Barrel-stave model or toroidal model? A case study on melittin pores

Transmembrane pores induced by amphiphilic peptides, including melittin, are often modeled with the barrel-stave model after the alamethicin pore. We examine this assumption on melittin by using two methods, oriented circular dichroism (OCD) for detecting the orientation of melittin helix and neutron scattering for detecting transmembrane pores. OCD spectra of melittin were systematically measured. Melittin can orient either perpendicularly or parallel to a lipid bilayer, depending on the physical condition and the composition of the bilayer. Transmembrane pores were detected when the helices oriented perpendicularly to the plane of the bilayers, not when the helices oriented parallel to the bilayers. The evidence that led to the barrel-stave model for alamethicin and that to the toroidal model for magainin were reviewed. The properties of melittin pores are closely similar to that of magainin but unlike that of alamethicin. We conclude that, among naturally produced peptides that we have investigated, only alamethicin conforms to the barrel-stave model. Other peptides, including magainins, melittin and protegrins, all appear to induce transmembrane pores that conform to the toroidal model in which the lipid monolayer bends continuously through the pore so that the water core is lined by both the peptides and the lipid headgroups.     

Evidence of pores and thinned lipid bilayers induced in oriented lipid membranes interacting with the antimicrobial peptides, magainin-2 and aurein-3.3

Dynamic structures of supramolecular lipid assemblies, such as toroidal pores and thinned bilayers induced in oriented lipid membranes, which are interacting with membrane-acting antimicrobial peptides (AMPs), magainin-2 and aurein-3.3, were explored by ³¹P and ²H solid-state NMR (ssNMR) spectroscopy. Various types of phospholipid systems, such as POPC-d₃₁, POPC-d₃₁/POPG, and POPC-d₃₁/cholesterol, were investigated to understand the membrane disruption mechanisms of magainin-2 and aurein-3.3 peptides at various peptide-to-lipid (P:L) ratios. The experimental lineshapes of anisotropic ³¹P and ²H ssNMR spectra measured on these peptide-lipid systems were simulated reasonably well by assuming the presence of supramolecular lipid assemblies, such as toroidal pores and thinned bilayers, in membranes. Furthermore, the observed decrease in the anisotropic frequency span of either ³¹P or ²H ssNMR spectra of oriented lipid bilayers, particularly when anionic POPG lipids are interacting with AMPs at high P:L ratios, can directly be explained by a thinned membrane surface model with fast lateral diffusive motions of lipids. The spectral analysis protocol we developed enables extraction of the lateral diffusion coefficients of lipids distributed on the curved surfaces of pores and thinned bilayers on a few nanometers scale.     

Conformational and orientation studies of artificial ion channels incorporated into lipid bilayers

The conformational and orientation studies in lipid bilayers of 21 amino acid peptides bearing six crown ethers are reported. The compounds were designed to form artificial ion channels by stacking the crown rings, and were shown to be functional in bilayer membranes. We used Fourier transform infrared spectroscopy and CD spectropolarimetry to study the conformation of the peptides in solution and in lipid bilayers. These studies revealed that hexacrown peptides retain their alpha-helical conformation when incorporated in a lipid bilayer environment. Attenuated total reflectance spectroscopy was used to investigate the orientation of the peptides in a lipid bilayer. Results demonstrated that the peptides are not oriented at a fixed angle in membrane, but rather are in incorporation equilibrium between an active state parallel to the lipid chain and an inactive state adsorbed at the surface of the bilayer. From these results, we propose a model for the channel activity and the gating mechanism of these hexacrown peptides in bilayer membranes.     

