The Structure of a Melittin-Stabilized Pore

Melittin has been reported to form toroidal pores under certain conditions, but the atomic-resolution structure of these pores is unknown. A 9-μs all-atom molecular-dynamics simulation starting from a closely packed transmembrane melittin tetramer in DMPC shows formation of a toroidal pore after 1 μs. The pore remains stable with a roughly constant radius for the rest of the simulation. Surprisingly, one or two melittin monomers frequently transition between transmembrane and surface states. All four peptides are largely helical. A simulation in a DMPC/DMPG membrane did not lead to a stable pore, consistent with the experimentally observed lower activity of melittin on anionic membranes. The picture that emerges from this work is rather close to the classical toroidal pore, but more dynamic with respect to the configuration of the 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.     

Lipid-controlled peptide topology and interactions in bilayers: structural insights into the synergistic enhancement of the antimicrobial activities of PGLa and magainin 2

To gain further insight into the antimicrobial activities of cationic linear peptides, we investigated the topology of each of two peptides, PGLa and magainin 2, in oriented phospholipid bilayers in the presence and absence of the other peptide and as a function of the membrane lipid composition. Whereas proton-decoupled ¹⁵N solid-state NMR spectroscopy indicates that magainin 2 exhibits stable in-plane alignments under all conditions investigated, PGLa adopts a number of different membrane topologies with considerable variations in tilt angle. Hydrophobic thickness is an important parameter that modulates the alignment of PGLa. In equimolar mixtures of PGLa and magainin 2, the former adopts transmembrane orientations in dimyristoyl-, but not 1-palmitoyl-2-oleoyl-, phospholipid bilayers, whereas magainin 2 remains associated with the surface in all cases. These results have important consequences for the mechanistic models explaining synergistic activities of the peptide mixtures and will be discussed. The ensemble of data suggests that the thinning of the dimyristoyl membranes caused by magainin 2 tips the topological equilibrium of PGLa toward a membrane-inserted configuration. Therefore, lipid-mediated interactions play a fundamental role in determining the topology of membrane peptides and proteins and thereby, possibly, in regulating their activities as well.     

Lipid saturation and head group composition have a pronounced influence on the membrane insertion equilibrium of amphipathic helical polypeptides

The histidine-rich peptides of the LAH4 family were designed using cationic antimicrobial peptides such as magainin and PGLa as templates. The LAH4 amphipathic helical sequences exhibit a multitude of interesting biological properties such as antimicrobial activity, cell penetration of a large variety of cargo and lentiviral transduction enhancement. The parent peptide associates with lipid bilayers where it changes from an orientation along the membrane interface into a transmembrane configuration in a pH-dependent manner. Here we show that LAH4 adopts a transmembrane configuration in fully saturated DMPC membranes already at pH 3.5, i.e. much below the pK_a of the histidines whereas the transition pH in POPC correlates closely with histidine neutralization. In contrast in POPG membranes the in-planar configuration is stabilized by about one pH unit. The differences in pH can be converted into energetic contributions for the in-plane to transmembrane transition equilibrium, where the shift in the transition pH due to lipid saturation corresponds to energies which are otherwise obtained by the exchange of several cationic with hydrophobic residues. A similar dependence on lipid saturation has also been observed when the PGLa and magainin antimicrobial peptides interact within lipid bilayers suggesting that the quantitative evaluation presented in this paper also applies to other membrane polypeptides.     

What Makes a Good Pore Former: A Study of Synthetic Melittin Derivatives

Pore formation by membrane-active peptides, naturally encountered in innate immunity and infection, could have important medical and technological applications. Recently, the well-studied lytic peptide melittin has formed the basis for the development of combinatorial libraries from which potent pore-forming peptides have been derived, optimized to work under different conditions. We investigate three such peptides, macrolittin70, which is most active at neutral pH; pHD15, which is active only at low pH; and MelP5_Δ6, which was rationally designed to be active at low pH but formed only small pores. There are three, six, and six acidic residues in macrolittin70, pHD15, and MelP5_Δ6, respectively. We perform multi-microsecond simulations in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) of hexamers of these peptides starting from transmembrane orientations at neutral pH (all residues at standard protonation), low pH (acidic residues and His protonated), and highly acidic environments in which C-termini are also protonated. Previous simulations of the parent peptides melittin and MelP5 in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) are repeated in POPC. We find that the most potent pore-forming peptides exhibit strong interpeptide interactions, including salt bridges, H-bonds, and polar interactions. Protonation of the C-terminus promotes helicity and pore size. The proximity of the peptides allows fewer lipid headgroups to line the pores than in previous simulations, making the pores intermediate between barrel stave and toroidal. Based on these structures and geometrical arguments, we attempt to rationalize the factors that under different conditions can increase or decrease pore stability and propose mutations that could be tested experimentally.     

