Paulo F. Almeida

Mechanisms of Antimicrobial, Cytolytic, and Cell-Penetrating Peptides: From Kinetics to Thermodynamics

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; however, they all form amphipathic alpha-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 experiments in model membranes. We now propose the hypothesis that it is the thermodynamics of the insertion of the peptide into the membrane, from a surface-bound state, that determine the mechanism.

Thermodynamics of lipid interactions in complex bilayers

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.

Investigation of Domain Formation in Sphingomyelin/Cholesterol/POPC Mixtures by Fluorescence Resonance Energy Transfer and Monte Carlo Simulations

We have recently proposed a phase diagram for mixtures of porcine brain sphingomyelin (BSM), cholesterol (Chol), and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) on the basis of kinetics of carboxyfluorescein efflux induced by the amphipathic peptide δ-lysin. Although that study indicated the existence of domains, phase separations in the micrometer scale have not been observed by fluorescence microscopy in BSM/Chol/POPC mixtures, though they have for some other sphingomyelins (SM). Here we examine the same BSM/Chol/POPC system by a combination of fluorescence resonance energy transfer (FRET) and Monte Carlo simulations. The results clearly demonstrate that domains are formed in this system. Comparison of the FRET experimental data with the computer simulations allows the estimate of lipid-lipid interaction Gibbs energies between SM/Chol, SM/POPC, and Chol/POPC. The latter two interactions are weakly repulsive, but the interaction between SM and Chol is favorable. Furthermore, those three unlike lipid interaction parameters between the three possible lipid pairs are sufficient for the existence of a closed loop in the ternary phase diagram, without the need to involve multibody interactions. The calculations also indicate that the largest POPC domains contain several thousand lipids, corresponding to linear sizes of the order of a few hundred nanometers.

Magainin 2 Revisited: A Test of the Quantitative Model for the All-or-None Permeabilization of Phospholipid Vesicles

The all-or-none kinetic model that we recently proposed for the antimicrobial peptide cecropin A is tested here for magainin 2. In mixtures of phosphatidylcholine (PC)/phosphatidylglycerol (PG) 50:50 and 70:30, release of contents from lipid vesicles occurs in an all-or-none fashion and the differences between PC/PG 50:50 and 70:30 can be ascribed mainly to differences in binding, which was determined independently and is approximately 20 times greater to PC/PG 50:50 than to 70:30. Only one variable parameter, beta, corresponding to the ratio of the rates of pore opening to pore closing, is used to fit dye release kinetics from these two mixtures, for several peptide/lipid ratios ranging from 1:25 to 1:200. However, unlike for cecropin A where it stays almost constant, beta increases five times as the PG content of the vesicles increases from 30 to 50%. Thus, magainin 2 is more sensitive to anionic lipid content than cecropin A. But overall, magainin follows the same all-or-none kinetic model as cecropin A in these lipid mixtures, with slightly different parameter values. When the PG content is reduced to 20 mol %, dye release becomes very low; the mechanism appears to change, and is consistent with a graded kinetic model. We suggest that the peptide may be inducing formation of PG domains. In either mechanism, no peptide oligomerization occurs and magainin catalyzes dye release in proportion to its concentration on the membrane in a peptide state that we call a pore. We envision this structure as a chaotic or stochastic type of pore, involving both lipids and peptides, not a well-defined, peptide-lined channel.

Molecular Dynamics Studies of Transportan 10 (Tp10) Interacting with a POPC Lipid Bilayer

We performed a series of molecular dynamics simu lations to study the nature of interactions between transportan 10 (tp10) and a zwitterionic POPC bilayer. Tp10 is an amphipathic cell-penetrating peptide with a net positive charge of +5 and is known to adopt an α-helical secondary structure on the surface of POPC membranes. The study showed that tp10 preferentially binds to the membrane surface with its hydrophobic side facing the hydrophobic lipid core. Such orientation allows Lys residues, with positively charged long side chains, to stay in the polar environment during the insertion process. The simulations revealed that the Lys-phosphate salt bridge is a key factor in determining the orientation of the peptide in the interfacial region as well as in stabilizing the peptide-membrane interaction. The electrostatic attraction between Lys and phosphate groups is also believed to be the main bottleneck for the translocation of tp10 across the membrane.

