Interaction of amphipathic peptides with an immobilised model membrane

The interaction of melittin and a truncated analogue of melittin with an immobilised phosphatidylcholine monolayer has been studied using dynamic elution techniques. The melittin analogue (21Q analogue) had five amino acids omitted from the C-terminal region of melittin. The influence of temperature and methanol concentration on the binding affinity of the two peptides was determined and compared to the binding behaviour of two control molecules N-acetyltryptophanamide and diphenylalanine. Both peptides exhibited non-linear dependence of affinity on % methanol at different temperatures, while N-acetyltryptophanamide and diphenylalanine exhibited linear behaviour. In addition, both melittin and the 21Q analogue exhibited significant band broadening under a range of experimental conditions, which was not evident for N-acetyltryptophanamide and diphenylalanine. As melittin is known to adopt a significant degree of -helical conformation in the presence of lipids, the results suggest that melittin and the 21Q analogue adopt different conformations and orientations upon binding to the immobilised phosphatidylcholine surface. Overall, the results of this study demonstrate that the immobilised lipid monolayer provides a powerful system to rapidly assess the affinity of peptides for different lipid surfaces.     

Peptide-membrane interactions and mechanisms of membrane destruction by amphipathic alpha-helical antimicrobial peptides

Antimicrobial peptides (AMPs) have received considerable interest as a source of new antibiotics with the potential for treatment of multiple-drug resistant infections. An important class of AMPs is composed of linear, cationic peptides that form amphipathic alpha-helices. Among the most potent of these are the cecropins and synthetic peptides that are hybrids of cecropin and the bee venom peptide, mellitin. Both cecropins and cecropin-mellitin hybrids exist in solution as unstructured monomers, folding into predominantly alpha-helical structures upon membrane binding with their long helical axis parallel to the bilayer surface. Studies using model membranes have shown that these peptides intercalate into the lipid bilayer just below the level of the phospholipid glycerol backbone in a location that requires expansion of the outer leaflet of the bilayer, and evidence from a variety of experimental approaches indicates that expansion and thinning of the bilayer are common characteristics during the early stages of antimicrobial peptide-membrane interactions. Subsequent disruption of the membrane permeability barrier may occur by a variety of mechanisms, leading ultimately to loss of cytoplasmic membrane integrity and cell death.     

Mechanisms for the modulation of membrane bilayer properties by amphipathic helical peptides

The amphipathic helix, in which hydrophobia and hydrophilic residues are grouped on opposing faces, is a structural mot if found in many peptides and proteins that bind to membranes. One of the physical properties of membranes that can be altered by the binding of amphipathic helices is membrane monolayer curvature strain. Class A amphipathic helices, which are present in exchangeable plasma lipoproteins, can stabilize membranes by reducing negative monolayer curvature strain; proline-punctuated class A amphipathic helical segments are particularly effective in this regard. This property is suggested to be associated with some of the beneficial biological effects of this protein. On the other hand, lytic amphipathic helical peptides can act by increasing negative curvature strain or by forming pores composed of helical clusters. Thus, different amphipathic helical peptides can be membrane stabilizing or be lytic to membranes, depending on the structural motif of the helix, which in turn determines the nature of its association with membranes. Features of these peptides that are responsible for their specific properties are discussed. © 1994 John Wiley & Sons, Inc.     

Mechanism of membrane damage induced by the amphipathic peptides gramicidin S and melittin

The action of gramicidin S and melittin on human erythrocytes, Staphylococcus aureus and Escherichia coli was studied as an extension of the previous study (Katsu, T., Ninomiya, C., Kuroko, M., Kobayashi, H., Hirota, T. and Fujita, Y. (1988) Biochim. Biophys. Acta 939, 57-63). These amphipathic peptides stimulated the release of membrane phospholipids outside cells in a concentration range causing permeability change. The shape change of erythrocytes from normal discoid to spiculate form was observed just prior to the release of membrane components. We have proposed the following action mechanism of gramicidin S and melittin. The peptide molecules were predominantly accumulated in the outer half of the bilayer, deforming the erythrocyte cell into crenature. A large accumulation made the membrane structure unstable, resulting in the release of membrane fragments and the simultaneous enhancement of permeability. The action mechanism of these peptides was compared with that of simple surfactants.     

