Two distinct amphipathic peptide antibiotics with systemic efficacy.

Antimicrobial peptides are important candidates for developing new classes of antibiotics because of their potency against antibiotic-resistant pathogens. Current research focuses on topical applications and it is unclear how to design peptides with systemic efficacy. To address this problem, we designed two potent peptides by combining database-guided discovery with structure-based design. When bound to membranes, these two short peptides with an identical amino acid composition can adopt two distinct amphipathic structures: A classic horizontal helix (horine) and a novel vertical spiral structure (verine). Their horizontal and vertical orientations on membranes were determined by solid-state 15N NMR data. While horine was potent primarily against gram-positive pathogens, verine showed broad-spectrum antimicrobial activity. Both peptides protected greater than 80% mice from infection-caused deaths. Moreover, horine and verine also displayed significant systemic efficacy in different murine models comparable to conventional antibiotics. In addition, they could eliminate resistant pathogens and preformed biofilms. Significantly, the peptides showed no nephrotoxicity to mice after intraperitoneal or intravenous administration for 1 wk. Our study underscores the significance of horine and verine in fighting drug-resistant pathogens.

Membrane interactions of Ocellatins. Where do antimicrobial gaps stem from?

The antimicrobial peptides Ocellatin-LB1, -LB2 and -F1, isolated from frogs, are identical from residue 1 to 22, which correspond to the -LB1 sequence, whereas -LB2 carries an extra N and -F1 additional NKL residues at their C-termini. Despite the similar sequences, previous investigations showed different spectra of activities and biophysical investigations indicated a direct correlation between both membrane-disruptive properties and activities, i.e., ocellatin-F1 > ocellatin-LB1 > ocellatin-LB2. This study presents experimental evidence as well as results from theoretical studies that contribute to a deeper understanding on how these peptides exert their antimicrobial activities and how small differences in the amino acid composition and their secondary structure can be correlated to these activity gaps. Solid-state NMR experiments allied to the simulation of anisotropic NMR parameters allowed the determination of the membrane topologies of these ocellatins. Interestingly, the extra Asn residue at the Ocellatin-LB2 C-terminus results in increased topological flexibility, which is mainly related to wobbling of the helix main axis as noticed by molecular dynamics simulations. Binding kinetics and thermodynamics of the interactions have also been assessed by Surface Plasmon Resonance and Isothermal Titration Calorimetry. Therefore, these investigations allowed to understand in atomic detail the relationships between peptide structure and membrane topology, which are in tune within the series -F1 > > -LB1 ≥ -LB2, as well as how peptide dynamics can affect membrane topology, insertion and binding.

Investigations of the Structure, Topology, and Interactions of the Transmembrane Domain of the Lipid-Sorting Protein p24 Being Highly Selective for Sphingomyelin-C18.

The p24 proteins play an important role in the secretory pathway where they selectively connect various cargo to other proteins, thereby being involved in the controlled assembly and disassembly of the coat protein complexes and lipid sorting. Recently, a highly selective lipid interaction motif has been identified within the p24 transmembrane domain (TMD) that recognizes the combination of the sphingomyelin headgroup and the exact length of the C18 fatty acyl chain (SM-C18). Here, we present investigations of the structure, dynamics, and sphingomyelin interactions of the p24 transmembrane region using circular dichroism, tryptophan fluorescence, and solid-state nuclear magnetic resonance (NMR) spectroscopies of the polypeptides and the surrounding lipids. Membrane insertion and/or conformation of the TMD is strongly dependent on the membrane lipid composition where the transmembrane helical insertion is strongest in the presence of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) and SM-C18. By analyzing solid-state NMR angular restraints from a large number of labeled sites, we have found a tilt angle of 19° for the transmembrane helical domain at a peptide-to-lipid ratio of 1 mol %. Only minor changes in the solid-state NMR spectra are observed due to the presence of SM-C18; the only visible alterations are associated with the SM-C18 recognition motif close to the carboxy-terminal part of the hydrophobic transmembrane region in the proximity of the SM headgroup. Finally, the deuterium order parameters of POPC- d31 were nearly unaffected by the presence of SM-C18 or the polypeptide alone but decreased noticeably when the sphingomyelin and the polypeptide were added in combination.

Trichogin GA IV Alignment and Oligomerization in Phospholipid Bilayers.

