Free-energy determinants of alpha-helix insertion into lipid bilayers.

A detailed treatment is provided of the various free-energy terms that contribute to the transfer of a polyalanine alpha-helix from the aqueous phase into lipid bilayers. In agreement with previous work, the hydrophobic effect is found to provide the major driving force for helix insertion. However, an opposing effect of comparable magnitude is also identified and is attributed to the large free-energy penalty associated with the desolvation of peptide hydrogen bonds on transfer to the low dielectric environment of the bilayer. Lipid perturbation effects as well as the entropy loss associated with helix immobilization in the bilayer are also evaluated. Two configurations of a membrane-bound 25mer polyalanine helix were found to be lower in free energy than the isolated helix in the aqueous phase. The first corresponds to the case of vertical insertion, in which a helix terminus protrudes from each side of the bilayer. The second minimum is for the case of horizontal insertion, for which the helix is adsorbed upon the surface of the bilayer. The calculated free-energy minima are found to be in good agreement with recent measurements of related systems. Large free-energy barriers resulting from desolvation of unsatisfied hydrogen-bonding groups at the helix termini are obtained for both insertion processes. The barriers for insertion are significantly reduced if the helix termini are assumed to be "capped" through the formation of hydrogen bonds with polar sidechains. For uncapped helices, our results support recently proposed models in which helices are inserted by first adsorbing on the membrane surface and then having one terminus "swing around" so as to penetrate the bilayer.     

Membrane insertion of a voltage sensor helix

Most membrane proteins contain a transmembrane (TM) domain made up of a bundle of lipid-bilayer-spanning α-helices. TM α-helices are generally composed of a core of largely hydrophobic amino acids, with basic and aromatic amino acids at each end of the helix forming interactions with the lipid headgroups and water. In contrast, the S4 helix of ion channel voltage sensor (VS) domains contains four or five basic (largely arginine) side chains along its length and yet adopts a TM orientation as part of an independently stable VS domain. Multiscale molecular dynamics simulations are used to explore how a charged TM S4 α-helix may be stabilized in a lipid bilayer, which is of relevance in the context of mechanisms of translocon-mediated insertion of S4. Free-energy profiles for insertion of the S4 helix into a phospholipid bilayer suggest that it is thermodynamically favorable for S4 to insert from water to the center of the membrane, where the helix adopts a TM orientation. This is consistent with crystal structures of Kv channels, biophysical studies of isolated VS domains in lipid bilayers, and studies of translocon-mediated S4 helix insertion. Decomposition of the free-energy profiles reveals the underlying physical basis for TM stability, whereby the preference of the hydrophobic residues of S4 to enter the bilayer dominates over the free-energy penalty for inserting charged residues, accompanied by local distortion of the bilayer and penetration of waters. We show that the unique combination of charged and hydrophobic residues in S4 allows it to insert stably into the membrane.     

Free-energy profiles of membrane insertion of the M2 transmembrane peptide from influenza A virus

The insertion of the M2 transmembrane peptide from influenza A virus into a membrane has been studied with molecular-dynamics simulations. This system is modeled by an atomically detailed peptide interacting with a continuum representation of a membrane bilayer in aqueous solution. We performed replica-exchange molecular-dynamics simulations with umbrella-sampling techniques to characterize the probability distribution and conformation preference of the peptide in the solution, at the membrane interface, and in the membrane. The minimum in the calculated free-energy surface of peptide insertion corresponds to a fully inserted, helical peptide spanning the membrane. The free-energy profile also shows that there is a significant barrier for the peptide to enter into this minimum in a nonhelical conformation. The sequence of the peptide is such that hydrophilic amino acid residues at the ends of the otherwise primarily hydrophobic peptide create a trapped, U-shaped conformation with the hydrophilic residues associated with the aqueous phase and the hydrophobic residues embedded in the membrane. Analysis of the free energy shows that the barrier to insertion is largely enthalpic in nature, whereas the membrane-spanning global minimum is favored by entropy.     