Synergistic Insertion of Antimicrobial Magainin-Family Peptides in?Membranes Depends on the Lipid Spontaneous Curvature

PGLa and magainin 2 (MAG2) are amphiphilic antimicrobial peptides from frog skin with known synergistic activity. The orientation of the two helices in membranes was studied using solid-state ¹⁵N-NMR, for each peptide alone and for a 1:1 mixture of the peptides, in a range of different lipid systems. Two types of orientational behavior emerged. 1), In lipids with negative spontaneous curvature, both peptides remain flat on the membrane surface, when assessed both alone and in a 1:1 mixture. 2), In lipids with positive spontaneous curvature, PGLa alone assumes a tilted orientation but inserts into the bilayer in a transmembrane alignment in the presence of MAG2, whereas MAG2 stays on the surface or gets only slightly tilted, when observed both alone and in the presence of PGLa. The behavior of PGLa alone is identical to that of another antimicrobial peptide, MSI-103, in the same lipid systems, indicating that the curvature-dependent helix orientation is a general feature of membrane-bound peptides and also influences their synergistic intermolecular interactions.The two antimicrobial peptides PGLa and magainin 2 (MAG2) from the African frog Xenopus laevis, which are active against Gram-positive and Gram-negative bacteria, show intriguing synergistic effects that are not yet well understood (1). Structural insights into this synergy may help in the development of a new antibiotic-combination therapy. Both peptides are known to form α-helices when bound to lipid bilayers (2–4). The orientation of such α-helices in a membrane can be readily determined from the ¹⁵N-NMR chemical shift in oriented lipid bilayers that are aligned with the sample normal parallel to the external magnetic field (5). If the ¹⁵N chemical shift is ∼90 ppm, the peptide lies flat on the membrane surface (in the so-called S-state). On the other hand, when the ¹⁵N chemical shift is ∼200 ppm, the peptide is fully inserted (the I-state) in a transmembrane alignment. For intermediate orientations, where the peptide is tilted (the T-state) with an angle of typically 30°–60° relative to the membrane normal, intermediate chemical shifts are expected, but the exact tilt angle cannot be determined from a single label in such cases (6). Using several selectively ²H- or ¹⁹F-labeled peptide analogs, more exact orientations can be obtained, since both the tilt and the azimuthal angles can be measured with high accuracy, and valuable information about dynamics also can be deduced (3,7–10).The orientation of PGLa and MAG2 in membranes has been extensively studied with solid-state NMR, and some clues about the synergistic mechanism have been observed. Notably, in DMPC/DMPG membranes, it has been shown that the peptides on their own are in the S-state or T-state, but when the peptides are mixed in a 1:1 molar ratio, PGLa changes to the I-state, whereas MAG2 stays on the membrane surface (1,11,12). Thus, in the mixed system, transmembrane pores, which would not be spontaneously formed by each peptide on its own, appear to be stable, which could be the basis for synergy. On the other hand, it was also reported that in POPC/POPG there is no change in orientation when PGLa and MAG2 are mixed, as both peptides stay always in the S-state (12). This observation was attributed to the greater hydrophobic thickness of the POPC/POPG bilayer, compared to the DMPC/DMPG bilayer, suggesting that the PGLa helix is so short that it can only insert into thin DMPC/DMPG membranes. However, we have recently shown for MSI-103, a designer-made antimicrobial peptide based on the PGLa sequence, that the orientation determined by ²H-NMR depends not on the bilayer thickness but rather on the intrinsic spontaneous curvature of the lipids (13). Accordingly, an insertion of PGLa and MAG2 into POPC/POPG should be prevented by the pronounced negative spontaneous curvature induced by the unsaturated acyl chains. However, a simple comparison of only two lipid systems does not yield an answer as to which of these two hypotheses is correct. Therefore, we have now collected data over a wide range of lipid systems, with systematic variations of acyl chain lengths (to address bilayer thickness) as well as chain saturations (to address lipid curvature) (see Table S1 in the Supporting Material). In this way, we found unambiguously that lipid curvature is also the decisive factor in the insertion of PGLa/MAG2.Here, ¹⁵N-NMR spectra of singly labeled peptides were recorded, and for each liquid-crystalline lipid system we prepared four oriented samples: ¹⁵N-MAG2 alone, ¹⁵N-MAG2 with PGLa, ¹⁵N-PGLa with MAG2, and ¹⁵N-PGLa alone. The total peptide/lipid molar ratio (P/L) was 1:50. ¹⁵N-NMR spectra are shown in Fig. 1, and the chemical shifts are listed in Fig. 2. The quality of each oriented sample was checked with ³¹P-NMR (see Fig. S1). Our previous detailed ²H- and ¹⁹F-NMR analysis of PGLa in DMPC and DMPC/DMPG showed that the helix realigns depending on the peptide concentration. Namely, at low concentration, PGLa is in an S-state with a tilt angle of ∼98° (3), but above a threshold concentration around P/L = 1:100, it flips into a tilted T-state with a tilt angle of ∼125° (9,14). In the presence of MAG2, it was found that PGLa inserts almost upright in an I-state with a tilt angle of ∼158° (11). The present ¹⁵N-NMR study confirms that in DMPC/DMPG (3:1), PGLa is in the I-state when mixed with MAG2, as indicated by the ¹⁵N chemical shift of 205 ppm. PGLa alone at P/L = 1:50 has a chemical shift of 116 ppm, which corresponds to a tilted orientation, as expected. MAG2 alone is found to be in the S-state (91 ppm), but when it is mixed with PGLa its signal moves to 105 ppm, indicating a small change in the alignment. MAG2 is, however, clearly not inserted like PGLa.Open in a separate windowFigure 1¹⁵N-NMR spectra of ¹⁵N-labeled PGLa or MAG2, alone or in a synergistic 1:1 mixture with the other peptide, in differently oriented lipids. Powder spectra