Trichogin GA IV is a short peptaibol with antimicrobial activity. This uncharged, but amphipathic, sequence is aligned at the membrane interface and undergoes a transition to an aggregated state that inserts more deeply into the membrane, an assembly that predominates at a peptide-to-lipid ratio (P/L) of 1:20. In this work, the natural trichogin sequence was prepared and reconstituted into oriented lipid bilayers. The 15 N NMR chemical shift is indicative of a well-defined alignment of the peptide parallel to the membrane surface at P/Ls of 1:120 and 1:20. When the P/L is increased to 1:8, an additional peptide topology is observed that is indicative of a heterogeneous orientation, with helix alignments ranging from around the magic angle to perfectly in-plane. The topological preference of the trichogin helix for an orientation parallel to the membrane surface was confirmed by attenuated total reflection FTIR spectroscopy. Furthermore, 19 F CODEX experiments were performed on a trichogin sequence with 19 F-Phe at position 10. The CODEX decay is in agreement with a tetrameric complex, in which the 19 F sites are about 9-9.5 Šapart. Thus, a model emerges in which the monomeric peptide aligns along the membrane surface. When the peptide concentration increases, first dimeric and then tetrameric assemblies form, made up from helices oriented predominantly parallel to the membrane surface. The formation of these aggregates correlates with the release of vesicle contents including relatively large molecules.

Solid-State NMR Investigations of the MHC II Transmembrane Domains: Topological Equilibria and Lipid Interactions.

The major histocompatibility complex class II (MHC II) membrane proteins are key players in the adaptive immune response. An aberrant function of these molecules is associated with a large number of autoimmune diseases such as diabetes type I and chronic inflammatory diseases. The MHC class II is assembled from DQ alpha 1 and DQ beta 1 which come together as a heterodimer through GXXXG-mediated protein-protein interactions and a highly specific protein-sphingomyelin-C18 interaction motif located on DQA1. This association can have important consequences in regulating the function of these membrane proteins. Here, we investigated the structure and topology of the DQA1 and DQB1 transmembrane helical domains by CD-, oriented 2H and 15N solid-state NMR spectroscopies. The spectra at peptide-to-lipid ratios of 0.5 to 2 mol% are indicative of a topological equilibrium involving a helix crossing the membrane with a tilt angle of about 20° and another transmembrane topology with around 30° tilt. The latter is probably representing a dimer. Furthermore, at the lowest peptide-to-lipid ratio, a third polypeptide population becomes obvious. Interestingly, the DQB1 and to a lesser extent the DQA1 transmembrane helical domains exhibit a strong fatty acyl chain disordering effect on the inner segments of the 2H-labelled palmitoyl chain of POPC bilayers. This phosphatidylcholine disordering requires the presence of sphingomyelin-C18 suggesting that the ensemble of transmembrane polypeptide and sphingolipid exerts positive curvature strain.

Dynamic Nuclear Polarization / solid-state NMR of membranes. Thermal effects and sample geometry.

Whereas specially designed dinitroxide biradicals, reconstitution protocols, oriented sample geometries and NMR probes have helped to much increase the DNP enhancement factors of membrane samples they still lag considerably behind those obtained from glasses made of protein solutions. Here we show that not only the MAS rotor material but also the distribution of the membrane samples within the NMR rotor have a pronounced effect on the DNP enhancement. These observations are rationalized with the cooling efficiency and the internal properties of the sample, monitored by their T1 relaxation, microwave ON versus OFF signal intensities and DNP effect. The data are suggestive that for membranes the speed of cooling has a pronounced effect on the membrane properties and concomitantly the distribution of biradicals within the sample.

Supramolecular Organization of Apolipoprotein-A-I-Derived Peptides within Disc-like Arrangements.

Apolipoprotein A-I is the major protein component of high-density lipoproteins and fulfils important functions in lipid metabolism. Its structure consists of a chain of tandem domains of amphipathic helices. Using this protein as a template membrane scaffolding protein, class A amphipathic helical peptides were designed to support the amphipathic helix theory and later as therapeutic tools in biomedicine. Here, we investigated the lipid interactions of two apolipoprotein-A-I-derived class A amphipathic peptides, 14A (Ac-DYLKA FYDKL KEAF-NH2) and 18A (Ac-DWLKA FYDKV AEKLK EAF- NH2), including the disc-like supramolecular structures they form with phospholipids. Thus, the topologies of 14A and 18A in phospholipid bilayers have been determined by oriented solid-state NMR spectroscopy. Whereas at a peptide-to-lipid ratio of 2 mol% the peptides align parallel to the bilayer surface, at 7.5 mol% disc-like structures are formed that spontaneously orient in the magnetic field of the NMR spectrometer. From a comprehensive data set of four 15N- or 2H-labeled positions of 14A, a tilt angle, which deviates from perfectly in-planar by 14°, and a model for the peptidic rim structure have been obtained. The tilt and helical pitch angles are well suited to cover the hydrophobic chain region of the bilayer when two peptide helices form a head-to-tail dimer. Thus, the detailed topology found in this work agrees with the peptides forming the rim of nanodiscs in a double belt arrangement.