Structure of (KIAGKIA)3 aggregates in phospholipid bilayers by solid-state NMR

The interchain (13)C-(19)F dipolar coupling measured in a rotational-echo double-resonance (REDOR) experiment performed on mixtures of differently labeled KIAGKIA-KIAGKIA-KIAGKIA (K3) peptides (one specifically (13)C labeled, and the other specifically (19)F labeled) in multilamellar vesicles of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (1:1) shows that K3 forms close-packed clusters, primarily dimers, in bilayers at a lipid/peptide molar ratio (L/P) of 20. Dipolar coupling to additional peptides is weaker than that within the dimers, consistent with aggregates of monomers and dimers. Analysis of the sideband dephasing rates indicates a preferred orientation between the peptide chains of the dimers. The combination of the distance and orientation information from REDOR is consistent with a parallel (N-N) dimer structure in which two K3 helices intersect at a cross-angle of approximately 20 degrees. Static (19)F NMR experiments performed on K3 in oriented lipid bilayers show that between L/P = 200 and L/P = 20, K3 chains change their absolute orientation with respect to the membrane normal. This result suggests that the K3 dimers detected by REDOR at L/P = 20 are not on the surface of the bilayer but are in a membrane pore.     

Coarse-grained simulation studies of peptide-induced pore formation

We investigate the interactions between lipid bilayers and amphiphilic peptides using a solvent-free coarse-grained simulation technique. In our model, each lipid is represented by one hydrophilic and three hydrophobic beads. The amphiphilic peptide is modeled as a hydrophobic-hydrophilic cylinder with hydrophilic caps. We find that with increasing peptide-lipid attraction the preferred state of the peptide changes from desorbed, to adsorbed, to inserted. A single peptide with weak attraction binds on the bilayer surface, while one with strong attraction spontaneously inserts into the bilayer. We show how several peptides, which individually bind only to the bilayer surface, cooperatively insert. Furthermore, hydrophilic strips along the peptide cylinder induce the formation of multipeptide pores, whose size and morphology depend on the peptides’ overall hydrophilicity, the distribution of hydrophilic residues, and the peptide-peptide interactions. Strongly hydrophilic peptides insert less readily, but prove to be more destructive to bilayer integrity.     

Investigating molecular recognition and biological function at interfaces using piscidins, antimicrobial peptides from fish

We studied amidated and non-amidated piscidins 1 and 3, amphipathic cationic antimicrobial peptides from fish, to characterize functional and structural similarities and differences between these peptides and better understand the structural motifs involved in biological activity and functional diversity among amidated and non-amidated isoforms. Antimicrobial and hemolytic assays were carried out to assess their potency and toxicity, respectively. Site-specific high-resolution solid-state NMR orientational restraints were obtained from (15)N-labeled amidated and non-amidated piscidins 1 and 3 in the presence of hydrated oriented lipid bilayers. Solid-state NMR and circular dichroism results indicate that the peptides are alpha-helical and oriented parallel to the membrane surface. This orientation was expected since peptide-lipid interactions are enhanced at the water-bilayer interface for amphipathic cationic antimicrobial peptides. (15)N solid-state NMR performed on oriented samples demonstrate that piscidin experiences fast, large amplitude backbone motions around an axis parallel to the bilayer normal. Under the conditions tested here, piscidin 1 was confirmed to be more antimicrobially potent than piscidin 3 and antimicrobial activity was not affected by amidation. In light of functional and structural similarities between piscidins 1 and 3, we propose that their topology and fast dynamics are related to their mechanism of action.     

Investigating molecular recognition and biological function at interfaces using piscidins, antimicrobial peptides from fish

We studied amidated and non-amidated piscidins 1 and 3, amphipathic cationic antimicrobial peptides from fish, to characterize functional and structural similarities and differences between these peptides and better understand the structural motifs involved in biological activity and functional diversity among amidated and non-amidated isoforms. Antimicrobial and hemolytic assays were carried out to assess their potency and toxicity, respectively. Site-specific high-resolution solid-state NMR orientational restraints were obtained from ¹⁵N-labeled amidated and non-amidated piscidins 1 and 3 in the presence of hydrated oriented lipid bilayers. Solid-state NMR and circular dichroism results indicate that the peptides are α-helical and oriented parallel to the membrane surface. This orientation was expected since peptide-lipid interactions are enhanced at the water-bilayer interface for amphipathic cationic antimicrobial peptides. ¹⁵N solid-state NMR performed on oriented samples demonstrate that piscidin experiences fast, large amplitude backbone motions around an axis parallel to the bilayer normal. Under the conditions tested here, piscidin 1 was confirmed to be more antimicrobially potent than piscidin 3 and antimicrobial activity was not affected by amidation. In light of functional and structural similarities between piscidins 1 and 3, we propose that their topology and fast dynamics are related to their mechanism of action.     