Structure and dynamics of cationic membrane peptides and proteins: insights from solid-state NMR

Many membrane peptides and protein domains contain functionally important cationic Arg and Lys residues, whose insertion into the hydrophobic interior of the lipid bilayer encounters significant energy barriers. To understand how these cationic molecules overcome the free energy barrier to insert into the lipid membrane, we have used solid-state NMR spectroscopy to determine the membrane-bound topology of these peptides. A versatile array of solid-state NMR experiments now readily yields the conformation, dynamics, orientation, depth of insertion, and site-specific protein-lipid interactions of these molecules. We summarize key findings of several Arg-rich membrane peptides, including β-sheet antimicrobial peptides, unstructured cell-penetrating peptides, and the voltage-sensing helix of voltage-gated potassium channels. Our results indicate the central role of guanidinium-phosphate and guanidinium-water interactions in dictating the structural topology of these cationic molecules in the lipid membrane, which in turn account for the mechanisms of this functionally diverse class of membrane peptides.     

Interpretation of H-NMR Experiments on the Orientation of the Transmembrane Helix WALP23 by Computer Simulations

Orientation, dynamics, and packing of transmembrane helical peptides are important determinants of membrane protein structure, dynamics, and function. Because it is difficult to investigate these aspects by studying real membrane proteins, model transmembrane helical peptides are widely used. NMR experiments provide information on both orientation and dynamics of peptides, but they require that motional models be interpreted. Different motional models yield different interpretations of quadrupolar splittings (QS) in terms of helix orientation and dynamics. Here, we use coarse-grained (CG) molecular dynamics (MD) simulations to investigate the behavior of a well-known model transmembrane peptide, WALP23, under different hydrophobic matching/mismatching conditions. We compare experimental ²H-NMR QS (directly measured in experiments), as well as helix tilt angle and azimuthal rotation (not directly measured), with CG MD simulation results. For QS, the agreement is significantly better than previously obtained with atomistic simulations, indicating that equilibrium sampling is more important than atomistic details for reproducing experimental QS. Calculations of helix orientation confirm that the interpretation of QS depends on the motional model used. Our simulations suggest that WALP23 can form dimers, which are more stable in an antiparallel arrangement. The origin of the preference for the antiparallel orientation lies not only in electrostatic interactions but also in better surface complementarity. In most cases, a mixture of monomers and antiparallel dimers provides better agreement with NMR data compared to the monomer and the parallel dimer. CG MD simulations allow predictions of helix orientation and dynamics and interpretation of QS data without requiring any assumption about the motional model.     

Effects of arginine density on the membrane-bound structure of a cationic antimicrobial peptide from solid-state NMR

Solid-state NMR spectroscopy is used to determine the membrane-bound topological structure of a cationic β-hairpin antimicrobial peptide in which the number of Arg residues has been halved. The parent peptide, PG-1, was previously found to form transmembrane β-barrels in anionic membranes where the Arg residues complex with the lipid phosphate groups to cause toroidal pore defects in the membrane. In comparison, the charge-attenuated and less active mutant studied here forms β-sheets that lie on the surface of the zwitterionic membrane and only partially insert into the anionic membrane. The mutant also exhibits much looser contact with the lipid headgroups. These results indicate that transmembrane insertion and tight Arg-phosphate association are two important elements for strong antimicrobial activities of this class of peptides. Comparison with other β-hairpin antimicrobial peptides studied so far further suggests a relative potency scale for the various mechanisms of action for the β-sheet family of antimicrobial peptides. The transmembrane insertion-toroidal pore mechanism is the most potent in disrupting the lipid bilayer, followed by the large-amplitude in-plane motional mechanism. The carpet model, where peptides aggregate on the membrane surface to cause lateral expansion and eventual micellization of the membrane, is a weaker mechanism of action.     

Interactions of hydrophobic peptides with lipid bilayers: Monte Carlo simulations with M2delta