Dynamic Nuclear Polarization/Solid-State NMR Spectroscopy of Membrane Polypeptides: Free-Radical Optimization for Matrix-Free Lipid Bilayer Samples.

Dynamic nuclear polarization (DNP) boosts the sensitivity of NMR spectroscopy by orders of magnitude and makes investigations previously out of scope possible. For magic-angle-spinning (MAS) solid-state NMR spectroscopy studies, the samples are typically mixed with biradicals dissolved in a glass-forming solvent and are investigated at cryotemperatures. Herein, we present new biradical polarizing agents developed for matrix-free samples such as supported lipid bilayers, which are systems widely used for the investigation of membrane polypeptides of high biomedical importance. A series of 11 biradicals with different structures, geometries, and physicochemical properties were comprehensively tested for DNP performance in lipid bilayers, some of them developed specifically for DNP investigations of membranes. The membrane-anchored biradicals PyPol-C16, AMUPOL-cholesterol, and bTurea-C16 were found to exhibit improved g-tensor alignment, inter-radical distance, and dispersion. Consequently, these biradicals show the highest signal enhancement factors so far obtained for matrix-free membranes or other matrix-free samples and may potentially shorten NMR acquisition times by three orders of magnitude. Furthermore, the optimal biradical-to-lipid ratio, sample deuteration, and membrane lipid composition were determined under static and MAS conditions. To rationalize biradical performance better, DNP enhancement was measured by using the 13 C and 15 N signals of lipids and a peptide as a function of the biradical concentration, DNP build-up time, resonance line width, quenching effect, microwave power, and MAS frequency.

Alamethicin Supramolecular Organization in Lipid Membranes from 19F Solid-State NMR.

Alamethicins (ALMs) are antimicrobial peptides of fungal origin. Their sequences are rich in hydrophobic amino acids and strongly interact with lipid membranes, where they cause a well-defined increase in conductivity. Therefore, the peptides are thought to form transmembrane helical bundles in which the more hydrophilic residues line a water-filled pore. Whereas the peptide has been well characterized in terms of secondary structure, membrane topology, and interactions, much fewer data are available regarding the quaternary arrangement of the helices within lipid bilayers. A new, to our knowledge, fluorine-labeled ALM derivative was prepared and characterized when reconstituted into phospholipid bilayers. As a part of these studies, C19F3-labeled compounds were characterized and calibrated for the first time, to our knowledge, for 19F solid-state NMR distance and oligomerization measurements by centerband-only detection of exchange (CODEX) experiments, which opens up a large range of potential labeling schemes. The 19F-19F CODEX solid-state NMR experiments performed with ALM in POPC lipid bilayers and at peptide/lipid ratios of 1:13 are in excellent agreement with molecular-dynamics calculations of dynamic pentameric assemblies. When the peptide/lipid ratio was lowered to 1:30, ALM was found in the dimeric form, indicating that the supramolecular organization is tuned by equilibria that can be shifted by changes in environmental conditions.

Crystal structure and functional mechanism of a human antimicrobial membrane channel.

Multicellular organisms fight bacterial and fungal infections by producing peptide-derived broad-spectrum antibiotics. These host-defense peptides compromise the integrity of microbial cell membranes and thus evade pathways by which bacteria develop rapid antibiotic resistance. Although more than 1,700 host-defense peptides have been identified, the structural and mechanistic basis of their action remains speculative. This impedes the desired rational development of these agents into next-generation antibiotics. We present the X-ray crystal structure as well as solid-state NMR spectroscopy, electrophysiology, and MD simulations of human dermcidin in membranes that reveal the antibiotic mechanism of this major human antimicrobial, found to suppress Staphylococcus aureus growth on the epidermal surface. Dermcidin forms an architecture of high-conductance transmembrane channels, composed of zinc-connected trimers of antiparallel helix pairs. Molecular dynamics simulations elucidate the unusual membrane permeation pathway for ions and show adjustment of the pore to various membranes. Our study unravels the comprehensive mechanism for the membrane-disruptive action of this mammalian host-defense peptide at atomistic level. The results may form a foundation for the structure-based design of peptide antibiotics.