Interaction of antimicrobial peptides with lipopolysaccharides

We study the interaction of antimicrobial peptides with lipopolysaccharide (LPS) bilayers to understand how antimicrobial peptides interact with the LPS monolayer on the outer membrane of Gram-negative bacteria. LPS in water spontaneously forms a multilamellar structure composed of symmetric bilayers. We performed X-ray lamellar diffraction and wide-angle in-plane scattering to study the physical characteristics of LPS multilayers. The multilayer alignment of LPS is comparable to phospholipids. Thus, it is suitable for the application of oriented circular dichroism (OCD) to study the state of peptides in LPS bilayers. At high hydration levels, the chain melting temperature in multilamella detected by X-ray diffraction is the same as that of LPS aqueous dispersions, as measured by calorimetry. LPS has a strong CD, but with a careful subtraction of the lipid background, the OCD of peptides in LPS is measurable. The method was tested successfully with melittin. It was then applied to two representative antimicrobial peptides, magainin and protegrin. At peptide concentrations comparable to the physiological conditions, both peptides penetrate transmembrane in LPS bilayers. The results imply that antimicrobial peptides readily penetrate the LPS monolayer of the outer membrane.     

Investigation of the mechanism of action of novel amphipathic peptides: Insights from solid-state NMR studies of oriented lipid bilayers

We have investigated in the present study the effect of both non-selective and selective cationic 14-mer peptides on the lipid orientation of DMPC bilayers by ³¹P solid-state nuclear magnetic resonance (NMR) spectroscopy. Depending on the position of substitution, these peptides adopt mainly either an α-helical structure able to permeabilize DMPC and DMPG vesicles (non-selective peptides) or an intermolecular β-sheet structure only able to permeabilize DMPG vesicles (selective peptides). Several systems have been investigated, namely bilayers mechanically oriented between glass plates as well as bicelles oriented with their normal perpendicular or parallel to the external magnetic field. The results have been compared with spectral simulations with the goal of elucidating the difference in the interaction of these two types of peptides with zwitterionic lipid bilayers. The results indicate that the perturbation induced by selective peptides is much greater than that induced by non-selective peptides in all the lipid systems investigated, and this perturbation has been associated to the aggregation of the selective β-sheet peptides in these systems. On the other hand, the oriented lipid spectra obtained in the presence of non-selective peptides suggest the presence of toroidal pores. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Molecular mechanism of antimicrobial peptides: the origin of cooperativity

Based on very extensive studies on four peptides (alamethicin, melittin, magainin and protegrin), we propose a mechanism to explain the cooperativity exhibited by the activities of antimicrobial peptides, namely, a non-linear concentration dependence characterized by a threshold and a rapid rise to saturation as the concentration exceeds the threshold. We first review the structural basis of the mechanism. Experiments showed that peptide binding to lipid bilayers creates two distinct states depending on the bound-peptide to lipid ratio P/L. For P/L below a threshold P/L*, all of the peptide molecules are in the S state that has the following characteristics: (1) there are no pores in the membrane, (2) the axes of helical peptides are oriented parallel to the plane of membrane, and (3) the peptide causes membrane thinning in proportion to P/L. As P/L increases above P/L*, essentially all of the excessive peptide molecules occupy the I state that has the following characteristics: (1) transmembrane pores are detected in the membrane, (2) the axes of helical peptides are perpendicular to the plane of membrane, (3) the membrane thickness remains constant for P/L> or =P/L*. The free energy based on these two states agrees with the data quantitatively. The free energy also explains why lipids of positive curvature (lysoPC) facilitate and lipids of negative curvature (PE) inhibit pore formation.     

