Antimicrobial peptides are known to form pores in cell membranes. We study this process in model bilayers of various lipid compositions. We use two of the best-studied peptides, alamethicin and melittin, to represent peptides making two types of pores, that is, barrel-stave pores and toroidal pores. In both cases, the key control variable is the concentration of the bound peptides in the lipid bilayers (expressed in the peptide-lipid molar ratio, P/L). The method of oriented circular dichroism (OCD) was used to monitor the peptide orientation in bilayers as a function of P/L. The same samples were scanned by X-ray diffraction to measure the bilayer thickness. In all cases, the bilayer thickness decreases linearly with P/L and then levels off after P/L exceeds a lipid-dependent critical value, (P/L)*. OCD spectra showed that the helical peptides are oriented parallel to the bilayers as long as P/L < (P/L)*, but as P/L increases over (P/L)*, an increasing fraction of peptides changed orientation to become perpendicular to the bilayer. We analyzed the data by assuming an internal membrane tension associated with the membrane thinning. The free energy containing this tension term leads to a relation explaining the P/L-dependence observed in the OCD and X-ray diffraction measurements. We extracted the experimental parameters from this thermodynamic relation. We believe that they are the quantities that characterize the peptide-lipid interactions related to the mechanism of pore formation. We discuss the meaning of these parameters and compare their values for different lipids and for the two different types of pores. These experimental parameters are useful for further molecular analysis and are excellent targets for molecular dynamic simulation studies.Membrane active peptides, including antimicrobials and toxins, are known to induce transmembrane pores. The first peptide discovered to do so is alamethicin (1, 2). At first, alamethicin was thought to induce pores (which were detected by ion conduction) only by a transmembrane electric potential (see review in ref 3). However, numerous experiments (4-8) indicated that alamethicin could insert into bilayers in the absence of an external field (see review in ref 9). Although it was believed that alamethicin insertion would create pores, a direct correlation with ion conduction was difficult to establish. Later, with the combination of oriented circular dichroism (10, 11) and neutron diffraction (12, 13), we showed the direct correlation between alamethicin insertion (without voltage) and transmembrane pores. Two other extensively studied peptides, bee venom toxin melittin (14) and frog peptide magainin (15), also exhibited similar behaviors. Pores were evidently formed by both melittin (16-18) and magainin (19,20) because they caused leakage of fluorescent dyes from lipid vesicles. In the last 15 years, a great variety of antimicrobial peptides have been shown to induce transmembrane pores in bacterial cells as well as in lipid vesicles (see reviews in refs 2...

We suggest a physical mechanism by which antimicrobial peptides spontaneously induce stable pores in membranes. Peptide binding to a lipid bilayer causes an internal stress, or internal membrane tension, that can be sufficiently strong to create pores. Like detergents, peptides have a high affinity for the rim of the pore. Binding to the rims reduces the line tension and decreases the number of peptides causing the internal membrane tension. Consequently, the pore radius is stable. The pore formation resembles a phase transition.

How antimicrobial peptides form pores in membranes is of interest as a fundamental membrane process. However, the underlying molecular mechanism, which has potential applications in therapeutics, nonviral gene transfer, and drug delivery, has been in dispute. We have resolved this mechanism by observing the time-dependent process of pore formation in individual giant unilamellar vesicles (GUVs) exposed to a melittin solution. An individual GUV first expanded its surface area at constant volume and then suddenly reversed to expanding its volume at constant area. The area expansion, the volume expansion, and the point of reversal all match the results of equilibrium measurements performed on peptide-lipid mixtures. The mechanism includes a negative feedback that makes peptide-induced pores stable with a well defined size, contrary to the suggestion that peptides disintegrate the membrane in a detergent-like manner.antimicrobial peptides ͉ membrane-thinning effect ͉ stable membrane pore ͉ peptide-induced pore ͉ single-membrane experiment M any water-soluble amphipathic peptides spontaneously bind to membranes and form transmembrane pores when the peptide concentrations exceed certain threshold values. Such pore-forming activities are of interest for many reasons. It is the common mode of action used by the ubiquitous antimicrobial peptides (1). A similar mechanism is used by pore-forming proteins, such as the apoptosis regulator Bcl-2-associated X protein (Bax), which activates pore formation in the outer mitochondria membrane to release the apoptotic factor cytochrome c (2). Understanding the relatively simple process of pore formation by small peptides is an important step toward unraveling more complex membranous conformational changes such as membrane fusion (3). Clarifying the pore-forming mechanism will also facilitate its applications, including developing antimicrobial molecules as human therapeutics (1) or smallmolecule agents for nonviral gene transfer and drug delivery. The physical effects caused by the binding of pore-forming peptides to lipid bilayers have been studied by x-ray and neutron diffraction on peptide-lipid mixtures (4-6). The results showed that peptide binding caused membrane thinning and pores appeared only when the thinning reached a critical fraction of the membrane thickness (4-7). However, the equivalent effects have not been demonstrated by kinetic experiments. This is important because pore formation in cell membranes caused by water-soluble peptides typically occurs as a kinetic process. Here, we report the observation of the time behavior of giant unilamellar vesicles (GUVs) exposed to the peptide melittin in solution. The observed time behavior of individual lipid vesicles, although complex, exhibits the physical effects seen in equilibrium experiments, thereby confirming that the mechanism of kinetic pore formation in single membranes is the same as that governing peptide-lipid interactions in the mixtures. It also implies that the same effect is likely to occur in cell membran...

