Spontaneous transmembrane pore formation by short-chain synthetic peptide

Amphiphilic β-peptides, which are synthetically designed short-chain helical foldamers of β-amino acids, are established potent biomimetic alternatives of natural antimicrobial peptides. An intriguing question is how the distinct molecular architecture of these short-chain and rigid synthetic peptides translates to its potent membrane-disruption ability. Here, we address this question via a combination of all-atom and coarse-grained molecular dynamics simulations of the interaction of mixed phospholipid bilayer with an antimicrobial 10-residue globally amphiphilic helical β-peptide at a wide range of concentrations. The simulation demonstrates that multiple copies of this synthetic peptide, initially placed in aqueous solution, readily self-assemble and adsorb at membrane interface. Subsequently, beyond a threshold peptide/lipid ratio, the surface-adsorbed oligomeric aggregate moves inside the membrane and spontaneously forms stable water-filled transmembrane pores via a cooperative mechanism. The defects induced by these pores lead to the dislocation of interfacial lipid headgroups, membrane thinning, and substantial water leakage inside the hydrophobic core of the membrane. A molecular analysis reveals that despite having a short architecture, these synthetic peptides, once inside the membrane, would stretch themselves toward the distal leaflet in favor of potential contact with polar headgroups and interfacial water layer. The pore formed in coarse-grained simulation was found to be resilient upon structural refinement. Interestingly, the pore-inducing ability was found to be elusive in a non-globally amphiphilic sequence isomer of the same β-peptide, indicating strong sequence dependence. Taken together, this work puts forward key perspectives of membrane activity of minimally designed synthetic biomimetic oligomers relative to the natural antimicrobial peptides.     

Venom peptides from solitary hunting wasps induce feeding disorder in lepidopteran larvae

The cell lytic activity and toxicity against lepidopteran larvae of 13 venom peptides (4 OdVPs and 9 EpVPs) from two solitary hunting wasps, Orancistrocerus drewseni and Eumenes pomiformis, were examined with mastoparan as a reference peptide. Of the 13 peptides, 7 were predicted to have α-helical structures that exhibit the typical character of amphipathic α-helical antimicrobial peptides. The remaining peptides exhibited coil structures; among these, EpVP5 possesses two Cys residues that form an internal disulfide bridge. All the helical peptides including mastoparan showed antimicrobial and insect cell lytic activities, whereas only two of them were hemolytic against human erythrocytes. The helical peptides induced a feeding disorder when injected into the vicinity of the head and thorax of Spodoptera exigua larvae, perhaps because their non-specific neurotoxic or myotoxic action induced cell lysis. At low concentrations, however, these helical peptides increased cell permeability without inducing cell lysis. These findings suggest that the helical venom peptides may function as non-specific neurotoxins or myotoxins and venom-spreading factors at low concentrations, as well as preservatives for long-term storage of the prey via antimicrobial, particularly antifungal, activities.     

Proteolysis of Human Thrombin Generates Novel Host Defense Peptides

The coagulation system is characterized by the sequential and highly localized activation of a series of serine proteases, culminating in the conversion of fibrinogen into fibrin, and formation of a fibrin clot. Here we show that C-terminal peptides of thrombin, a key enzyme in the coagulation cascade, constitute a novel class of host defense peptides, released upon proteolysis of thrombin in vitro, and detected in human wounds in vivo. Under physiological conditions, these peptides exert antimicrobial effects against Gram-positive and Gram-negative bacteria, mediated by membrane lysis, as well as immunomodulatory functions, by inhibiting macrophage responses to bacterial lipopolysaccharide. In mice, they are protective against P. aeruginosa sepsis, as well as lipopolysaccharide-induced shock. Moreover, the thrombin-derived peptides exhibit helical structures upon binding to lipopolysaccharide and can also permeabilize liposomes, features typical of “classical” helical antimicrobial peptides. These findings provide a novel link between the coagulation system and host-defense peptides, two fundamental biological systems activated in response to injury and microbial invasion.     