Peptide-lipid interactions of the beta-hairpin antimicrobial peptide tachyplesin and its linear derivatives from solid-state NMR

The peptide-lipid interaction of a beta-hairpin antimicrobial peptide tachyplesin-1 (TP-1) and its linear derivatives are investigated to gain insight into the mechanism of antimicrobial activity. (31)P and (2)H NMR spectra of uniaxially aligned lipid bilayers of varying compositions and peptide concentrations are measured to determine the peptide-induced orientational disorder and the selectivity of membrane disruption by tachyplesin. The disulfide-linked TP-1 does not cause any disorder to the neutral POPC and POPC/cholesterol membranes but induces both micellization and random orientation distribution to the anionic POPE/POPG membranes above a peptide concentration of 2%. In comparison, the anionic POPC/POPG bilayer is completely unaffected by TP-1 binding, suggesting that TP-1 induces negative curvature strain to the membrane as a mechanism of its action. Removal of the disulfide bonds by substitution of Cys residues with Tyr and Ala abolishes the micellization of POPE/POPG bilayers but retains the orientation randomization of both POPC/POPG and POPE/POPG bilayers. Thus, linear tachyplesin derivatives have membrane disruptive abilities but use different mechanisms from the wild-type peptide. The different lipid-peptide interactions between TP-1 and other beta-hairpin antimicrobial peptides are discussed in terms of their molecular structure.     

Two states of cyclic antimicrobial peptide RTD-1 in lipid bilayers

RTD-1 is a recently discovered cyclic peptide that, like other well-studied antimicrobial peptides, appears to bind to the lipid matrix of cell membrane in the initial stage of activity. We studied the states of RTD-1 bound to lipid bilayers by two methods: oriented circular dichroism and X-ray diffraction. RTD-1 shows two physically distinct bound states in lipid bilayers like magainins, protegrins, alamethicin, and melittin that were previously studied. However, the nature of transition between the two states is different for RTD-1 as compared with the aforementioned peptides. In one of the two states, RTD-1 is oriented with its backbone ring parallel to the plane of the bilayer. Only in this state RTD-1 induces membrane thinning. But the effect of membrane thinning is much weaker than all other peptides, suggesting that the mechanism of RTD-1 may be different from the other peptides.     

Oblique membrane insertion of viral fusion peptide probed by neutron diffraction

Fusion peptides mimic the membrane fusion activities of the larger viral proteins from which they derive their sequences. A possible mode of activity involves their oblique insertion into lipid bilayers, causing membrane disruption by promoting highly curved hemifusion intermediates, leading to fusion. We have determined the location and orientation of the simian immunodeficiency virus (SIV) fusion peptide in planar lipid bilayers using neutron lamellar diffraction. The helical axis of the peptide adopts an angle of 55 degrees relative to the membrane normal, while it positions itself nearest the lipid bilayer surface. This is the first direct observation of the structural interaction between a fusion peptide and a phospholipid bilayer.     

Membrane thinning due to antimicrobial peptide binding: an atomic force microscopy study of MSI-78 in lipid bilayers

The interaction of an antimicrobial peptide, MSI-78, with phospholipid bilayers has been investigated using atomic force microscopy, circular dichroism, and nuclear magnetic resonance (NMR). Binding of amphipathic peptide helices with their helical axis parallel to the membrane surface leads to membrane thinning. Atomic force microscopy of supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers in the presence of MSI-78 provides images of the membrane thinning process at a high spatial resolution. This data reveals that the membrane thickness is not reduced uniformly over the entire bilayer area. Instead, peptide binding leads to the formation of distinct domains where the bilayer thickness is reduced by 1.1 +/- 0.2 nm. The data is interpreted using a previously published geometric model for the structure of the peptide-lipid domains. In this model, the peptides reside at the hydrophilic-hydrophobic boundary in the lipid headgroup region, which leads to an increased distance between lipid headgroups. This picture is consistent with concentration-dependent 31P and 2H NMR spectra of MSI-78 in mechanically aligned DMPC bilayers. Furthermore, 2H NMR experiments on DMPC-d54 multilamellar vesicles indicate that the acyl chains of DMPC are highly disordered in the presence of the peptide as is to be expected for the proposed structure of the peptide-lipid assembly.     