We introduce here a novel Monte Carlo simulation method for studying the interactions of hydrophobic peptides with lipid membranes. Each of the peptide's amino acids is represented as two interaction sites: one corresponding to the backbone alpha-carbon and the other to the side chain, with the membrane represented as a hydrophobic profile. Peptide conformations and locations in the membrane and changes in the membrane width are sampled using the Metropolis criterion, taking into account the underlying energetics. Using this method we investigate the interactions between the hydrophobic peptide M2delta and a model membrane. The simulations show that starting from an extended conformation in the aqueous phase, the peptide first adsorbs onto the membrane surface, while acquiring an ordered helical structure. This is followed by formation of a helical-hairpin and insertion into the membrane. The observed path is in agreement with contemporary understanding of peptide insertion into biological membranes. Two stable orientations of membrane-associated M2delta were obtained: transmembrane (TM) and surface, and the value of the water-to-membrane transfer free energy of each of them is in agreement with calculations and measurements on similar cases. M2delta is most stable in the TM orientation, where it assumes a helical conformation with a tilt of 14 degrees between the helix principal axis and the membrane normal. The peptide conformation agrees well with the experimental data; average root-mean-square deviations of 2.1 A compared to nuclear magnetic resonance structures obtained in detergent micelles and supported lipid bilayers. The average orientation of the peptide in the membrane in the most stable configurations reported here, and in particular the value of the tilt angle, are in excellent agreement with the ones calculated using the continuum-solvent model and the ones observed in the nuclear magnetic resonance studies. This suggests that the method may be used to predict the three-dimensional structure of TM peptides.     

Dermaseptin S9, an alpha-helical antimicrobial peptide with a hydrophobic core and cationic termini

The dermaseptins S are closely related peptides with broad-spectrum antibacterial activity that are produced by the skin of the South American hylid frog, Phyllomedusa sauvagei. These peptides are polycationic (Lys-rich), alpha-helical, and amphipathic, with their polar/charged and apolar amino acids on opposing faces along the long axis of the helix cylinder. The amphipathic alpha-helical structure is believed to enable the peptides to interact with membrane bilayers, leading to permeation and disruption of the target cell. We have identified new members of the dermaseptin S family that do not resemble any of the naturally occurring antimicrobial peptides characterized to date. One of these peptides, designated dermaseptin S9, GLRSKIWLWVLLMIWQESNKFKKM, has a tripartite structure that includes a hydrophobic core sequence encompassing residues 6-15 (mean hydrophobicity, +4.40, determined by the Liu-Deber scale) flanked at both termini by cationic and polar residues. This structure is reminiscent of that of synthetic peptides originally designed as transmembrane mimetic models and that spontaneously become inserted into membranes [Liu, L., and Deber, C. M. (1998) Biopolymers 47, 41-62]. Dermaseptin S9 is a potent antibacterial, acting on gram-positive and gram-negative bacteria. The structure of dermaseptin S9 in aqueous solution and in TFE/water mixtures was analyzed by circular dichroism and two-dimensional NMR spectroscopy combined with molecular dynamics calculations. Dermaseptin S9 is aggregated in water, but a monomeric nonamphipathic alpha-helical conformation, mostly in residues 6-21, is stabilized by the addition of TFE. These results, combined with membrane permeabilization assays and surface plasmon resonance analysis of the peptide binding to zwitterionic and anionic phospholipid bilayers, demonstrate that spatial segregation of hydrophobic and hydrophilic/charged residues on opposing faces along the long axis of a helix is not essential for the antimicrobial activity of cationic alpha-helical peptides.     

Molecular dynamics methods to predict peptide locations in membranes: LAH4 as a stringent test case

Determining the structure of membrane-active peptides inside lipid bilayers is essential to understand their mechanism of action. Molecular dynamics simulations can easily provide atomistic details, but need experimental validation. We assessed the reliability of self-assembling (or “minimum-bias”) and potential of mean force (PMF) approaches, using all-atom (AA) and coarse-grained (CG) force-fields. The LAH4 peptide was selected as a stringent test case, since it is known to attain different orientations depending on the protonation state of its four histidine residues.In all simulations the histidine side-chains inserted in the membrane when neutral, while they interacted with phospholipid headgroups in their charged state. This led to transmembrane orientations for neutral-His LAH4 in all minimum-bias AA simulations and in most CG trajectories. By contrast, the charged-His peptide stabilized membrane defects in AA simulations, whereas it was located at the membrane surface in some CG trajectories, and interacted with both lipid leaflets in others. This behavior is consistent with the higher antimicrobial activity and membrane-permeabilizing behavior of the charged-His LAH4. In addition, good agreement with solid-state NMR orientational data was observed in AA simulations.PMF calculations correctly predicted a higher membrane affinity for the neutral-His peptide. Interestingly, the structures and relative populations of PMF local free-energy minima corresponded to those determined in the less computationally demanding minimum-bias simulations.These data provide an indication about the possible membrane-perturbation mechanism of the charged-His LAH4 peptide: by interacting with lipid headgroups of both leaflets through its cationic side-chains, it could favor membrane defects and facilitate translocation across the bilayer.     