Probing the Huntingtin 1-17 membrane anchor on a phospholipid bilayer by using all-atom simulations.

Mislocalization and aggregation of the huntingtin protein are related to Huntington's disease. Its first exon-more specifically the first 17 amino acids (Htt17)-is crucial for the physiological and pathological functions of huntingtin. It regulates huntingtin's activity through posttranslational modifications and serves as an anchor to membrane-containing organelles of the cell. Recently, structure and orientation of the Htt17 membrane anchor were determined using a combined solution and solid-state NMR approach. This prompted us to refine this model by investigating the dynamics and thermodynamics of this membrane anchor on a POPC bilayer using all-atom, explicit solvent molecular dynamics and Hamiltonian replica exchange. Our simulations are combined with various experimental measurements to generate a high-resolution atomistic model for the huntingtin Htt17 membrane anchor on a POPC bilayer. More precisely, we observe that the single α-helix structure is more stable in the phospholipid membrane than the NMR model obtained in the presence of dodecylphosphocholine detergent micelles. The resulting Htt17 monomer has its hydrophobic plane oriented parallel to the bilayer surface. Our results further unveil the key residues interacting with the membrane in terms of hydrogen bonds, salt-bridges, and nonpolar contributions. We also observe that Htt17 equilibrates at a well-defined insertion depth and that it perturbs the physical properties-order parameter, thickness, and area per lipid-of the bilayer in a manner that could favor its dimerization. Overall, our observations reinforce and refine the NMR measurements on the Htt17 membrane anchor segment of huntingtin that is of fundamental importance to its biological functions.

Lipid interactions of LAH4, a peptide with antimicrobial and nucleic acid transfection activities.

The cationic amphipathic designer peptide LAH4 exhibits potent antimicrobial, nucleic acid transfection and cell penetration activities. Closely related derivatives have been developed to enhance viral transduction for gene therapeutic assays. LAH4 contains four histidines and, consequently, its overall charge and membrane topology in lipid bilayers are strongly pH dependent. In order to better understand the differential interactions of this amphipathic peptide with negatively-charged membranes its interactions, topologies, and penetration depth were investigated in the presence of lipid bilayers as a function of pH, buffer, phospholipid head group, and fatty acyl chain composition using a combination of oriented synchrotron radiation circular dichroism spectroscopy as well as oriented and non-oriented solid-state NMR spectroscopy. This combination of methods indicates that in the presence of lipids with phosphatidylglycerol head groups, the topological equilibria of LAH4 is shifted towards more in-plane configurations even at neutral pH. In contrast, a transmembrane alignment is promoted when LAH4 interacts with membranes made of dimyristoyl phospholipids rather than palmitoyl-oleoyl-phospholipids. Finally, the addition of citrate buffer favours LAH4 transmembrane alignments, even at low pH, probably by complex formation with the cationic charges of the peptide. In summary, this study has revealed that the membrane topology of this peptide is readily modulated by the environmental conditions.

On the design of supramolecular assemblies made of peptides and lipid bilayers.

Peptides confer interesting properties to materials, supramolecular assemblies and to lipid membranes and are used in analytical devices or within delivery vehicles. Their relative ease of production combined with a high degree of versatility make them attractive candidates to design new such products. Here, we review and demonstrate how CD- and solid-state NMR spectroscopic approaches can be used to follow the reconstitution of peptides into membranes and to describe some of their fundamental characteristics. Whereas CD spectroscopy is used to monitor secondary structure in different solvent systems and thereby aggregation properties of the highly hydrophobic domain of p24, a protein involved in vesicle trafficking, solid-state NMR spectroscopy was used to deduce structural information and the membrane topology of a variety of peptide sequences found in nature or designed. (15)N chemical shift solid-state NMR spectroscopy indicates that the hydrophobic domain of p24 as well as a designed sequence of 19 hydrophobic amino acid residues adopt transmembrane alignments in phosphatidylcholine membranes. In contrast, the amphipathic antimicrobial peptide magainin 2 and the designed sequence LK15 align parallel to the bilayer surface. Additional angular information is obtained from deuterium solid-state NMR spectra of peptide sites labelled with (2)H3-alanine, whereas (31)P and (2)H solid-state NMR spectra of the lipids furnish valuable information on the macroscopic order and phase properties of the lipid matrix. Using these approaches, peptides and reconstitution protocols can be elaborated in a rational manner, and the analysis of a great number of peptide sequences is reviewed. Finally, a number of polypeptides with membrane topologies that are sensitive to a variety of environmental conditions such as pH, lipid composition and peptide-to-lipid ratio will be presented.