How type II diabetes-related islet amyloid polypeptide damages lipid bilayers

A leading hypothesis for the decimation of insulin-producing β-cells in type 2 diabetes attributes the cause to islet amyloid polypeptide (IAPP) for its deleterious effects on the cell membranes. This idea has produced extensive investigations on human IAPP (hIAPP) and its interactions with lipid bilayers. However, it is still difficult to correlate the peptide-lipid interactions with its effects on islet cells in culture. The hIAPP fibrils have been shown to interact with lipids and damage lipid bilayers, but appear to have no effect on islet cells in culture. Thus, a modified amyloid hypothesis assumes that the toxicity is caused by hIAPP oligomers, which are not preamyloid fibrils or protofibrils. However, so far such oligomers have not been isolated or identified. The hIAPP monomers also bind to lipid bilayers, but the mode of interaction is not clear. Here, we performed two types of experiments that, to our knowledge, have not been done before. We used x-ray diffraction, in conjunction with circular dichroism measurement, to reveal the location of the peptide bound to a lipid bilayer. We also investigated the effects of hIAPP on giant unilamellar vesicles at various peptide concentrations. We obtained the following qualitative results. Monomeric hIAPP binds within the headgroup region and expands the membrane area of a lipid bilayer. At low concentrations, such binding causes no leakage or damage to the lipid bilayer. At high concentrations, the bound peptides transform to β-aggregates. The aggregates exit the headgroup region and bind to the surface of lipid bilayers. The damage by the surface bound β-aggregates depends on the aggregation size. The initial aggregation extracts lipid molecules, which probably causes ion permeation, but no molecular leakage. However, the initial β-aggregates serve as the seed for larger fibrils, in the manner of the Jarrett-Lansbury seeded-polymerization model, that eventually disintegrate lipid bilayers by electrostatic and hydrophobic interactions.     

Lipid composition regulates the conformation and insertion of the antimicrobial peptide maculatin 1.1

Antimicrobial peptides interact with cell membranes and their selectivity is contingent on the nature of the constituent lipids. Eukaryotic and bacterial membranes are comprised of different proportions of a range of lipid species with different physical properties. Hence, characterisation of antimicrobial peptides with respect to the magnitude of their interactions with model membranes of different lipid types is needed. Maculatin 1.1 is a short antimicrobial peptide secreted from the skin of several Australian tree-frog species. Circular dichroism spectroscopy (CD) was used to explore the interaction of maculatin 1.1 with a wide range of model membrane systems of different head group and acyl chain characteristics. For neutral phosphatidylcholine (PC), unlike anionic phospholipids, the magnitude of the peptide interactions was dependent on the length and degree of saturation of the constituent acyl chains. Oriented circular dichroism (OCD) data indicated that helical structure was likely promoted by peptide insertion into the hydrophobic core of PC bilayers. The addition of cholesterol (30% mol/mol) tended to decrease the membrane interaction of maculatin 1.1. Anionic lipids locked maculatin 1.1 via electrostatic interactions onto the surface of oriented bilayers as seen in OCD spectra. Furthermore, increasing the membrane curvature by reducing the vesicle radii only slightly reduced the proportion of helical structure in all systems by approximately 10%. The peptide-lipid interaction was strongly dependent on both the lipid chain length and head group, which highlights the importance of the lipid composition used to mimic different cell types. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Conformation and membrane orientation of amphiphilic helical peptides by oriented circular dichroism

Oriented circular dichroism (OCD) was used to characterize and compare in a quantitative manner the secondary structure and concentration dependent realignment of the antimicrobial peptides PGLa and MSI-103, and of the structurally related cell-penetrating peptide MAP in aligned phospholipid bilayers. All these peptides adopt an amphiphilic α-helical conformation, and from solid-state NMR analysis they are known to bind to membranes in two distinct orientations depending on their concentration. At low peptide/lipid (P/L) ratio the helices are aligned parallel to membrane surface (S-state), but with increasing concentration they realign to a tilted orientation (T-state), getting immersed into the membrane with an oblique angle supposedly as a result of dimer-formation. In macroscopically aligned liquid crystalline 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers the two limiting states are represented by distinct OCD spectra, and all spectra at intermediate peptide concentrations can be described by a linear combination of these two line shapes. The corresponding fraction of molecules occupying the T-state was determined by fitting the intermediate spectra with a superposition of the two extreme line shapes. By plotting this fraction versus 1/(P/L), the threshold P/L* ratio for realignment was extracted for each of the three related peptides. Despite their structural similarity distinctly different thresholds were obtained, namely for MSI-103 realignment starts already at a low P/L of ∼1:236, for a MAP derivative (using a nonaggregating analog containing a D-amino acid) the transition begins at P/L ∼1:156, whereas PGLa needs the highest concentration to flip into T-state at P/L ∼1:85. Analysis of the original MAP sequence (containing only L-amino acids) gave OCD spectra compatible with β-pleated conformation, suggesting that this peptide starts to aggregate with increasing concentration, unlike the other helical peptides. All these changes in peptide conformation and membrane alignment observed here by OCD seem to be functionally relevant, as they can be correlated with the membrane perturbing activities of the three antimicrobial and cell-penetrating sequences.     