are shown in the top row. Red, green, and blue lines indicate chemical shifts associated with the S-, T-, and I-state orientations, respectively.Open in a separate windowFigure 2Schematic overview of the orientation of PGLa (red), MAG2 (green), and MSI-103 (orange (13)) in different lipids. The corresponding ¹⁵N-NMR chemical shifts (in ppm) of the spectra in Fig. 1 are indicated beneath each peptide.In thin DLPC bilayers (12 carbon atoms in the chains) we see a behavior similar to that in DMPC (14 carbons), even though the exact chemical shifts are slightly different. PGLa alone is in the T-state, but in combination with MAG2, it flips into the I-state. MAG2, on the other hand, stays in the S-state with and without PGLa. In DPPC bilayers (16 carbons) also, the behavior is similar. PGLa alone is in the T-state but flips into the I-state in the presence of MAG2. MAG2 is slightly more tilted than in DMPC, but it never reaches the I-state.In contrast, in unsaturated lipids, both peptides are always in the S-state, both alone and in the presence of the synergistic partner. In POPC/POPG (9:1), the ¹⁵N chemical shifts of both PGLa and MAG2, alone and in the 1:1 mixture, are between 84 and 89 ppm, clearly indicating a flat alignment on the bilayer surface. As there are no changes in chemical shift with or without the other peptide, this could indicate that there are no interactions between them, in contrast to the situation in saturated lipids. Also, in thin DMoPC bilayers (with 14 carbon atoms and a double bond), the chemical shifts of all samples show that both peptides remain always in the S-state, whether alone or mixed.These results clearly demonstrate that the hydrophobic membrane thickness is not a critical factor for the insertion of PGLa in the presence of MAG2. In DMoPC (thinner than DMPC), there is no insertion, whereas in DPPC (thicker than POPC) insertion occurs. On the other hand, the results fully support the lipid-curvature hypothesis, which states that peptides remain on the surface in membranes composed of lipids with a negative spontaneous curvature, but are more easily tilted or inserted when the lipids have a positive spontaneous curvature (13).In a special lipid mixture, POPE/POPG/TOCL (72:23:5), often used to mimic the composition of the inner membrane of Escherichia coli (15), the result is practically the same as in POPC/POPG (9:1). Also here, chemical shifts of ∼84 ppm indicate that PGLa and MAG2 are always in the S-state, both alone and as a mixture. This behavior is in accordance with the curvature hypothesis, since PE and CL both have a strong negative curvature. On the other hand, when lyso-MPC is added to DMPC to increase the positive curvature, the chemical shift of MAG2 increases to 117 ppm, indicating a more tilted orientation in the membrane with enhanced curvature compared to DMPC or DMPC/DMPG, both with and without PGLa. PGLa alone gives a somewhat larger chemical shift but stays in the T-state, whereas PGLa together with MAG2 flips again into the I-state.We can now compare the results presented here with those from our previous study on the related peptide MSI-103 (13) to find strong correlations. Fig. 2 gives an overview of all results, illustrating the peptide orientations in the different lipid systems. PGLa on its own behaves just like MSI-103 and assumes the same S-state or T-state in the same systems, in full accordance with the lipid-curvature hypothesis. MAG2 alone behaves similarly but seems to have a higher concentration threshold to flip from the S-state to the T-state. In DMPC and DMPC/DMPG, where PGLa is already in the T-state, MAG2 is still in the S-state at P/L = 1:50. However, at P/L = 1:10 (Fig. S2), MAG2 has also reached the T-state. Since MAG2 is charged at both termini, whereas PGLa and MSI-103 are amidated and thus uncharged on the C terminus, it is indeed expected that MAG2 should not start to tilt as easily as PGLa or MSI-103. The polar sector of MAG2 is also larger (Fig. S3).When PGLa and MAG2 are mixed 1:1, their behavior correlates well with that of the individual peptides. In systems where PGLa and MSI-103 are in the S-state, the mixture of PGLa and MAG2 also remains in the S-state. Only when PGLa alone prefers the T-state does it get fully pushed into the I-state by the presence of MAG2. Thus, the model of MAG2-assisted insertion of PGLa proposed previously (12), which suggested that MAG2 would facilitate a thinning of the membrane such that PGLa would be able to insert into it, cannot be correct. We can instead conclude that only lipid systems that encourage peptide insertion per se show the MAG2-induced I-state of PGLa. The relationship between lipid shape and the tendency of peptides to insert into the membrane, as previously discussed (13), is illustrated in Fig. S4. Interestingly, common bacterial lipids like PE and CL have a negative spontaneous curvature and should thus not support peptide insertion and stable pores. However, pores could still be transient in native membranes, or other components like membrane proteins could influence the overall spontaneous curvature.In conclusion, we propose several criteria that encourage a peptide to insert from the surface-bound S-state more deeply into the membrane (i.e., into a T-state or I-state): 1), positive lipid spontaneous curvature, which is enhanced by large headgroups and ordered lipid chains (due to saturation, but also found at low temperatures close to the gel-to-liquid-crystalline phase transition); 2), a narrow polar sector and uncharged termini of the peptide; and 3), the presence of another peptide. The other peptide might have an indirect effect by changing the membrane properties via crowding. However, for PGLa/MAG2, a distinct synergistic activity has been demonstrated, indicating more specific interactions between these two peptides. The present ¹⁵N-NMR analysis shows that the two partner peptides are not aligned side-by-side as a dimer. Further solid-state NMR distance measurements will be required to clarify their detailed mode of assembly.     

