The technique of fluorescence recovery after photobleaching is used to perform an extensive study of the lateral diffusion of a phospholipid probe in the binary mixture dimyristoylphosphatidylcholine/cholesterol, above the melting temperature of the phospholipid. In the regions of the phase diagram where a single liquid phase exists, diffusion can be quantitatively described by free volume theory, using a modified Macedo-Litovitz hybrid equation. In the liquid-liquid immiscibility region, the temperature dependence of the diffusion coefficient is in excellent agreement with current theories of generalized diffusivities in composite two-phase media. A consistent interpretation of the diffusion data can be provided based essentially on the idea that the primary effect of cholesterol addition to the bilayer is to occupy free volume. On this basis, a general interpretation of the phase behavior of this mixture is also proposed.

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 mutual interactions between lipids in bilayers are reviewed, including mixtures of phospholipids, and mixtures of phospholipids and cholesterol (Chol). Binary mixtures and ternary mixtures are considered, with special emphasis on membranes containing Chol, an ordered phospholipid, and a disordered phospholipid. Typically the ordered phospholipid is a sphingomyelin (SM) or a long-chain saturated phosphatidylcholine (PC), both of which have high phase transitions temperatures; the disordered phospholipid is 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) or dioleoylphosphatidylcholine (DOPC). The unlike nearest-neighbor interaction free energies (omega(AB)) between lipids (including Chol), obtained by an variety of unrelated methods, are typically in the range of 0-400 cal/mol in absolute value. Most are positive, meaning that the interaction is unfavorable, but some are negative, meaning it is favorable. It is of special interest that favorable interactions occur mainly between ordered phospholipids and Chol. The interpretation of domain formation in complex mixtures of Chol and phospholipids in terms of phase separation or condensed complexes is discussed in the light of the values of lipid mutual interactions.

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.