Molecular dynamics simulations of hydrophilic pores in lipid bilayers

Hydrophilic pores are formed in peptide free lipid bilayers under mechanical stress. It has been proposed that the transport of ionic species across such membranes is largely determined by the existence of such meta-stable hydrophilic pores. To study the properties of these structures and understand the mechanism by which pore expansion leads to membrane rupture, a series of molecular dynamics simulations of a dipalmitoylphosphatidylcholine (DPPC) bilayer have been conducted. The system was simulated in two different states; first, as a bilayer containing a meta-stable pore and second, as an equilibrated bilayer without a pore. Surface tension in both cases was applied to study the formation and stability of hydrophilic pores inside the bilayers. It is observed that below a critical threshold tension of approximately 38 mN/m the pores are stabilized. The minimum radius at which a pore can be stabilized is 0.7 nm. Based on the critical threshold tension the line tension of the bilayer was estimated to be approximately 3 x 10(-11) N, in good agreement with experimental measurements. The flux of water molecules through these stabilized pores was analyzed, and the structure and size of the pores characterized. When the lateral pressure exceeds the threshold tension, the pores become unstable and start to expand causing the rupture of the membrane. In the simulations the mechanical threshold tension necessary to cause rupture of the membrane on a nanosecond timescale is much higher in the case of the equilibrated bilayers, as compared with membranes containing preexisting pores.     

Hydrophobic mismatch between helices and lipid bilayers

alpha-Helical transmembrane peptides, named WALP, with a hydrophobic sequence of leucine and alanine of varying length bordered at both ends by two tryptophans as membrane anchors, were synthesized to study the effect of hydrophobic matching in lipid bilayers. WALPs of 13-, 16-, and 19-residues were incorporated into 1,2-dilauroyl-sn-glycero-3-phosphocholine (12C), 1,2-tridecanoyl-sn-glycero-3-phosphocholine (13C), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (14C) bilayers in the form of oriented multilayers. Oriented circular dichroism spectra and x-ray diffraction patterns showed that the peptides were homogenously distributed in the lipid bilayers with the helical axes oriented approximately normal to the plane of bilayers. But in all cases, x-ray diffraction showed that the peptides did not alter the thickness of the bilayer. This is contrary to the case of gramicidin where 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dilauroyl-sn-glycero-3-phosphocholine clearly thinned and thickened, respectively, to approach the hydrophobic thickness of the gramicidin channels. The result seems to indicate that the packing of lipid chains around a single helix is fundamentally different from the way the chains pack against a large protein surface.     

Structure of the alamethicin pore reconstructed by x-ray diffraction analysis

We reconstructed the electron density profile of the alamethicin-induced transmembrane pore by x-ray diffraction. We prepared fully hydrated multiple bilayers of alamethicin-lipid mixtures in a condition where pores were present, as established previously by neutron in-plane scattering in correlation with oriented circular dichroism. At dehydrated conditions, the interbilayer distance shortened and the interactions between bilayers caused the membrane pores to become long-ranged correlated and form a periodically ordered lattice of rhombohedral symmetry. To resolve the phase problem of diffraction, we used a brominated lipid and performed multiwavelength anomalous diffraction at the bromine K edge. The result unambiguously shows that the alamethicin pore is of the barrel-stave type consisting of eight alamethicin helices. This pore structure corresponds to the stable pores detected by neutron in-plane scattering in fully hydrated fluid bilayers at high peptide/lipid ratios, which are the conditions at which alamethicin was tested for its antibacterial activity.