Interactions involved in the realignment of membrane-associated helices. An investigation using oriented solid-state NMR and attenuated total reflection Fourier transform infrared spectroscopies

A series of histidine-containing peptides (LAH4X6) was designed to investigate the membrane interactions of selected side chains. To this purpose, their pH-dependent transitions from in-plane to transmembrane orientations were investigated by attenuated total reflection Fourier transform infrared and oriented solid-state NMR spectroscopies. Peptides of the same family have previously been shown to exhibit antibiotic and DNA transfection activities. Solution NMR spectroscopy indicates that these peptides form amphipathic helical structures in membrane environments, and the technique was also used to characterize the pK values of all histidines in the presence of detergent micelles. Whereas one face of the amphipathic helix is clearly hydrophobic, the opposite side is flanked by four histidines surrounding six leucine, alanine, glycine, tryptophan, or tyrosine residues, respectively. This diversity in peptide composition causes pronounced shifts in the midpoint pH of the in-plane to transmembrane helical transition, which is completely abolished for the peptides carrying the most hydrophilic amino acid residues. These properties open up a conceptually new approach to study in a quantitative manner the hydrophobic as well as specific interactions of amino acids in membranes. Notably, the resulting scale for whole residue transitions from the bilayer interface to the hydrophobic membrane interior is obtained from extended helical sequences in lipid bilayers.     