Intestinal sterol absorption mediated by scavenger receptors is competitively inhibited by amphipathic peptides and proteins

Exchangeable serum apolipoproteins and amphipathic alpha-helical peptides are effective inhibitors of sterol (free and esterified cholesterol) uptake at the small-intestinal brush border membrane. The minimal structural requirement of an inhibitor is an amphipathic alpha-helix of 18 amino acids. The inhibition is competitive, indicating that the inhibitor binds to scavenger receptor class B type I (SR-BI) present in the brush border membrane and responsible for sterol uptake. Binding of apolipoprotein A-I to SR-BI of rabbit brush border membrane is cooperative, characterized by a dissociation constant K(d) = 0.45 microM and a Hill coefficient of n = 2.8. The cooperativity of the interaction is due to binding of the inhibitor molecule to a dimeric or oligomeric form of SR-BI held together by disulfide bridges. Consistent with the competitive nature of the inhibition, the K(d) value agrees within experimental error with the IC(50) value of inhibition and with the inhibition constant K(I). After proteinase K treatment of brush border membrane vesicles, the affinity of the interaction of apolipoprotein A-I expressed as K(d) is reduced by a factor of 20, and the cooperativity is lost. The interaction of proteinase K-treated brush border membrane vesicles with apolipoprotein A-I is nonspecific partitioning of the apolipoprotein into the lipid bilayer of brush border membrane vesicles.     

Orientations of amphipathic helical peptides in membrane bilayers determined by solid-state NMR spectroscopy

Summary Solid-state NMR spectroscopy was used to determine the orientations of two amphipathic helical peptides associated with lipid bilayers. A single spectral parameter provides sufficient orientational information for these peptides, which are known, from other methods, to be helical. The orientations of the peptides were determined using the¹⁵N chemical shift observed for specifically labeled peptide sites. Magainin, an antibiotic peptide from frog skin, was found to lie in the plane of the bilayer. M2, a helical segment of the nicotinic acetylcholine receptor, was found to span the membrane, perpendicular to the plane of the bilayer. These findings have important implications for the mechanisms of biological functions of these peptides.     

Ion channels formed by amphipathic helical peptides

Channel forming peptides (CFPs) are amphipathic peptides, of length ca. 20 residues, which adopt an -helical conformation in the presence of lipid bilayers and form ion channels with electrophysiological properties comparable to those of ion channel proteins. We have modelled CFP channels as bundles of parallel trans-bilayer helices surrounding a central ion-permeable pore. Ion-channel interactions have been explored via accessible surface area calculations, and via evaluation of changes in van der Waals and electrostatic energies as a K⁺ ion is translated along the length of the pore. Two CFPs have been modelled: (a) zervamicin-A1-16, a synthetic apolar peptaibol related to alamethicin, and (b) -toxin from Staphylococcus aureus. Both of these CFPs have previously been shown to form ion channels in planar lipid bilayers, and have been shown to have predominantly helical conformations. Zervamicin-A1-16 channels were modelled as bundles of 4 to 8 parallel helices. Two related helix bundle geometries were explored. K⁺channel interactions have been shown to involve exposed backbone carbonyl oxygen atoms. -Toxin channels were modelled as bundles of 6 parallel helices. Residues Q3, D11 and D18 generate favourable K⁺-channel interactions. Rotation of W15 about its C-C bond has been shown to be capable of occluding the central pore, and is discussed as a possible model for sidechain conformational changes in relation to ion channel gating.     

Action mechanism of amphipathic peptides gramicidin S and melittin on erythrocyte membrane

Amphipathic peptides gramicidin S and melittin caused a characteristic colloid-osmotic hemolysis on human erythrocytes; that is, the peptides produced initially a small membrane lesion in erythrocyte membrane, followed by the release of hemoglobin. The size of membrane lesion increased with an increase in the concentration of peptide. Under the conditions causing membrane lesion, we observed the release of membrane fragments containing phospholipids. The present results show that both the peptides have the ability to stimulate the release of membrane fragments out of the cells and this brings about the perforation of molecules of small size, leading to a colloid-osmotic hemolysis.     