Solution synthesis, conformational analysis, and antimicrobial activity of three alamethicin F50/5 analogs bearing a trifluoroacetyl label.

We prepared, by solution-phase methods, and fully characterized three analogs of the membrane-active peptaibiotic alamethicin F50/5, bearing a single trifluoroacetyl (Tfa) label at the N-terminus, at position 9 (central region) or at position 19 (C-terminus), and with the three Gln at positions 7, 18, and 19 replaced by Glu(OMe) residues. To add the Tfa label at position 9 or 19, a γ-trifluoroacetylated α,γ-diaminobutyric acid (Dab) residue was incorporated as a replacement for the original Val(9) or Glu(OMe)(19) amino acid. We performed a detailed conformational analysis of the three analogs (using FT-IR absorption, CD, 2D-NMR, and X-ray diffraction), which clearly showed that Tfa labeling does not introduce any dramatic backbone modification in the predominantly α-helical structure of the parent peptaibiotic. The results of an initial solid-state (19)F-NMR study on one of the analogs favor the conclusion that the Tfa group is a very promising reporter for the analysis of peptaibioticmembrane interactions. Finally, we found that the antimicrobial activities of the three newly synthesized analogs depend on the position of the Tfa label in the peptide sequence.

Structure and topology of the huntingtin 1-17 membrane anchor by a combined solution and solid-state NMR approach.

The very amino-terminal domain of the huntingtin protein is directly located upstream of the protein's polyglutamine tract, plays a decisive role in several important properties of this large protein and in the development of Huntington's disease. This huntingtin 1-17 domain is on the one hand known to markedly increase polyglutamine aggregation rates and on the other hand has been shown to be involved in cellular membrane interactions. Here, we determined the high-resolution structure of huntingtin 1-17 in dodecyl phosphocholine micelles and the topology of its helical domain in oriented phosphatidylcholine bilayers. Using two-dimensional solution NMR spectroscopy the low-energy conformations of the polypeptide were identified in the presence of dodecyl phosphocholine detergent micelles. In a next step a set of four solid-state NMR angular restraints was obtained from huntingtin 1-17 labeled with (15)N and (2)H at selected sites. Of the micellar ensemble of helical conformations only a limited set agrees in quantitative detail with the solid-state angular restraints of huntingtin 1-17 obtained in supported planar lipid bilayers. Thereby, the solid-state NMR data were used to further refine the domain structure in phospholipid bilayers. At the same time its membrane topology was determined and different motional regimes of this membrane-associated domain were explored. The pronounced structural transitions of huntingtin 1-17 upon membrane-association result in a α-helical conformation from K6 to F17, i.e., up to the very start of the polyglutamine tract. This amphipathic helix is aligned nearly parallel to the membrane surface (tilt angle ∼77°) and is characterized by a hydrophobic ridge on one side and an alternation of cationic and anionic residues that run along the hydrophilic face of the helix. This arrangement facilitates electrostatic interactions between huntingtin 1-17 domains and possibly with the proximal polyglutamine tract.

Membrane interactions of the amphipathic amino terminus of huntingtin.

The amino-terminal domain of huntingtin (Htt17), located immediately upstream of the decisive polyglutamine tract, strongly influences important properties of this large protein and thereby the development of Huntington's disease. Htt17 markedly increases polyglutamine aggregation rates and the level of huntingtin's interactions with biological membranes. Htt17 adopts a largely helical conformation in the presence of membranes, and this structural transition was used to quantitatively analyze membrane association as a function of lipid composition. The apparent membrane partitioning constants increased in the presence of anionic lipids but decreased with increasing amounts of cholesterol. When membrane permeabilization was tested, a pronounced dye release was observed from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles and 75:25 (molar ratio) POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine vesicles but not across bilayers that better mimic cellular membranes. Solid-state nuclear magnetic resonance structural investigations indicated that the Htt17 α-helix adopts an alignment parallel to the membrane surface, and that the tilt angle (∼75°) was nearly constant in all of the membranes that were investigated. Furthermore, the addition of Htt17 resulted in a decrease in the lipid order parameter in all of the membranes that were investigated. The lipid interactions of Htt17 have pivotal implications for membrane anchoring and functional properties of huntingtin and concomitantly the development of the disease.