Synthesis of novel unnatural amino acid as a building block and its incorporation into an antimicrobial peptide

Considering the biological mechanism and in vivo stability of antimicrobial peptides, we designed and synthesized novel unnatural amino acids with more positively charged and bulky side chain group than lysine residue. The unusual amino acids, which were synthesized by either solution phase or solid phase, were incorporated into an antimicrobial peptide. Its effect on the stability, activity, and the structure of the peptide was studied to evaluate the potential of these novel unnatural amino acids as a building block for antimicrobial peptides. The incorporation of this unusual amino acid increased the resistance of the peptide against serum protease more than three times without a decrease in the activity. Circular dichroism spectra of the peptides indicated that all novel unnatural amino acids must have lower helical forming propensities than lysine. Our results indicated that the unnatural amino acids synthesized in this study could be used not only as a novel building block for combinatorial libraries of antimicrobial peptides, but also for structure–activity relationship studies about antimicrobial peptides.     

A Sensory Complex Consisting of an ATP-binding Cassette Transporter and a Two-component Regulatory System Controls Bacitracin Resistance in Bacillus subtilis

Resistance against antimicrobial peptides in many Firmicutes bacteria is mediated by detoxification systems that are composed of a two-component regulatory system (TCS) and an ATP-binding cassette (ABC) transporter. The histidine kinases of these systems depend entirely on the transporter for sensing of antimicrobial peptides, suggesting a novel mode of signal transduction where the transporter constitutes the actual sensor. The aim of this study was to investigate the molecular mechanisms of this unusual signaling pathway in more detail, using the bacitracin resistance system BceRS-BceAB of Bacillus subtilis as an example. To analyze the proposed communication between TCS and the ABC transporter, we characterized their interactions by bacterial two-hybrid analyses and could show that the permease BceB and the histidine kinase BceS interact directly. In vitro pulldown assays confirmed this interaction, which was found to be independent of bacitracin. Because it was unknown whether BceAB-type transporters could detect their substrate peptides directly or instead recognized the peptide-target complex in the cell envelope, we next analyzed substrate binding by the transport permease, BceB. Direct and specific binding of bacitracin by BceB was demonstrated by surface plasmon resonance spectroscopy. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the autophosphorylation activity of the histidine kinase. Taken together, our findings clearly show the existence of a sensory complex composed of TCS and ABC transporters and provide the first functional insights into the mechanisms of stimulus perception, signal transduction, and antimicrobial resistance employed by Bce-like detoxification systems.     

EcDBS1R6: A novel cationic antimicrobial peptide derived from a signal peptide sequence

BackgroundBacterial infections represent a major worldwide health problem the antimicrobial peptides (AMPs) have been considered as potential alternative agents for treating these infections. Here we demonstrated the antimicrobial activity of EcDBS1R6, a peptide derived from a signal peptide sequence of Escherichia coli that we previously turned into an AMP by making changes through the Joker algorithm.MethodsAntimicrobial activity was measured by broth microdilution method. Membrane integrity was measured using fluorescent probes and through scanning electron microscopy imaging. A sliding window of truncated peptides was used to determine the EcDBS1R6 active core. Molecular dynamics in TFE/water environment was used to assess the EcDBS1R6 structure.ResultsSignal peptides are known to naturally interact with membranes; however, the modifications introduced by Joker transformed this peptide into a membrane-active agent capable of killing bacteria. The C-terminus was unable to fold into an α-helix whereas its fragments showed poor or no antimicrobial activity, suggesting that the EcDBS1R6 antibacterial core was located at the helical N-terminus, corresponding to the signal peptide portion of the parent peptide.ConclusionThe strategy of transforming signal peptides into AMPs appears to be promising and could be used to produce novel antimicrobial agents.General significanceThe process of transforming an inactive signal peptide into an antimicrobial peptide could open a new venue for creating new AMPs derived from signal peptides.     

