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.

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.

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.

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.

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.

Peptides derived from the C-terminal domain of HIV-1 Viral Protein R in lipid bilayers: Structure, membrane positioning and gene delivery.

Viral protein R (Vpr) is a small accessory protein of 96 amino acids that is present in Human and simian immunodeficiency viruses. Among the very different properties that Vpr possesses we can find cell penetrating capabilities. Based on this and on its capacity to interact with nucleic acids we previously investigated the DNA transfection properties of Vpr and subfragments thereof. We found that fragments of the C-terminal helical domain of Vpr are able to deliver efficiently plasmid DNA into different cell lines. As the amphipathic helix may play a role in the interactions with membranes, we investigated whether insertion of a proline residue in the α-helix modifies the transfection properties of Vpr. Unexpectedly, we found that the resulting Vpr55-82 Pro70 peptide was even more efficient than wild type Vpr55-82 in the gene delivery assays. Using circular dichroism, light scattering and solid-state NMR techniques, we characterized the secondary structure and interactions of Vpr and several mutants with model membranes. A model is proposed where the proline shifts the dissociation equilibrium of the peptide-cargo complex and thereby its endosomal release.

Structure, Topology, and Dynamics of Membrane-Inserted Polypeptides and Lipids by Solid-State NMR Spectroscopy: Investigations of the Transmembrane Domains of the DQ Beta-1 Subunit of the MHC II Receptor and of the COP I Protein p24.

MHC class II receptors carry important function in adaptive immunity and their malfunctioning is associated with diabetes type I, chronic inflammatory diseases and other autoimmune diseases. The protein assembles from the DQ alpha-1 and DQ beta-1 subunits where the transmembrane domains of these type I membrane proteins have been shown to be involved in homo- and heterodimer formation. Furthermore, the DQ alpha 1 chain carries a sequence motif that has been first identified in the context of p24, a protein involved in the formation of COPI vesicles of the intracellular transport machinery, to specifically interact with sphingomyelin-C18 (SM-C18). Here we investigated the membrane interactions and dynamics of DQ beta-1 in liquid crystalline POPC phospholipid bilayers by oriented 15N solid-state NMR spectroscopy. The 15N resonances are indicative of a helical tilt angle of the membrane anchor sequence around 20°. Two populations can be distinguished by their differential dynamics probably corresponding the DQ beta-1 mono- and homodimer. Whereas, this equilibrium is hardly affected by the addition of 5 mole% SM-C18 a single population is visible in DMPC lipid bilayers suggesting that the lipid saturation is an important parameter. Furthermore, the DQ alpha-1, DQ beta-1 and p24 transmembrane helical domains were reconstituted into POPC or POPC/SM-C18 lipid bilayers where the fatty acyl chain of either the phosphatidylcholine or of the sphingolipid have been deuterated. Interestingly in the presence of both sphingolipid and polypeptide a strong decrease in the innermost membrane order of the POPC palmitoyl chain is observed, an effect that is strongest for DQ beta-1. In contrast, for the first time the polypeptide interactions were monitored by deuteration of the stearoyl chain of SM-C18. The resulting 2H solid-state NMR spectra show an increase in order for p24 and DQ alpha-1 which both carry the SM recognition motif. Thereby the data are suggestive that SM-C18 together with the transmembrane domains form structures imposing positive curvature strain on the surrounding POPC lipids. This effect is attenuated when SM-C18 is recognized by the protein.

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.

Solid-State NMR Approaches to Study Protein Structure and Protein-Lipid Interactions.