Pore Structure and Synergy in Antimicrobial Peptides of the Magainin Family

Magainin 2 and PGLa are among the best-studied cationic antimicrobial peptides. They bind preferentially to negatively charged membranes and apparently cause their disruption by the formation of transmembrane pores, whose detailed structure is still unclear. Here we report the results of 5–9 μs all-atom molecular dynamics simulations starting from tetrameric transmembrane helical bundles of these two peptides, as well as their stoichiometric mixture, and the analog MG-H2 in DMPC or 3:1 DMPC/DMPG membranes. The simulations produce pore structures that appear converged, although some effect of the starting peptide arrangement (parallel vs. antiparallel) is still observed on this timescale. The peptides remain mostly helical and adopt tilted orientations. The calculated tilt angles for PGLa are in excellent agreement with recent solid state NMR experiments. The antiparallel dimer structure in the magainin 2 simulations resembles previously determined NMR and crystal structures. More transmembrane orientations and a larger and more ordered pore are seen in the 1:1 heterotetramer with an antiparallel helix arrangement. Insights into the mechanism of synergy between these two peptides are obtained via implicit solvent modeling of homo- and heterodimers and analysis of interactions in the atomistic simulations. This analysis suggests stronger pairwise interactions in the heterodimer than in the two homodimers.     

Peptide mimics of the M13 coat protein transmembrane segment. Retention of helix-helix interaction motifs

Sequence-specific noncovalent helix-helix interactions between transmembrane (TM) segments in proteins are investigated by incorporating selected TM sequences into synthetic peptides using the construct CKKK-TM-KKK. The peptides are of suitable hydrophobicity for spontaneous membrane insertion, whereas formation of an N-terminal S-S bond can bring pairs of TM helices into proximity and promote their parallel orientation. Using the propensity of the protein to undergo thermally induced alpha-helix --> beta-sheet transitions as a parameter for helix stability, we compared the wild type and mutant (V29A and V31A) bacteriophage M13 coat proteins with their corresponding TM peptide constructs (M13 residues 24-42). Our results demonstrated that the relevant helix-helix tertiary contacts found in the intact proteins persist in the peptide mimics. Molecular dynamics simulations support the tight "two in-two out" dimerization motif for V31A consistent with mutagenesis data. The overall results reinforce the notion of TM segments as autonomous folding domains and suggest that the generic peptide construct provides a viable reductionist system for membrane protein structural and computational analysis.     

Exploring peptide-membrane interactions with coarse-grained MD simulations

The interaction of α-helical peptides with lipid bilayers is central to our understanding of the physicochemical principles of biological membrane organization and stability. Mutations that alter the position or orientation of an α-helix within a membrane, or that change the probability that the α-helix will insert into the membrane, can alter a range of membrane protein functions. We describe a comparative coarse-grained molecular dynamics simulation methodology, based on self-assembly of a lipid bilayer in the presence of an α-helical peptide, which allows us to model membrane transmembrane helix insertion. We validate this methodology against available experimental data for synthetic model peptides (WALP23 and LS3). Simulation-based estimates of apparent free energies of insertion into a bilayer of cystic fibrosis transmembrane regulator-derived helices correlate well with published data for translocon-mediated insertion. Comparison of values of the apparent free energy of insertion from self-assembly simulations with those from coarse-grained molecular dynamics potentials of mean force for model peptides, and with translocon-mediated insertion of cystic fibrosis transmembrane regulator-derived peptides suggests a nonequilibrium model of helix insertion into bilayers.     