Structural determinants of antimicrobial and antiplasmodial activity and selectivity in histidine-rich amphipathic cationic peptides

Designed histidine-rich amphipathic cationic peptides, such as LAH4, have enhanced membrane disruption and antibiotic properties when the peptide adopts an alignment parallel to the membrane surface. Although this was previously achieved by lowering the pH, here we have designed a new generation of histidine-rich peptides that adopt a surface alignment at neutral pH. In vitro, this new generation of peptides are powerful antibiotics in terms of the concentrations required for antibiotic activity; the spectrum of target bacteria, fungi, and parasites; and the speed with which they kill. Further modifications to the peptides, including the addition of more hydrophobic residues at the N terminus, the inclusion of a helix-breaking proline residue or using D-amino acids as building blocks, modulated the biophysical properties of the peptides and led to substantial changes in toxicity to human and parasite cells but had only a minimal effect on the antibacterial and antifungal activity. Using a range of biophysical methods, in particular solid-state NMR, we show that the peptides are highly efficient at disrupting the anionic lipid component of model membranes. However, we also show that effective pore formation in such model membranes may be related to, but is not essential for, high antimicrobial activity by cationic amphipathic helical peptides. The information in this study comprises a new layer of detail in the understanding of the action of cationic helical antimicrobial peptides and shows that rational design is capable of producing potentially therapeutic membrane active peptides with properties tailored to their function.     

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/.     

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.     

Antimicrobial activity and conformation of gaegurin-6 amide and its analogs

A function of the intra-disulfide bridge located at the C-terminal of Rana peptides has not been extensively studied. To investigate the function of the disulfide bridge related to the activity and the structure, we chose Gaegurin-6, isolated from Rana rugosa as a model peptide and synthesized linear analogs. The reduction of the disulfide bridge resulted in the complete loss of antimicrobial activity while replacements of cysteines by serines retained antimicrobial activity. Circular dichroism spectra from a titration of the peptides in sodium dodecyl sulfate indicated that the disulfide bridge of Gaegurin-6 might stabilize the induction of an helical structure in lipid membranes and the helical forming propensity of the peptides correlated with antimicrobial activity.     

Transmembrane peptide effects on bacterial membrane integrity and organization

As the bacterial multidrug resistance crisis continues, membrane-active antimicrobial peptides are being explored as an alternate treatment to conventional antibiotics. In contrast to antimicrobial peptides, which function by a nonspecific membrane disruption mechanism, here we describe a series of transmembrane (TM) peptides that are designed to act as drug efflux inhibitors by aligning with and out-competing a conserved TM4-TM4 homodimerization motif within bacterial small multidrug resistance proteins. The peptides contain two terminal tags: a C-terminal lysine tag to direct the peptides toward the negatively charged bacterial membrane, and an uncharged N-terminal sarcosine (N-methyl-glycine) tag to promote membrane insertion. While effective at inhibiting efflux activity, ostensibly through their designed mechanism of action, the impact of the peptides on the bacterial inner membrane remains undetermined. To evaluate the extant peptide-membrane interactions, we performed a series of biophysical measurements. Circular dichroism spectroscopy and Trp fluorescence showed that the peptides insert into the membrane generally in helical form. Interestingly, differential scanning calorimetry of the peptides added to bacterial-like membranes (POPE:POPG 3:1) revealed the peptides’ ability to demix the POPE and POPG lipids, creating two pools, one of which is likely a peptide-POPG conglomerate, and the other a POPE-rich component where the native POPG content has been depleted. However, dye leakage assays confirmed that these events occur without causing significant membrane disruption both in vitro and in vivo, indicating that the peptides can target the small multidrug resistance TM4-TM4 motif without nonspecific membrane disruption. In related studies, DiOC₂(3) fluorescence indicated moderate peptide-mediated reduction of the proton motive force for all peptides, including control peptides that did not display inhibitory activity. The overall findings suggest that peptides designed with suitable tags, sequence hydrophobicity, and charge distribution can be directed more generally to impact proteins whose function involves membrane-embedded protein-protein interactions.     

Alpha-helical antimicrobial peptides—Using a sequence template to guide structure-activity relationship studies

An important class of cytolytic antimicrobial peptides (AMPs) assumes an amphipathic, α-helical conformation that permits efficient interaction with biological membranes. Host defence peptides of this type are widespread in nature, and numerous synthetic model AMPs have been derived from these or designed de novo based on their characteristics. In this review we provide an overview of the ‘sequence template’ approach which we have used to design potent artificial helical AMPs, to guide structure-activity relationship studies aimed at their optimization, and to help identify novel natural AMP sequences. Combining this approach with the rational use of natural and non-proteinogenic amino acid building blocks has allowed us to probe the individual effects on the peptides' activity of structural and physico-chemical parameters such as the size, propensity for helical structuring, amphipathic hydrophobicity, cationicity, and hydrophobic or polar sector characteristics. These studies furthermore provided useful insights into alternative modes of action for natural membrane-active helical peptides.     