Solid-state NMR spectroscopy has been developed for the investigation of membrane-associated polypeptides and remains one of the few techniques to reveal high-resolution structural information in liquid-disordered phospholipid bilayers. In particular, oriented samples have been used to investigate the structure, dynamics and topology of membrane polypeptides. Much of the previous solid-state NMR work has been developed and performed on peptides but the technique is constantly expanding towards larger membrane proteins. Here, a number of protocols are presented describing among other the reconstitution of membrane proteins into oriented membranes, monitoring membrane alignment by 31P solid-state NMR spectroscopy, investigations of the protein by one- and two-dimensional 15N solid-state NMR and measurements of the lipid order parameters using 2H solid-state NMR spectroscopy. Using such methods solid-state NMR spectroscopy has revealed a detailed picture of the ensemble of both lipids and proteins and their mutual interdependence in the bilayer environment.

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.

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.

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.

Solid-state NMR structural investigations of peptide-based nanodiscs and of transmembrane helices in bicellar arrangements.

The membrane topology of the peptide 18A, a derivative of apolipoprotein A-I, is investigated in structural detail. Apolipoprotein A-I is the dominant protein component of high density lipoproteins with important functions in cholesterol metabolism. 18A (Ac-DWLKA FYDKV AEKLK EAF- NH2) was designed to mimic the structure of tandem domains of class A amphipathic helices and has served as a lead peptide for biomedical applications. At low peptide-to-lipid ratios 18A partitions into phosphatidylcholine membranes with helix topologies parallel to the membrane surface, an alignment that is maintained when disc-like bicelles form at higher peptide-to-lipid ratios. Notably, the bicelles interact cooperatively with the magnetic field of the NMR spectrometer, thus the bilayer normal is oriented perpendicular to the magnetic field direction. A set of peptides that totals four 15N or 2H labelled positions of 18A allowed the accurate analysis of tilt and azimuthal angles relative to the membrane surface under different conditions. The topology agrees with a double belt arrangement forming a rim that covers the hydrophobic fatty acyl chains of the bicelles. In another set of experiments, it was shown that POPC nanodiscs prepared in the presence of diisobutylene/maleic acid (DIBMA) polymers can also be made to align in the magnetic field. Finally, the transmembrane domains of the DQ alpha-1 and DQ beta-1 subunits of the major histocomptability complex (MHC) class II have been prepared and reconstituted into magnetically oriented bicelles for NMR structural analysis.

Lipid bilayer position and orientation of novel carprofens, modulators of γ-secretase in Alzheimer's disease.

γ-Secretase is an integral membrane protein complex and is involved in the cleavage of the amyloid precursor protein APP to produce amyloid-ß peptides. Amyloid-ß peptides are considered causative agents for Alzheimer's disease and drugs targeted at γ-secretase are investigated as therapeutic treatments. We synthesized new carprofen derivatives, which showed γ-secretase modulating activity and determined their precise position, orientation, and dynamics in lipid membranes by combining neutron diffraction, solid-state NMR spectroscopy, and molecular dynamics simulations. Our data indicate that the carprofen derivatives are inserted into the membrane interface, where the exact position and orientation depends on the lipid phase. This knowledge will help to understand the docking of carprofen derivatives to γ-secretase and in the design of new potent drugs. The approach presented here promises to serve as a general guideline how drug/target interactions in membranes can be analyzed in a comprehensive manner.

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.

Structural Characterization of the Amyloid Precursor Protein Transmembrane Domain and Its γ-Cleavage Site.

Alzheimer's disease is the most common form of dementia that affects about 50 million of sufferers worldwide. A major role for the initiation and progression of Alzheimer's disease has been associated with the amyloid ß-peptide (Aß), which is a protease cleavage product of the amyloid precursor protein. The amyloid precursor protein is an integral membrane protein with a single transmembrane domain. Here, we assessed the structural integrity of the transmembrane domain within oriented phosphatidylcholine lipid bilayers and determined the tilt angle distribution and dynamics of various subdomains using solid-state NMR and attenuated total reflectance Fourier transform infrared spectroscopies. Although the overall secondary structure of the transmembrane domain is α-helical, pronounced conformational and topological heterogeneities were observed for the γ- and, to a lesser extent, the ζ-cleavage site, with pronounced implications for the production of Aß and related peptides, the development of the disease, and pharmaceutical innovation.

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.