Structure, topology, and tilt of cell-signaling peptides containing nuclear localization sequences in membrane bilayers determined by solid-state NMR and molecular dynamics simulation studies

Cell-signaling peptides have been extensively used to transport functional molecules across the plasma membrane into living cells. These peptides consist of a hydrophobic sequence and a cationic nuclear localization sequence (NLS). It has been assumed that the hydrophobic region penetrates the hydrophobic lipid bilayer and delivers the NLS inside the cell. To better understand the transport mechanism of these peptides, in this study, we investigated the structure, orientation, tilt of the peptide relative to the bilayer normal, and the membrane interaction of two cell-signaling peptides, SA and SKP. Results from CD and solid-state NMR experiments combined with molecular dynamics simulations suggest that the hydrophobic region is helical and has a transmembrane orientation with the helical axis tilted away from the bilayer normal. The influence of the hydrophobic mismatch, between the hydrophobic length of the peptide and the hydrophobic thickness of the bilayer, on the tilt angle of the peptides was investigated using thicker POPC and thinner DMPC bilayers. NMR experiments showed that the hydrophobic domain of each peptide has a tilt angle of 15 +/- 3 degrees in POPC, whereas in DMPC, 25 +/- 3 degree and 30 +/- 3 degree tilts were observed for SA and SKP peptides, respectively. These results are in good agreement with molecular dynamics simulations, which predict a tilt angle of 13.3 degrees (SA in POPC), 16.4 degrees (SKP in POPC), 22.3 degrees (SA in DMPC), and 31.7 degrees (SKP in DMPC). These results and simulations on the hydrophobic fragment of SA or SKP suggest that the tilt of helices increases with a decrease in bilayer thickness without changing the phase, order, and structure of the lipid bilayers.     

3D Hydrophobic Moment Vectors as a Tool to Characterize the Surface Polarity of Amphiphilic Peptides

The interaction of membranes with peptides and proteins is largely determined by their amphiphilic character. Hydrophobic moments of helical segments are commonly derived from their two-dimensional helical wheel projections, and the same is true for β-sheets. However, to the best of our knowledge, there exists no method to describe structures in three dimensions or molecules with irregular shape. Here, we define the hydrophobic moment of a molecule as a vector in three dimensions by evaluating the surface distribution of all hydrophilic and lipophilic regions over any given shape. The electrostatic potential on the molecular surface is calculated based on the atomic point charges. The resulting hydrophobic moment vector is specific for the instantaneous conformation, and it takes into account all structural characteristics of the molecule, e.g., partial unfolding, bending, and side-chain torsion angles. Extended all-atom molecular dynamics simulations are then used to calculate the equilibrium hydrophobic moments for two antimicrobial peptides, gramicidin S and PGLa, under different conditions. We show that their effective hydrophobic moment vectors reflect the distribution of polar and nonpolar patches on the molecular surface and the calculated electrostatic surface potential. A comparison of simulations in solution and in lipid membranes shows how the peptides undergo internal conformational rearrangement upon binding to the bilayer surface. A good correlation with solid-state NMR data indicates that the hydrophobic moment vector can be used to predict the membrane binding geometry of peptides. This method is available as a web application on http://www.ibg.kit.edu/HM/.     

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.     

Synergistic transmembrane insertion of the heterodimeric PGLa/magainin 2 complex studied by solid-state NMR

The skin secretions of amphibians are a rich source of antimicrobial peptides. The two antimicrobial peptides PGLa and magainin 2, isolated from the African frog Xenopus laevis, have been shown to act synergistically by permeabilizing the membranes of microorganisms. In this report, the literature on PGLa is extensively reviewed, with special focus on its synergistically enhanced activity in the presence of magainin 2. Our recent solid state ²H NMR studies of the orientation of PGLa in lipid membranes alone and in the presence of magainin 2 are described in detail, and some new data from 3,3,3-²H₃-L-alanine labeled PGLa are included in the analysis.     