Synthetic oligoureas of metaphenylenediamine mimic host defence peptides in their antimicrobial behaviour

Oligomeric ureas of m-phenylenediamine target anionic DMPG (dimyristoylphosphatidylglycerol) and possess promise as antimicrobial agents. Their similar size, shape and hydrophobicity to helical antimicrobial peptides (AMPs) may be important for activity to exist and the ability of these compounds to insert into a well ordered lipid environment.     

Importance of residue 13 and the C-terminus for the structure and activity of the antimicrobial peptide aurein 2.2

Previous studies on aurein 2.2 and 2.3 in DMPC/DMPG and POPC/POPG membranes have shown that bilayer thickness and phosphatidylglycerol content have a significant impact on the interaction of these peptides with membrane bilayers. Further examination with the DiSC₃5 assay has indicated that aurein 2.2 induces greater membrane leakage than aurein 2.3 in Staphylococcus aureus C622. The only difference between these peptides is a Leu to Ile mutation at residue 13. To better understand the importance of this residue, the structure and activity of the L13A, L13F, and L13V mutants were investigated. In addition, we investigated a number of peptides with truncations at the C-terminus to determine whether the C-terminus, which contains residue 13, is crucial for antimicrobial activity. Solution circular dichroism results demonstrated that the L13F mutation and the truncation of the C-terminus by six residues resulted in decreased helical content, whereas the L13A or L13V mutation and the truncation of the C-terminus by three residues showed little to no effect on the structure. Oriented circular dichroism results demonstrated that only an extensive C-terminal truncation reduced the ability of the peptide to insert into lipid bilayers. ³¹P NMR spectroscopy showed that all peptides disorder the headgroups. The implications of these results in terms of antimicrobial activity and the ability of these peptides to induce leakage in S. aureus are discussed. The results suggest that the presence of the 13th residue in aurein 2.2 is important for structure and activity, but the exact nature of residue 13 is less important as long as it is a hydrophobic residue.     

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.     

Expression,Folding, and Proton Transport Activity of Human Uncoupling Protein-1 (UCP1) in Lipid Membranes: EVIDENCE FOR ASSOCIATED FUNCTIONAL FORMS*

Uncoupling protein-1 (UCP1) is abundantly expressed in the mitochondrial inner membrane of brown adipose tissues and has an important role in heat generation, mediated by its proton transport function. The structure and function of UCP1 are not fully understood, partially due to the difficulty in obtaining native-like folded proteins in vitro. In this study, using the auto-induction method, we have successfully expressed UCP1 in Escherichia coli membranes in high yield. Overexpressed UCP1 in bacterial membranes was extracted using mild detergents and reconstituted into phospholipid bilayers for biochemical studies. UCP1 was folded in octyl glucoside, as indicated by its high helical content and binding to ATP, a known UCP1 proton transport inhibitor. Reconstituted UCP1 in phospholipid vesicles also exhibited highly helical structures and proton transport that is activated by fatty acids and inhibited by purine nucleotides. Self-associated functional forms of UCP1 in lipid membranes were observed for the first time. The self-assembly of UCP1 into tetramers was unambiguously characterized by circular dichroism and fluorescence spectroscopy, analytical ultracentrifugation, and semi-native gel electrophoresis. In addition, the mitochondrial lipid cardiolipin stabilized the structure of associated UCP1 and enhanced the proton transport activity of the protein. The existence of the functional oligomeric states of UCP1 in the lipid membranes has important implications for understanding the structure and proton transport mechanism of this protein in brown adipose tissues as well as structure-function relationships of other mammalian UCPs in other tissues.     