Hydrophobic hydration of amphipathic peptides

Biomolecular surfaces and interfaces are commonly found with apolar character. The hydrophobic effect thus plays a crucial role in processes involving association with biomolecular surfaces in the cellular environment. By computer simulation, we compared the hydrogen bonding structures and energetics of the proximal hydration shells of the monomer and dimer from a recent study of an extrinsic membrane peptide, melittin. The two peptides were studied in their amphipathic alpha-helical forms, which possess extended hydrophobic surfaces characterized by different topography. The topography of the peptide-water interface was found to be critical in determining the enthalpic nature of hydrophobic hydration. This topographical dependence has far-reaching implications in the regulation of bioactivities in the presence of amphipathicity. This result also engenders reconsideration of the validity of using free energy parameters that depend solely on the chemical nature of constituent moieties in characterizing hydrophobic hydration of proteins and biomolecules in general.     

Lipid shape is a key factor for membrane interactions of amphipathic helical peptides

The membrane alignment of the amphiphilic α-helical model peptide MSI-103 (sequence [KIAGKIA](3)-NH(2)) was examined by solid state (2)H-NMR in different lipid systems by systematically varying the acyl chain length and degree of saturation, the lipid head group type, and the peptide-to-lipid molar ratio. In liquid crystalline phosphatidylcholine (PC) lipids with saturated chains, the amphiphilic helix changes its orientation from a surface-bound S-state to a tilted T-state with increasing peptide concentration. In PC lipids with unsaturated chains, on the other hand, the S-state is found throughout all concentrations. Using phosphatidylethanolamine lipids with a small head group or by addition of lyso-lipids with only one acyl chain, the spontaneous curvature of the bilayer was purposefully changed. In the first case with a negative curvature only the S-state was found, whereas in systems with a positive curvature the peptide preferred the obliquely immersed T-state at high concentration. The orientation of MSI-103 thus correlates very well with the shape of the lipid molecules constituting the membrane. Lipid charge, on the other hand, was found to affect only the initial electrostatic attraction to the membrane surface but not the alignment preferences. In bilayers that are sealed with 20% cholesterol, MSI-103 cannot bind in a well-oriented manner and forms immobilized aggregates instead. We conclude that the curvature properties of a membrane are a key factor in the interactions of amphiphilic helical peptides in general, whose re-alignment and immersion preferences may thus be inferred in a straightforward manner from the lipid-shape concept.     

Stabilization of bilayer structure of raft due to elastic deformations of membrane

Line tension of the boundary of specific domains (rafts) rich in sphingomyelin was calculated. The line tension was calculated based on macroscopic theory of elasticity under assumption that the bilayer in raft is thicker than in the surrounding membrane. The calculations took into account the possibility of lateral shift of the domain boundaries located in different monolayers of the membrane. The line tension was associated with the energy of elastic deformations appearing in the vicinity of the boundary in order to compensate for the difference in the thickness of the monolayers. Spatial distribution of deformations and the line tension was calculated by minimization of elastic free energy of the system. Dependence of the line tension on the distance between the domains boundaries located in different monolayers was obtained. It was shown that the line tension is minimal if the distance is about 4 nm. Thus, membrane deformations stabilize the bilayer structure of rafts observed experimentally. The calculated value of line tension is about 0.6 pN for the difference between the monolayer thickness of raft and surrounding membrane of about 0.5 nm, which is in agreement with the experimental data available.     

Osmotically induced membrane tension modulates membrane permeabilization by class L amphipathic helical peptides: nucleation model of defect formation.