Structures of the dimeric and monomeric variants of magainin antimicrobial peptides (MSI-78 and MSI-594) in micelles and bilayers, determined by NMR spectroscopy

Magainins are antimicrobial peptides that selectively disrupt bacterial cell membranes. In an effort to determine the propensity for oligomerization of specific highly active magainin analogues in membrane mimetic systems, we studied the structures and lipid interactions of two synthetic variants of magainins (MSI-78 and MSI-594) originally designed by Genaera Corp. Using NMR experiments on these peptides solubilized in dodecylphosphocholine (DPC) micelles, we found that the first analogue, MSI-78, forms an antiparallel dimer with a "phenylalanine zipper" holding together two highly helical protomers, whereas the second analogue, MSI-594, whose phenylalanines 12 and 16 were changed into glycine and valine, respectively, does not dimerize under our experimental conditions. In addition, magic angle spinning solid-state NMR experiments carried out on multilamellar vesicles were used to corroborate the helical conformation of the peptides found in detergent micelles and support the existence of a more compact structure for MSI-78 and a pronounced conformational heterogeneity for MSI-594. Since magainin activity is modulated by oligomerization within the membrane bilayers, this study represents a step forward in understanding the role of self-association in determining magainin function.     

Raman spectroscopy of synthetic antimicrobial frog peptides magainin 2a and PGLa

Magainin and PGLa are 23- and 21-residue peptides isolated from the skin of the African clawed frog Xenopus laevis. They protect the frog from infection and exhibit a broad-spectrum antimicrobial activity in vitro. The mechanism of this activity involves the interaction of magainin with microbial membranes. We have measured the secondary structure and membrane-perturbing ability of these peptides to obtain information about this mechanism. Our results show that mgn2a forms a helix with an average length of less than 20 A upon binding to liposomes. At high concentrations (50 mg/mL) mgn2a spontaneously solubilizes phosphatidylcholine liposomes at temperatures above the gel-liquid-crystalline phase transition. Mgn2a appears to bind to the surface of liposomes made of negatively charged lipids without spontaneously penetrating the bilayer. Finally, mgn2a and PGLa interact together with liposomes in a synergistic way that enhances the helix content of one or both of the peptides and allows the peptides to more easily penetrate the bilayer. PGLa mixed with a small nonperturbing amount of magainin 2 amide is 25-43 times as potent as PGLa alone at inducing the release of carboxyfluorescein from liposomes. The results suggest that the mechanism of antimicrobial activity does not involve a channel formed by transmembrane helical peptides.     

Effects of Peptide Charge,Orientation, and Concentration on Melittin Transmembrane Pores

Melittin is a short cationic peptide that exerts cytolytic effects on bacterial and eukaryotic cells. Experiments suggest that in zwitterionic membranes, melittin forms transmembrane toroidal pores supported by four to eight peptides. A recently constructed melittin variant with a reduced cationic charge, MelP5, is active at 10-fold lower concentrations. In previous work, we performed molecular dynamics simulations on the microsecond timescale to examine the supramolecular pore structure of a melittin tetramer in zwitterionic and partially anionic membranes. We now extend that study to include the effects of peptide charge, initial orientation, and number of monomers on the pore formation and stabilization processes. Our results show that parallel transmembrane orientations of melittin and MelP5 are more consistent with experimental data. Whereas a MelP5 parallel hexamer forms a large stable pore during the 5-μs simulation time, a melittin hexamer and an octamer are not fully stable, with several monomers dissociating during the simulation time. Interaction-energy analysis shows that this difference in behavior between melittin and MelP5 is not due to stronger electrostatic repulsion between neighboring melittin peptides but to peptide-lipid interactions that disfavor the isolated MelP5 transmembrane monomer. The ability of melittin monomers to diffuse freely in the 1,2-dimyristoyl-SN-glycero-3-phosphocholine membrane leads to dynamic pores with varying molecularity.     