Design and activity of novel lactoferrampin analogues against O157:H7 enterohemorrhagic escherichia coli

Lactoferrampin 265–284 (LFampin 265–284) is a peptide consisting of residues 265–284 of N1‐domain of bovine Lactoferrin (LF). This peptide has several cationic groups in the C‐terminal lobe, exhibiting an antibacterial activity against a wide range of microorganisms. However, LFampin 265–284 exhibits low antimicrobial activity against the O157:H7 enterohaemorrhagic Escherichia coli (EHEC O157:H7) when compared with Lactoferrin chimera and Lactoferricin. Here, we have designed three analogues of LFampin 265–284 based on the distribution of cationic groups, hydrophobicity, size, and sequence. Analogues were synthesized by solid phase chemistry using Fmoc methodology obtaining peptides with 95% purity. All peptides maintain the ability to adopt helical conformations (checked by circular dichroism spectra and molecular simulations). Some of these analogues exhibited a significant increase in antimicrobial activity by counting colony forming units against EHEC O157:H7 compared to native LFampin 265–284, with MIC of 10 and 40 µM for 264G‐D265K and 264G‐D265K/S272R, respectively. The incorporation of a GKLI sequence in the N‐terminal lobe increased dramatically its antibacterial activity, an effect which has been attributed to the addition of cationic groups in the N‐terminal side that may stabilize the helical conformation of the new designed peptides. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 319–328, 2014.     

Novel antimicrobial peptides from the venom of the eusocial bee Halictus sexcinctus (Hymenoptera: Halictidae) and their analogs

Two novel antimicrobial peptides, named halictines, were isolated from the venom of the eusocial bee Halictus sexcinctus. Their primary sequences were established by ESI-QTOF mass spectrometry, Edman degradation and enzymatic digestion as Gly-Met-Trp-Ser-Lys-Ile-Leu-Gly-His-Leu-Ile-Arg-NH₂ (HAL-1), and Gly-Lys-Trp-Met-Ser-Leu-Leu-Lys–His-Ile-Leu-Lys-NH₂ (HAL-2). Both peptides exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria but also noticeable hemolytic activity. The CD spectra of HAL-1 and HAL-2 measured in the presence of trifluoroethanol or SDS showed ability to form an amphipathic α-helical secondary structure in an anisotropic environment such as bacterial cell membrane. NMR spectra of HAL-1 and HAL-2 measured in trifluoroethanol/water confirmed formation of helical conformation in both peptides with a slightly higher helical propensity in HAL-1. Altogether, we prepared 51 of HAL-1 and HAL-2 analogs to study the effect of such structural parameters as cationicity, hydrophobicity, α-helicity, amphipathicity, and truncation on antimicrobial and hemolytic activities. The potentially most promising analogs in both series are those with increased net positive charge, in which the suitable amino acid residues were replaced by Lys. This improvement basically relates to the increase of antimicrobial activity against pathogenic Pseudomonas aeruginosa and to the mitigation of hemolytic activity.     

H1 helix of colicin U causes phospholipid membrane permeation

In light of an increasing number of antibiotic-resistant bacterial strains, it is essential to understand an action imposed by various antimicrobial agents on bacteria at the molecular level. One of the leading mechanisms of killing bacteria is related to the alteration of their plasmatic membrane. We study bio-inspired peptides originating from natural antimicrobial proteins colicins, which can disrupt membranes of bacterial cells. Namely, we focus on the α-helix H1 of colicin U, produced by bacterium Shigella boydii, and compare it with analogous peptides derived from two different colicins. To address the behavior of the peptides in biological membranes, we employ a combination of molecular simulations and experiments. We use molecular dynamics simulations to show that all three peptides are stable in model zwitterionic and negatively charged phospholipid membranes. At the molecular level, their embedment leads to the formation of membrane defects, membrane permeation for water, and, for negatively charged lipids, membrane poration. These effects are caused by the presence of polar moieties in the considered peptides. Importantly, simulations demonstrate that even monomeric H1 peptides can form toroidal pores. At the macroscopic level, we employ experimental co-sedimentation and fluorescence leakage assays. We show that the H1 peptide of colicin U incorporates into phospholipid vesicles and disrupts their membranes, causing leakage, in agreement with the molecular simulations. These insights obtained for model systems seem important for understanding the mechanisms of antimicrobial action of natural bacteriocins and for future exploration of small bio-inspired peptides able to disrupt bacterial membranes.     

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.