Structure and alignment of the membrane-associated peptaibols ampullosporin A and alamethicin by oriented 15N and 31P solid-state NMR spectroscopy

Ampullosporin A and alamethicin are two members of the peptaibol family of antimicrobial peptides. These compounds are produced by fungi and are characterized by a high content of hydrophobic amino acids, and in particular the α-tetrasubstituted amino acid residue α-aminoisobutyric acid. Here ampullosporin A and alamethicin were uniformly labeled with ¹⁵N, purified and reconstituted into oriented phophatidylcholine lipid bilayers and investigated by proton-decoupled ¹⁵N and ³¹P solid-state NMR spectroscopy. Whereas alamethicin (20 amino acid residues) adopts transmembrane alignments in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes the much shorter ampullosporin A (15 residues) exhibits comparable configurations only in thin membranes. In contrast the latter compound is oriented parallel to the membrane surface in 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine and POPC bilayers indicating that hydrophobic mismatch has a decisive effect on the membrane topology of these peptides. Two-dimensional ¹⁵N chemical shift - ¹H-¹⁵N dipolar coupling solid-state NMR correlation spectroscopy suggests that in their transmembrane configuration both peptides adopt mixed α-/3₁₀-helical structures which can be explained by the restraints imposed by the membranes and the bulky α-aminoisobutyric acid residues. The ¹⁵N solid-state NMR spectra also provide detailed information on the helical tilt angles. The results are discussed with regard to the antimicrobial activities of the peptides.     

Configuration of influenza hemagglutinin fusion peptide monomers and oligomers in membranes

The 20 N-terminal residues of the HA2 subunit of influenza hemagglutinin (HA), known as the fusion peptide, play a crucial role in membrane fusion. Molecular dynamics simulations with implicit solvation are employed here to study the structure and orientation of the fusion peptide in membranes. As a monomer the alpha-helical peptide adopts a shallow, slightly tilted orientation along the lipid tail-head group interface. The average angle of the peptide with respect to membrane plane is 12.4 degrees . We find that the kinked structure proposed on the basis of NMR data is not stable in our model because of the high energy cost related to the membrane insertion of polar groups. Because hemagglutinin-mediated membrane fusion is promoted by low pH, we examined the effect of protonation of the Glu and Asp residues. The configurations of the protonated peptides were slightly deeper in the membrane but at similar angles. Finally, because HA is a trimer, we modeled helical fusion peptide trimers. We find that oligomerization affects the insertion depth of the peptide and its orientation with respect to the membrane: a trimer exhibits equally favorable configurations in which some or all of the helices in the bundle insert obliquely deep into the membrane.     

Configuration of influenza hemagglutinin fusion peptide monomers and oligomers in membranes

The 20 N-terminal residues of the HA2 subunit of influenza hemagglutinin (HA), known as the fusion peptide, play a crucial role in membrane fusion. Molecular dynamics simulations with implicit solvation are employed here to study the structure and orientation of the fusion peptide in membranes. As a monomer the α-helical peptide adopts a shallow, slightly tilted orientation along the lipid tail-head group interface. The average angle of the peptide with respect to membrane plane is 12.4 °. We find that the kinked structure proposed on the basis of NMR data is not stable in our model because of the high energy cost related to the membrane insertion of polar groups. Because hemagglutinin-mediated membrane fusion is promoted by low pH, we examined the effect of protonation of the Glu and Asp residues. The configurations of the protonated peptides were slightly deeper in the membrane but at similar angles. Finally, because HA is a trimer, we modeled helical fusion peptide trimers. We find that oligomerization affects the insertion depth of the peptide and its orientation with respect to the membrane: a trimer exhibits equally favorable configurations in which some or all of the helices in the bundle insert obliquely deep into the membrane.     

Solution NMR structures of the antimicrobial peptides phylloseptin-1, -2, and -3 and biological activity: the role of charges and hydrogen bonding interactions in stabilizing helix conformations

Phylloseptins are antimicrobial peptides of 19-20 residues which are found in the skin secretions of the Phyllomedusa frogs that inhabit the tropical forests of South and Central Americas. The peptide sequences of PS-1, -2, and -3 carry an amidated C-terminus and they exhibit 74% sequence homology with major variations of only four residues close to the C-terminus. Here we investigated and compared the structures of the three phylloseptins in detail by CD- and two-dimensional NMR spectroscopies in the presence of phospholipid vesicles or in membrane-mimetic environments. Both CD and NMR spectroscopies reveal a high degree of helicity in the order PS-2> or =PS-1>PS-3, where the differences accumulate at the C-terminus. The conformational variations can be explained by taking into consideration electrostatic interactions of the negative ends of the helix dipoles with potentially cationic residues at positions 17 and 18. Whereas two are present in the sequence of PS-1 and -2 only one is present in PS-3. In conclusion, the antimicrobial phylloseptin peptides adopt alpha-helical conformations in membrane environments which are stabilized by electrostatic interactions of the helix dipole as well as other contributions such hydrophobic and capping interactions.     

