The mechanisms of six different antimicrobial, cytolytic, and cell-penetrating peptides, including some of their variants, are discussed and compared. The specificity of these polypeptides varies, but they all form amphipathic α-helices when bound to membranes, and there are no striking differences in their sequences. We have examined the thermodynamics and kinetics of their interaction with phospholipid vesicles, namely binding and peptide-induced dye efflux. The thermodynamics of binding calculated using the Wimley-White interfacial hydrophobicity scale are in good agreement with the values derived from experiment. The generally accepted view that binding affinity determines functional specificity is also supported by experiment in model membranes. We now propose the hypothesis that it is the thermodynamics of peptide insertion into the membrane, from a surface-bound state, that determines the mechanism.During the past three decades, a vast number of antimicrobial peptides (1,2) and other related cytolytic peptides (3) have been discovered and their mechanisms examined. More recently, several cell-penetrating peptides have been described, which allow for transport of large molecules, such as proteins or DNA fragments, into cells (4-8). Perhaps surprisingly, many of these antimicrobial, cytolytic, and cell-penetrating peptides fall into the same structural class: they form an amphipathic α-helix of some 14-40 residues, when bound to a membrane surface. Yet, they show remarkable specificity regarding the target membrane or organism. What has befuddled researchers for a long time is the absence of a correlation between sequence and function or mechanism. The only element that appears to separate antimicrobial from cytolytic peptides is that antimicrobials are usually cationic. This provides a simple explanation for their specificity because cationic peptides should bind better to the anionic membranes of most bacteria than to the neutral membranes of eukaryotic cells (9).We now critically review results obtained over the past several years on a set of representative antimicrobial, cytolytic, and cell-penetrating peptides. The interactions of these peptides with model membranes were all studied with the same methods and under similar conditions. Experiments using small unilamellar vesicles (SUV) 1 were common in the past but we purposely exclude them because of the strained nature of those vesicles, concentrating instead on studies that use unstrained vesicles, such as large (LUV) or giant unilamellar vesicles (GUV). The results are, therefore, directly comparable. On the basis of a quantitative analysis of the kinetics and thermodynamics of these interactions, we propose the hypothesis that the peptide sequence only specifies the mechanism indirectly, through the thermodynamics of peptide insertion into the bilayer medium from the surface-bound state. This would explain the lack of direct correlation between sequence and mechanism. † This work was supported by National Institutes of Health Grant GM072507....
The mechanism of the interaction between the cell-penetrating peptide transportan 10 (tp10) and phospholipid membranes was investigated. Tp10 induces graded release of the contents of phospholipid vesicles. The kinetics of peptide association with vesicles and peptide-induced dye efflux from the vesicle lumen were examined experimentally by stopped-flow fluorescence. The experimental kinetics were analyzed by directly fitting to the data the numerical solution of mathematical kinetic models. A very good global fit was obtained using a model in which tp10 binds to the membrane surface and perturbs it because of the mass imbalance thus created across the bilayer. The perturbed bilayer state allows peptide monomers to insert transiently into its hydrophobic core and cross the membrane, until the peptide mass imbalance is dissipated. In that transient state tp10 "catalyzes" dye efflux from the vesicle lumen. These conclusions are consistent with recent reports that used molecular dynamics simulations to study the interactions between peptide antimicrobials and phospholipid bilayers. A thermodynamic analysis of tp10 binding and insertion in the bilayer using water-membrane transfer hydrophobicity scales is entirely consistent with the model proposed. A small bilayer perturbation is both necessary and sufficient to achieve very good agreement with the model, indicating that the role of the lipids must be included to understand the mechanism of cell-penetrating and antimicrobial peptides.
