507. *To whom correspondence should be addressed: Tel: (910) 962-...

In Vitro Activity of Telavancin in Combination with Colistin versus Gram-Negative Bacterial Pathogens

“…Mutations in either the LPS biosynthetic genes or the regulators of two-component systems involved in their expression have also been shown to lead to acquired COL resistance in a number of Gram-negative species (1, 11), making it unlikely that a TLV-COL combination would be active against these isolates as well. A number of other cell-permeabilizing peptides have been shown to be active against COL-resistant organisms, notably mastaparan (24,28), and it may prove useful to investigate the activities of glycopeptides in combination with these compounds. The combination was also only found to be synergistic versus P. aeruginosa NCTC 27853 in the time-kill assay, despite the strain appearing to be fully susceptible to COL when we used static methods (Etest or broth microtiter dilution).…”

Lipid-packing perturbation of model membranes by pH-responsive antimicrobial peptides

“…The lytic activity is analyzed using a doseresponse curve, % leakage versus peptide concentration, and by the kinetic of fluorescence intensity change due to the leakage (Jin et al 2005;Rautenbach et al 2006;Wang et al 2012). Alternatively, the fluorescent dye, 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), can be entrapped with its quencher, p-xylenebis-pyridinum bromide (DPX), and, upon release, it can be re-quenched by further addition of the quencher (Yandek et al 2009). This method allows the amount of dye released and the amount that remained entrapped after the peptide action to be monitored.…”

Strategies for Exploring Electrostatic and Nonelectrostatic Contributions to the Interaction of Helical Antimicrobial Peptides with Model Membranes

“…In their experiments they used stopped flow for the rapid mixture of peptide and lipid vesicles containing a fluorescent dye (carboxyfluorescein) or a dye and its quencher (ANTS and DPX). They monitored changes in the fluorescence intensity due to the lytic activity, and fluorescence resonant energy transfer between the peptide fluorophore and a fluorescently labeled lipid to assess peptide-membrane binding [59,112]. In this method all possible interactions are considered: peptide self-assemble and disassemble, peptide-membrane association and dissociation, and translocation of the peptide through the membrane.…”

“…It is noteworthy that the sum of the adsorption free energy with the energy for transferring the folded peptide from the bilayer interface to octanol (ΔG oct ), that is the free energy for peptide insertion (ΔG ins ) on the lipid bilayer, gives information about the type of leakage process [59,112]. For MP1, ΔG oct ¼ 12.5 kcal/mol resulting in ΔG ins between 20.0 and 21.0 kcal/mol for the lipid compositions used in the experiments showed by Leite et al [134].…”

“…When the dye 8-aminonaphthalene-1,3,6 trisulfonic acid (ANTS) is entrapped with its quencher, p-xylene-bispyridinium bromide (DPX), it is possible to monitor the amount of dye released and of the fraction that remained entrapped in the vesicles simultaneously. These experiments provide information on the nature of dye release as all-or-none [59], whose mechanism was described by Parente et al [26], or as graded, whose mechanism was named sinking rafts [30].…”