Translocation of cationic amphipathic peptides across the membranes of pure phospholipid giant vesicles

The ability of amphipathic polypeptides with substantial net positive charges to translocate across lipid membranes is a fundamental problem in physical biochemistry. These peptides should not passively cross the bilayer nonpolar region, but they do. Here we present a method to measure peptide translocation and test it on three representative membrane-active peptides. In samples of giant unilamellar vesicles (GUVs) prepared by electroformation, some GUVs enclose inner vesicles. When these GUVs are added to a peptide solution containing a membrane-impermeant fluorescent dye (carboxyfluorescein), the peptide permeabilizes the outer membrane, and dye enters the outer GUV, which then exhibits green fluorescence. The inner vesicles remain dark if the peptide does not cross the outer membrane. However, if the peptide translocates, it permeabilizes the inner vesicles as well, which then show fluorescence. We also measure translocation, simultaneously on the same GUV, by the appearance of fluorescently labeled peptides on the inner vesicle membranes. All three peptides examined are able to translocate, but to different extents. Peptides with smaller Gibbs energies of insertion into the membrane translocate more easily. Further, translocation and influx occur broadly over the same period, but with very different kinetics. Translocation across the outer membrane follows approximately an exponential rise, with a characteristic time of 10 min. Influx occurs more abruptly. In the outer vesicle, influx happens before most of the translocation. However, some peptides cross the membrane before any influx is observed. In the inner vesicles, influx occurs abruptly sometime during peptide translocation across the membrane of the outer vesicle.

Wasp mastoparans follow the same mechanism as the cell-penetrating peptide transportan 10.

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 seven 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.

Hydrogen-bond energetics drive helix formation in membrane interfaces

The free energy cost ΔG of partitioning many unfolded peptides into membrane interfaces is unfavorable due to the cost of partitioning backbone peptide bonds. The partitioning cost is dramatically reduced if the peptide bonds participate in hydrogen bonds. The reduced cost underlies secondary structure formation by amphiphilic peptides partitioned into membrane interfaces through a process referred to as partitioning-folding coupling. This coupling is characterized by the free energy reduction per residue, ∆G(res) that drives folding. There is some debate about the correct value of ∆G(res) and its dependence on the hydrophobic moment (μ(H)) of amphiphilic α-helical peptides. We show how to compute ∆G(res) correctly. Using published data for two families of peptides with different hydrophobic moments and charges, we find that ∆G(res) does not depend upon μ(H). The best estimate of ∆G(res) is -0.37 ± 0.02 kcal mol(-1). This article is part of a Special Issue entitled: Membrane protein structure and function.

A Simple Thermodynamic Model of the Liquid-Ordered State and the Interactions between Phospholipids and Cholesterol

A theoretical model is proposed to describe the heat capacity function and the phase behavior of binary mixtures of phospholipids and cholesterol. The central idea is that the liquid-ordered state (L(o)) is a thermodynamic state or an ensemble of conformations of the phospholipid, characterized by enthalpy and entropy functions that are intermediate between those of the solid and the liquid-disordered (L(d)) states. The values of those thermodynamic functions are such that the L(o) state is not appreciably populated in the pure phospholipid, at any temperature, because either the solid or the L(d) state have much lower free energies. Cholesterol stabilizes the L(o) state by nearest-neighbor interactions, giving rise to the appearance of the L(o) phase. The model is studied by Monte Carlo simulations on a lattice with nearest-neighbor interactions, which are derived from experiment as much as possible. The calculated heat capacity function closely resembles that obtained by calorimetry. The phase behavior produced by the model is also in agreement with experimental data. The simulations indicate that separation between solid and L(o) phases occurs below the melting temperature of the phospholipid (T(m)). Above T(m), small L(d) and L(o) domains do exist, but there is no phase separation.

A Thermodynamic Approach to the Mechanism of Cell-Penetrating Peptides in Model Membranes

We report a first test of the hypothesis that the mechanism of antimicrobial, cytolytic, and amphipathic cell-penetrating peptides in model membranes is determined by the thermodynamics of insertion of the peptide into the lipid bilayer from the surface-associated state. Three peptides were designed with minimal mutations relative to the sequence of TP10W, the Y3W variant of transportan 10, which is a helical, amphipathic cell-penetrating peptide previously studied. Binding to 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) membranes and release of dye from those vesicles were assessed by stopped-flow fluorescence, and the secondary structure of the peptides on the membrane was determined by circular dichroism. The Gibbs energy of binding determined experimentally was in excellent agreement with that calculated using the Wimley-White interfacial hydrophobicity scale, taking into account the helical content of the membrane-associated peptide. Release of dye from POPC vesicles remained graded, as predicted by the hypothesis. More significantly, as the Gibbs energy of insertion into the bilayer became more unfavorable, which was estimated using the Wimley-White octanol hydrophobicity scale, dye release became slower, in quantitative agreement with the prediction.