The mechanism of the all-or-none release of the contents of phospholipid vesicles induced by the antimicrobial peptide cecropin A was investigated. A detailed experimental study of the kinetics of dye release showed that the rate of release increases with the ratio of peptide bound per vesicle and, at constant concentration, with the fraction of the anionic lipid phosphatidylglycerol in neutral, phosphatidylcholine membranes. Direct measurement of the kinetics of peptide binding and dissociation from vesicles revealed that the on-rate is almost independent of vesicle composition, whereas the off-rate decreases by orders of magnitude with increasing content of anionic lipid. A simple, exact model fits all experimental kinetic data quantitatively. This is the first time that an exact kinetic model is implemented for a peptide with an all-or-none mechanism. In this model, cecropin A binds reversibly to vesicles, which at a certain point enter an unstable state. In this state, a pore probably opens and all vesicle contents are released, consistent with the all-or-none mechanism. This pore state is a state of the whole vesicle, but does not necessarily involve all peptides on that vesicle. No peptide oligomerization on the vesicle is involved; alternative models that assume oligomerization are inconsistent with the experimental data. Thus, formation of well-defined, peptide-lined pores is unlikely.
Delta-lysin is a 26-residue, amphipathic, alpha-helical peptide of bacterial origin. Its specificity is to some extent complementary to that of antimicrobial peptides. Therefore, understanding its mechanism is important for the more general goal of understanding the interaction of amphipathic peptides with membranes. In this article, we show that delta-lysin induces graded efflux of the contents of phosphatidylcholine vesicles. In view of this finding, carboxyfluorescein efflux kinetics were re-examined. In addition, peptide-induced lipid flip-flop was directly measured using fluorescence energy transfer between two lipid fluorophores initially placed on opposite leaflets of the bilayer. Carboxyfluorescein efflux and lipid flip-flop occur with essentially identical rate constants. On the basis of a detailed, quantitative analysis of the kinetics of peptide-vesicle interactions, we conclude that the peptide translocates across the bilayer as a small, transient aggregate, most likely a trimer. Dye efflux and lipid flip-flop occur concomitantly with the transient peptide-induced perturbation of the membrane. The experimental data are interpreted by comparing the predictions of the available models for the mechanism of action of amphipathic alpha-helical peptides. We demonstrate how the combination of the quantitative kinetic analysis, graded efflux, and reversibility of the peptide-vesicle interaction can be used to reject several models for this particular peptide. Two models are compatible with the data, the toroidal pore model and the sinking raft model. On the basis of the small aggregate size, a trimer, the latter appears to be more plausible. Some significant modifications are introduced in the sinking raft model to take into account the new finding of graded dye release. Furthermore, we present an explanation for the phenomenon of graded release in general, which, contrary to all-or-none efflux, has not been well-understood.
Delta-lysin is a 26 amino acid, hemolytic peptide toxin secreted by Staphylococcus aureus. It has been reported to form an amphipathic helix upon binding to lipid bilayers and is often cited as a typical example of the barrel-stave model for pore formation in lipid bilayer membranes. However, the exact mechanism by which it lyses cells and the physical basis of its target specificity are still unknown. Moreover, the evidence for delta-lysin insertion and pore formation in the membrane stems largely from theoretical modeling of the toxin and lacks experimental confirmation. We investigated binding and insertion of delta-lysin into phospholipid bilayer vesicles. The kinetics of these processes were studied by stopped-flow fluorescence with two types of experiments: (a) carboxyfluorescein release from the vesicles upon peptide-vesicle interaction, with concomitant relief of dye self-quenching; (b) fluorescence energy transfer from the intrinsic tryptophan of the peptide to a membrane-bound lipid probe. We formulated a detailed kinetic mechanism with explicit molecular rate constants for peptide binding, association, and insertion, obtaining a quantitative description of the experimental results. delta-Lysin insertion is strongly dependent on the peptide-to-lipid ratio, suggesting that association of a critical number of monomers on the membrane is required for activity. However, we found no evidence for a stable membrane-inserted pore. Rather, the peptide appears to cross the membrane rapidly and reversibly and cause release of the lipid vesicle contents in this process.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.