The mechanism of action of lytic peptides on membranes is widely studied and is important in view of potential medical applications. Previously (I. V. Polozov, A. I. Polozova, E. M. Tytler, G. M. Anantharamaiah, J. P. Segrest, G. A. Woolley, and R. M., Biochemistry, 36:9237--9245) we analyzed the mechanism of membrane permeabilization by 18L, the archetype lytic peptide featuring the class L amphipathic alpha-helix, according to the classification of Segrest et al. (J. P. Segrest, G. de Loof, J. G. Dohlman, C. G. Brouillette, and G. M. Anantharamaiah, 1990, Proteins, 8:103--117). We concluded that the 18L peptide destabilizes membranes, leading to a transient formation of large defects that result in contents leakage and, in the presence of bilayer-bilayer contact, could lead to vesicle fusion. Here we report that this defect formation is strongly enhanced by the membrane tension induced by osmotic swelling of vesicles. Even below standard leakage-inducing peptide/lipid ratios, membrane resistance to osmotic tension drops from hundreds to tens of milliosmoles. The actual decrease is dependent on the peptide/lipid ratio and on the type of lipid. We propose that under membrane tension a peptidic pore serves as a nucleation site for the transient formation of a lipidic pore. The tension is released upon pore expansion with inclusion of more peptides and lipids into the pore lining. This tension modulation of leakage was observed for other class L peptides (mastoparan, K18L) and thus may be of general applicability for the action of membrane active lytic peptides.     

Lecithin:cholesterol acyltransferase activation by synthetic amphipathic peptides

The amphipathic helical theory of Segrest and colleagues (FEBS Lett.:38:247-253, 1974) proposes that the lipid-binding segments of serum apolipoproteins are in an alpha helical conformation. Furthermore the helices have a hydrophobic face and a hydrophilic face with a specific distribution of positively and negatively charged residues. The importance of the pattern of the charged residues in the lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation by the segments is still debated. We designed a 30-residue peptide, GALA, which in the alpha helical conformation has a hydrophilic face composed of glutamic acid residues (Sabbarao et al.: Biochemistry 26:2964-2972, 1987). GALA behaves like the serum apolipoproteins in its interaction with dimyristoylphosphatidylcholine (DMPC) at neutral pH; the amino terminal tryptophan of GALA undergoes a blue shift in its fluorescence emission spectrum, and the circular dichroism (CD) spectrum indicates that GALA acquires alpha helical structure in the presence of DMPC. A DMPC-GALA:19/1 (molar ratio) complex can be isolated by gel-permeation chromatography. This complex has a discoidal structure with the approximate dimensions of 44-A edge thickness and a 170- to 350-A diameter. GALA activates LCAT with DMPC but not with unsaturated phospholipids as the substrate. The apparent partition coefficient of GALA into DMPC vesicles is 100-fold larger than into egg phosphatidylcholine vesicles. The interaction of GALA with unsaturated lipids at neutral pH is so weak that no detectable change in the spectroscopic properties of GALA or the structure of the liposomes can be detected under the conditions used here. The sequence of GALA differs from previously studied model Apo A1 peptides by the absence of positively charged residues on the hydrophilic face. This indicates that positive charges in Apo A1-like peptides are not required in order to form discoidal structures with saturated phospholipids or to activate LCAT with such lipid substrates.     

Interaction of 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes

We investigated the interaction of six 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes. The net charge of the peptides at neutral pH varies from −1 to +6. Circular dichroism spectra indicate that peptides with a high net positive charge tend to fold into a helical conformation in the presence of negatively charged lipid vesicles. In helical conformation, their average hydrophobic moment and hydrophobicity would render them surface-active. The composition of amino acids on the polar face of the helix in the peptides is considerably different. The peptides show variations in their ability to permeabilise zwitterionic and anionic lipid vesicles. Whereas increased net positive charge favours greater permeabilisation, the distribution of charged residues in the polar face also plays a role in determining membrane activity. The distribution of amino acids in the polar face of the helix in the peptides that were investigated do not fall into the canonical classes described. Amphipathic helices, which are part of proteins, with a pattern of amino acid distribution different from those observed in class L, A and others, could help in providing newer insights into peptide-membrane interactions.     

Cationic amphipathic histidine-rich peptides for gene delivery

Besides being a useful tool in research, gene transfer has a high potential as treatment for a variety of genetic and acquired diseases. However, in order to enable a gene to become a pharmaceutical, efficient and safe methods of delivery have to be developed. We recently found that cationic amphipathic histidine-rich peptide antibiotics can efficiently deliver DNA into mammalian cells. Our lead compound, LAH4 (KKALLALALHHLAHLALHLALALKKA), demonstrated in vitro transfection efficiencies comparable to those of commercially available reagents. Synthesis and evaluation of LAH mutants provided evidence that the transfection efficiency depends on the number and positioning of histidine residues in the peptide as well as on the pH at which the in-plane to transmembrane transition takes place. Moreover, recent results suggest that binding of the DNA complexes to the plasma membrane is mediated by heparan sulfate proteoglycans and that anionic phospholipids may be involved in the endosomal destabilization process. Finally, we also describe in this review the rationale that led to the development of LAH4 as a DNA carrier as well as the biophysical methods that have allowed us to propose a model which could explain the way this peptide destabilizes the endosomal bilayer.     