Three-dimensional structure of the two peptides that constitute the two-peptide bacteriocin plantaricin EF

The three-dimensional structures of the two peptides plantaricin E (plnE; 33 residues) and plantaricin F (plnF; 34 residues) constituting the two-peptide bacteriocin plantaricin EF (plnEF) have been determined by nuclear magnetic resonance (NMR) spectroscopy in the presence of DPC micelles. PlnE has an N-terminal alpha-helix (residues 10-21), and a C-terminal alpha-helix-like structure (residues 25-31). PlnF has a long central alpha-helix (residues 7-32) with a kink of 38+/-7 degrees at Pro20. There is some flexibility in the helix in the kink region. Both helices in plnE are amphiphilic, while the helix in plnF is polar in its N-terminal half and amphiphilic in its C-terminal half. The alpha-helical content obtained by NMR spectroscopy is in agreement with CD studies. PlnE has two GxxxG motifs which are putative helix-helix interaction motifs, one at residues 5 to 9 and one at residues 20 to 24, while plnF has one such motif at residues 30 to 34. The peptides are flexible in these GxxxG regions. It is suggested that the two peptides lie parallel in a staggered fashion relative to each other and interact through helix-helix interactions involving the GxxxG motifs.     

Effects of alterations of the amino-terminal glycine of influenza hemagglutinin fusion peptide on its structure,organization and membrane interactions

Mutations of the glycine residue at the amino terminus of HA2 have been shown to have a large effect on the fusion activity of HA2, the extent of which apparently correlates with the side chain bulkiness of the substituting amino acids. To investigate into the cause of abrogation in fusogenicity and virus-promoted fusion mechanism, we synthesized several peptides in which this glycine was substituted by serine, glutamic acid, or lysine. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl sn-glycero-3-phosphoglycerol (DMPG) were used as model membranes in the fluorescence, circular dichroism (CD), and FTIR measurements while sodium dodecyl sulfate was used in NMR studies. We found that, for the less active variants, affinity to membrane, degree of solvent dehydration, lipid perturbation, depth of insertion, and helicity were less. Comparison of affinity to membrane bilayer among these analogs revealed that binding of the fusion peptide is determined largely by the hydrophobic effect. Additionally, the orientation is closer to the membrane normal for the wild-type fusion peptide in the helix form while the inactive analogs inserted more parallel to the membrane surface.     

Three-dimensional structure/hydrophobicity of latarcins specifies their mode of membrane activity

Latarcins, linear peptides from the Lachesana tarabaevi spider venom, exhibit a broad-spectrum antimicrobial activity, likely acting on the bacterial cytoplasmic membrane. We study their spatial structures and interaction with model membranes by a combination of experimental and theoretical methods to reveal the structure-activity relationship. In this work, a 26 amino acid peptide, Ltc1, was investigated. Its spatial structure in detergent micelles was determined by (1)H nuclear magnetic resonance (NMR) and refined by Monte Carlo simulations in an implicit water-octanol slab. The Ltc1 molecule was found to form a straight uninterrupted amphiphilic helix comprising 8-23 residues. A dye-leakage fluorescent assay and (31)P NMR spectroscopy established that the peptide does not induce the release of fluorescent marker nor deteriorate the bilayer structure of the membranes. The voltage-clamp technique showed that Ltc1 induces the current fluctuations through planar membranes when the sign of the applied potential coincides with the one across the bacterial inner membrane. This implies that Ltc1 acts on the membranes via a specific mechanism, which is different from the carpet mode demonstrated by another latarcin, Ltc2a, featuring a helix-hinge-helix structure with a hydrophobicity gradient along the peptide chain. In contrast, the hydrophobic surface of the Ltc1 helix is narrow-shaped and extends with no gradient along the axis. We have also disclosed a number of peptides, structurally homologous to Ltc1 and exhibiting similar membrane activity. This indicates that the hydrophobic pattern of the Ltc1 helix and related antimicrobial peptides specifies their activity mechanism. The latter assumes the formation of variable-sized lesions, which depend upon the potential across the membrane.