The condensing effect of cholesterol on phospholipid bilayers was systematically investigated for saturated and unsaturated chains, as a function of cholesterol concentration. X-ray lamellar diffraction was used to measure the phosphate-to-phosphate distances, PtP, across the bilayers. The measured PtP increases nonlinearly with the cholesterol concentration until it reaches a maximum. With further increase of cholesterol concentration, the PtP remains at the maximum level until the cholesterol content reaches the solubility limit. The data in all cases can be quantitatively explained with a simple model that cholesterol forms complexes with phospholipids in the bilayers. The phospholipid molecules complexed with cholesterol are lengthened and this lengthening effect extends into the uncomplexed phospholipids surrounding the cholesterol complexes. This long-range thickening effect is similar to the effect of gramicidin on the thickness of lipid bilayers due to hydrophobic matching.

Antimicrobial peptides have two binding states in a lipid bilayer, a surface state S and a pore-forming state I. The transition from the S state to the I state has a sigmoidal peptide-concentration dependence indicating cooperativity in the peptide-membrane interactions. In a previous paper, we reported the transition of alamethicin measured in three bilayer conditions. The data were explained by a free energy that took into account the membrane thinning effect induced by the peptides. In this paper, the full implications of the free energy were tested by including another type of peptide, melittin, that forms toroidal pores, instead of barrel-stave pores as in the case of alamethicin. The S-to-I transitions were measured by oriented circular dichroism. The membrane thinning effect was measured by x-ray diffraction. All data were in good agreement with the theory, indicating that the membrane thinning effect is a plausible mechanism for the peptide-induced pore formations.

The transition of the state of alamethicin from its inactive state to its active state of pore formation was measured as a function of the peptide concentration in three different membrane conditions. In each case the fraction of the alamethicin molecules occupying the active state, phi, showed a sigmoidal concentration dependence that is typical of the activities of antimicrobial peptides. Such a concentration dependence is often interpreted as due to peptide aggregation. However, we will show that a simple effect of aggregation cannot explain the data. We will introduce a model based on the elasticity of membrane, taking into consideration the membrane-thinning effect due to protein inclusion. The elastic energy of membrane provides an additional driving force for aggregation. The model produces a relation that not only predicts the correct concentration dependence but also explains qualitatively how the dependence changes with membrane conditions. The result shows that the membrane-mediated interactions between monomers and aggregates are essential for the strong cooperativity shown in pore formation.

Interaction of curcumin with lipid bilayers is not well understood. A recent experiment showed that curcumin significantly affected the single-channel lifetime of gramicidin in a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayer without affecting its single-channel conductance. We performed two experiments to understand this result. By isothermal titration calorimetry, we measured the partition coefficient of curcumin binding to DOPC bilayers. By x-ray lamellar diffraction, we measured the thickness change of DOPC bilayers as a function of the curcumin/lipid ratio. A nonlinear membrane-thinning effect by curcumin was discovered. The gramicidin data were qualitatively interpreted by the combination of isothermal titration calorimetry and x-ray results. We show that not only does curcumin thin the lipid bilayer, it might also weaken its elasticity moduli. The result implies that curcumin may affect the function of membrane proteins by modifying the properties of the host membrane.

Recently we have shown that the free energy for pore formation induced by antimicrobial peptides contains a term representing peptide-peptide interactions mediated by membrane thinning. This many-body effect gives rise to the cooperative concentration dependence of peptide activities. Here we performed oriented circular dichroism and x-ray diffraction experiments to study the lipid dependence of this many-body effect. In particular we studied the correlation between lipid's spontaneous curvature and peptide's threshold concentration for pore formation by adding phosphatidylethanolamine and lysophosphocholine to phosphocholine bilayers. Previously it was argued that this correlation exhibited by magainin and melittin supported the toroidal model for the pores. Here we found similar correlations exhibited by melittin and alamethicin. We found that the main effect of varying the spontaneous curvature of lipid is to change the degree of membrane thinning, which in turn influences the threshold concentration for pore formation. We discuss how to interpret the lipid dependence of membrane thinning.