Effect of antimicrobial peptides from Australian tree frogs on anionic phospholipid membranes

Skin secretions of numerous Australian tree frogs contain antimicrobial peptides that form part of the host defense mechanism against bacterial infection. The mode of action of these antibiotics is thought to be lysis of infectious organisms via cell membrane disruption, on the basis of vesicle-encapsulated dye leakage data [Ambroggio et al. (2005) Biophys. J. 89, 1874-1881]. A detailed understanding of the interaction of these peptides with bacterial membranes at a molecular level, however, is critical to their development as novel antibacterial therapeutics. We focus on four of these peptides, aurein 1.2, citropin 1.1, maculatin 1.1, and caerin 1.1, which exist as random coil in aqueous solution but have alpha-helical secondary structure in membrane mimetic environments. In our earlier solid-state NMR studies, only neutral bilayers of the zwitterionic phospholipid dimyristoylphosphatidylcholine (DMPC) were used. Deuterated DMPC ( d 54-DMPC) was used to probe the effect of the peptides on the order of the lipid acyl chains and dynamics of the phospholipid headgroups by deuterium and (31)P NMR, respectively. In this report we demonstrate several important differences when anionic phospholipid is included in model membranes. Peptide-membrane interactions were characterized using surface plasmon resonance (SPR) spectroscopy and solid-state nuclear magnetic resonance (NMR) spectroscopy. Changes in phospholipid motions and membrane binding information provided additional insight into the action of these antimicrobial peptides. While this set of peptides has significant C- and N-terminal sequence homology, they vary in their mode of membrane interaction. The longer peptides caerin and maculatin exhibited properties that were consistent with transmembrane insertion while citropin and aurein demonstrated membrane disruptive mechanisms. Moreover, aurein was unique with greater perturbation of neutral versus anionic membranes. The results are consistent with a surface interaction for aurein 1.2 and pore formation rather than membrane lysis by the longer peptides.     

Control and role of pH in peptide–lipid interactions in oriented membrane samples

To understand the molecular mechanisms of amphiphilic membrane-active peptides, one needs to study their interactions with lipid bilayers under ambient conditions. However, it is difficult to control the pH of the sample in biophysical experiments that make use of mechanically aligned multilamellar membrane stacks on solid supports. HPLC-purified peptides tend to be acidic and can change the pH in the sample significantly. Here, we have systematically studied the influence of pH on the lipid interactions of the antimicrobial peptide PGLa embedded in oriented DMPC/DMPG bilayers. Using solid-state NMR (³¹P, ²H, ¹⁹F), both the lipid and peptide components were characterized independently, though in the same oriented samples under typical conditions of maximum hydration. The observed changes in lipid polymorphism were supported by DSC on multilamellar liposome suspensions. On this basis, we can present an optimized sample preparation protocol and discuss the challenges of performing solid-state NMR experiments under controlled pH. DMPC/DMPG bilayers show a significant up-field shift and broadening of the main lipid phase transition temperature when lowering the pH from 10.0 to 2.6. Both, strongly acidic and basic pH, cause a significant degree of lipid hydrolysis, which is exacerbated by the presence of PGLa. The characteristic re-alignment of PGLa from a surface-bound to a tilted state is not affected between pH of 7 to 4 in fluid bilayers. On the other hand, in gel-phase bilayers the peptide remains isotropically mobile under acidic conditions, displays various co-existing orientational states at pH 7, and adopts an unknown structural state at basic pH.     

Orientation and Dynamics of Peptides in Membranes Calculated from H-NMR Data

Solid-state ²H-NMR is routinely used to determine the alignment of membrane-bound peptides. Here we demonstrate that it can also provide a quantitative measure of the fluctuations around the distinct molecular axes. Using several dynamic models with increasing complexity, we reanalyzed published ²H-NMR data on two representative α-helical peptides: 1), the amphiphilic antimicrobial peptide PGLa, which permeabilizes membranes by going from a monomeric surface-bound to a dimeric tilted state and finally inserting as an oligomeric pore; and 2), the hydrophobic WALP23, which is a typical transmembrane segment, although previous analysis had yielded helix tilt angles much smaller than expected from hydrophobic mismatch and molecular dynamics simulations. Their ²H-NMR data were deconvoluted in terms of the two main helix orientation angles (representing the time-averaged peptide tilt and azimuthal rotation), as well as the amplitudes of fluctuation about the corresponding molecular axes (providing the dynamic picture). The mobility of PGLa is found to be moderate and to correlate well with the respective oligomeric states. WALP23 fluctuates more vigorously, now in better agreement with the molecular dynamics simulations and mismatch predictions. The analysis demonstrates that when ²H-NMR data are fitted to extract peptide orientation angles, an explicit representation of the peptide rigid-body angular fluctuations should be included.     