The Mechanism of Action of SAAP-148 Antimicrobial Peptide as Studied with NMR and Molecular Dynamics Simulations.

BACKGROUND: SAAP-148 is an antimicrobial peptide derived from LL-37. It exhibits excellent activity against drug-resistant bacteria and biofilms while resisting degradation in physiological conditions. Despite its optimal pharmacological properties, its mechanism of action at the molecular level has not been explored. METHODS: The structural properties of SAAP-148 and its interaction with phospholipid membranes mimicking mammalian and bacterial cells were studied using liquid and solid-state NMR spectroscopy as well as molecular dynamics simulations. RESULTS: SAAP-148 is partially structured in solution and stabilizes its helical conformation when interacting with DPC micelles. The orientation of the helix within the micelles was defined by paramagnetic relaxation enhancements and found similar to that obtained using solid-state NMR, where the tilt and pitch angles were determined based on 15N chemical shift in oriented models of bacterial membranes (POPE/POPG). Molecular dynamic simulations revealed that SAAP-148 approaches the bacterial membrane by forming salt bridges between lysine and arginine residues and lipid phosphate groups while interacting minimally with mammalian models containing POPC and cholesterol. CONCLUSIONS: SAAP-148 stabilizes its helical fold onto bacterial-like membranes, placing its helix axis almost perpendicular to the surface normal, thus probably acting by a carpet-like mechanism on the bacterial membrane rather than forming well-defined pores.

Effects of antimicrobial peptides on membrane dynamics: A comparison of fluorescence and NMR experiments.

Antimicrobial peptides (AMPs) represent a promising class of compounds to fight resistant infections. They are commonly thought to kill bacteria by perturbing the permeability of their cell membranes. However, bacterial killing requires a high coverage of the cell surface by bound peptides, at least in the case of cationic and amphipathic AMPs. Therefore, it is conceivable that peptide accumulation on the bacterial membranes might interfere with vital cellular functions also by perturbing bilayer dynamics, a hypothesis that has been termed "sand in the gearbox". Here we performed a systematic study of such possible effects, for two representative peptides (the cationic cathelicidin PMAP-23 and the peptaibol alamethicin), employing fluorescence and NMR spectroscopies. These approaches are commonly applied to characterize lipid order and dynamics, but sample different time-scales and could thus report on different membrane properties. In our case, fluorescence anisotropy measurements on liposomes labelled with probes localized at different depths in the bilayer showed that both peptides perturb membrane fluidity and order. Pyrene excimer-formation experiments showed a peptide-induced reduction in lipid lateral mobility. Finally, laurdan fluorescence indicated that peptide binding reduces water penetration below the headgroups region. Comparable effects were observed also in fluorescence experiments performed directly on live bacterial cells. By contrast, the fatty acyl chain order parameters detected by deuterium NMR spectroscopy remained virtually unaffected by addition of the peptides. The apparent discrepancy between the two techniques confirms previous sporadic observations and is discussed in terms of the different characteristic times of the two approaches. The perturbation of membrane dynamics in the ns timescale, indicated by the multiple fluorescence approaches reported here, could contribute to the antimicrobial activity of AMPs, by affecting the function of membrane proteins, which is strongly dependent on the physicochemical properties of the bilayer.

Effect of lipid saturation on the topology and oligomeric state of helical membrane polypeptides.

Natural liquid crystalline membranes are made up of many different lipids carrying a mixture of saturated and unsaturated fatty acyl chains. Whereas in the past considerable attention has been paid to cholesterol content, the phospholipid head groups and the membrane surface charge the detailed fatty acyl composition was often considered less important. However, recent investigations indicate that the detailed fatty acyl chain composition has pronounced effects on the oligomerization of the transmembrane helical anchoring domains of the MHC II receptor or the membrane alignment of the cationic antimicrobial peptide PGLa. In contrast the antimicrobial peptides magainin 2 and alamethicin are less susceptible to lipid saturation. Using histidine-rich LAH4 designer peptides the high energetic contributions of lipid saturation in stabilizing transmembrane helical alignments are quantitatively evaluated. These observations can have important implications for the biological regulation of membrane proteins and should be taken into considerations during biophysical or structural experiments.

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 pKa 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.