Cationic amphipathic histidine-rich peptides for gene delivery

Besides being a useful tool in research, gene transfer has a high potential as treatment for a variety of genetic and acquired diseases. However, in order to enable a gene to become a pharmaceutical, efficient and safe methods of delivery have to be developed. We recently found that cationic amphipathic histidine-rich peptide antibiotics can efficiently deliver DNA into mammalian cells. Our lead compound, LAH4 (KKALLALALHHLAHLALHLALALKKA), demonstrated in vitro transfection efficiencies comparable to those of commercially available reagents. Synthesis and evaluation of LAH mutants provided evidence that the transfection efficiency depends on the number and positioning of histidine residues in the peptide as well as on the pH at which the in-plane to transmembrane transition takes place. Moreover, recent results suggest that binding of the DNA complexes to the plasma membrane is mediated by heparan sulfate proteoglycans and that anionic phospholipids may be involved in the endosomal destabilization process. Finally, we also describe in this review the rationale that led to the development of LAH4 as a DNA carrier as well as the biophysical methods that have allowed us to propose a model which could explain the way this peptide destabilizes the endosomal bilayer.     

Cationic amphipathic peptides accumulate sialylated proteins and lipids in the plasma membrane of eukaryotic host cells

Cationic antimicrobial peptides (CAMPs) selectively target bacterial membranes by electrostatic interactions with negatively charged lipids. It turned out that for inhibition of microbial growth a high CAMP membrane concentration is required, which can be realized by the incorporation of hydrophobic groups within the peptide. Increasing hydrophobicity, however, reduces the CAMP selectivity for bacterial over eukaryotic host membranes, thereby causing the risk of detrimental side-effects. In this study we addressed how cationic amphipathic peptides-in particular a CAMP with Lysine-Leucine-Lysine repeats (termed KLK)-affect the localization and dynamics of molecules in eukaryotic membranes. We found KLK to selectively inhibit the endocytosis of a subgroup of membrane proteins and lipids by electrostatically interacting with negatively charged sialic acid moieties. Ultrastructural characterization revealed the formation of membrane invaginations representing fission or fusion intermediates, in which the sialylated proteins and lipids were immobilized. Experiments on structurally different cationic amphipathic peptides (KLK, 6-MO-LF11-322 and NK14-2) indicated a cooperation of electrostatic and hydrophobic forces that selectively arrest sialylated membrane constituents.     

Detergent-like actions of linear amphipathic cationic antimicrobial peptides

Antimicrobial peptides have raised much interest as pathogens become resistant against conventional antibiotics. We review biophysical studies that have been performed to better understand the interactions of linear amphipathic cationic peptides such as magainins, cecropins, dermaseptin, delta-lysin or melittin. The amphipathic character of these peptides and their interactions with membranes resemble the properties of detergent molecules and analogies between membrane-active peptide and detergents are presented. Several models have been suggested to explain the pore-forming, membrane-lytic and antibiotic activities of these peptides. Here we suggest that these might be 'special cases' within complicated phase diagrams describing the morphological plasticity of peptide/lipid supramolecular assemblies.     

Detergent-like actions of linear amphipathic cationic antimicrobial peptides

Antimicrobial peptides have raised much interest as pathogens become resistant against conventional antibiotics. We review biophysical studies that have been performed to better understand the interactions of linear amphipathic cationic peptides such as magainins, cecropins, dermaseptin, δ-lysin or melittin. The amphipathic character of these peptides and their interactions with membranes resemble the properties of detergent molecules and analogies between membrane-active peptide and detergents are presented. Several models have been suggested to explain the pore-forming, membrane-lytic and antibiotic activities of these peptides. Here we suggest that these might be ‘special cases’ within complicated phase diagrams describing the morphological plasticity of peptide/lipid supramolecular assemblies.