Synergistic transmembrane alignment of the antimicrobial heterodimer PGLa/magainin

The antimicrobial activity of amphipathic alpha-helical peptides is usually attributed to the formation of pores in bacterial membranes, but direct structural information about such a membrane-bound state is sparse. Solid state (2)H-NMR has previously shown that the antimicrobial peptide PGLa undergoes a concentration-dependent realignment from a surface-bound S-state to a tilted T-state. The corresponding change in helix tilt angle from 98 to 125 degrees was interpreted as the formation of PGLa/magainin heterodimers residing on the bilayer surface. Under no conditions so far, has an upright membrane-inserted I-state been observed in which a transmembrane helix alignment would be expected. Here, we have demonstrated that PGLa is able to assume such an I-state in a 1:1 mixture with magainin 2 at a peptide-to-lipid ratio as low as 1:100 in dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol model membranes. This (2)H-NMR analysis is based on seven orientational constraints from Ala-3,3,3-d(3) substituted in a non-perturbing manner for four native Ala residues as well as two Ile and one Gly. The observed helix tilt of 158 degrees is rationalized by the formation of heterodimers. This structurally synergistic effect between the two related peptides from the skin of Xenopus laevis correlates very well with their known functional synergistic mode of action. To our knowledge, this example of PGLa is the first case where an alpha-helical antimicrobial peptide is directly shown to assume a transmembrane state that is compatible with the postulated toroidal wormhole pore structure.     

Membrane thickening by the antimicrobial peptide PGLa

Using x-ray diffraction, solid-state ²H-NMR, differential scanning calorimetry, and dilatometry, we have observed a perturbation of saturated acyl chain phosphatidylglycerol bilayers by the antimicrobial peptide peptidyl-glycylleucine-carboxyamide (PGLa) that is dependent on the length of the hydrocarbon chain. In the gel phase, PGLa induces a quasi-interdigitated phase, previously reported also for other peptides, which is most pronounced for C18 phosphatidylglycerol. In the fluid phase, we found an increase of the membrane thickness and NMR order parameter for C14 and C16 phosphatidylglycerol bilayers, though not for C18. The data is best understood in terms of a close hydrophobic match between the C18 bilayer core and the peptide length when PGLa is inserted with its helical axis normal to the bilayer surface. The C16 acyl chains appear to stretch to accommodate PGLa, whereas tilting within the bilayer seems to be energetically favorable for the peptide when inserted into bilayers of C14 phosphatidylglycerol. In contrast to the commonly accepted membrane thinning effect of antimicrobial peptides, the data demonstrate that pore formation does not necessarily relate to changes in the overall bilayer structure.     

Molecular Dynamics Simulations Reveal the HIV-1 Vpu Transmembrane Protein to Form Stable Pentamers

The human immunodeficiency virus type I (HIV-1) Vpu protein is 81 residues long and has two cytoplasmic and one transmembrane (TM) helical domains. The TM domain oligomerizes to form a monovalent cation selective ion channel and facilitates viral release from host cells. Exactly how many TM domains oligomerize to form the pore is still not understood, with experimental studies indicating the existence of a variety of oligomerization states. In this study, molecular dynamics (MD) simulations were performed to investigate the propensity of the Vpu TM domain to exist in tetrameric, pentameric, and hexameric forms. Starting with an idealized α-helical representation of the TM domain, a thorough search for the possible orientations of the monomer units within each oligomeric form was carried out using replica-exchange MD simulations in an implicit membrane environment. Extensive simulations in a fully hydrated lipid bilayer environment on representative structures obtained from the above approach showed the pentamer to be the most stable oligomeric state, with interhelical van der Waals interactions being critical for stability of the pentamer. Atomic details of the factors responsible for stable pentamer structures are presented. The structural features of the pentamer models are consistent with existing experimental information on the ion channel activity, existence of a kink around the Ile17, and the location of tetherin binding residues. Ser23 is proposed to play an important role in ion channel activity of Vpu and possibly in virus propagation.