232 research outputs found
Magainin 2 in phospholipid bilayers: peptide orientation and lipid chain ordering studied by X-ray diffraction
No full textAbstractWe present a structural study of biomimetic lipid bilayers interacting with the antimicrobial peptide magainin 2 amide, using grazing incidence X-ray diffraction and reciprocal space mapping (RSM) techniques. The short-range order of lipid chains in lecithin is found to be strongly reduced by the peptides. From the scattering intensity of the chain correlation peak, we can quantify the lateral length scale R over which the bilayer structure is affected by peptide binding. The non-local perturbation of the bilayer is discussed in the framework of bilayer elasticity theory
Grazing incidence X-ray diffraction of highly aligned phospholipid membranes containing the antimicrobial peptide magainin 2
No full textWe present the first study of grazing incidence X-ray diffraction on a model system of phospholipid membranes and antimicrobial peptides. For this purpose, highly oriented multilamellar samples have been prepared on solid substrates. By this technique, the short-range order of the lipid chains in the fluid Lα phase can be investigated quantitatively, including not only the mean distance between acyl chains, but also the associated correlation length. The short-range order in lecithin is found to be severely affected by the amphiphilic peptide magainin 2
Hydration- and Temperature-Dependent Fluorescence Spectra of Laurdan Conformers in a DPPC Membrane
No full textInternational audienceThe widely used Laurdan probe has two conformers, resulting in different optical properties when embedded in a lipid bilayer membrane, as demonstrated by our previous simulations. Up to now, the two conformers’ optical responses have, however, not been investigated when the temperature and the phase of the membrane change. Since Laurdan is known to be both a molecular rotor and a solvatochromic probe, it is subject to a profound interaction with both neighboring lipids and water molecules. In the current study, molecular dynamics simulations and hybrid Quantum Mechanics/Molecular Mechanics calculations are performed for a DPPC membrane at eight temperatures between 270K and 320K, while the position, orientation, fluorescence lifetime and fluorescence anisotropy of the embedded probes are monitored. The importance of both conformers is proven through a stringent comparison with experiments, which corroborates the theoretical findings. It is seen that for Conf-I, the excited state lifetime is longer than the relaxation of the environment, while for Conf-II, the surroundings are not yet adapted when the probe returns to the ground state. Throughout the temperature range, the lifetime and anisotropy decay curves can be used to identify the different membrane phases. The current work might, therefore, be of importance for biomedical studies on diseases, which are associated with cell membrane transformations
New methodologies of solid state NMR and biophysical studies of antimicrobial and designed peptides in model and natural membranes
No full textLa spectroscopie de Résonance Magnétique Nucléaire des solides est un outil puissant pour l’étude structurale des peptides membranaires. L'angle d'inclinaison par rapport à la membrane de peptides structurés en hélice peut être obtenu par l’analyse des spectres de poudre provenant de systèmes non-orientés par rapport au champ magnétique statique. Cette approche permet un meilleur contrôle sur plusieurs paramètres par rapport aux systèmes orientés. Cependant, les spectres de poudre de ces systèmes présentent d’importantes distorsions à proximité du déplacement chimique isotrope (Magic Angle Hole, MAH) qui empêchent d'atteindre une qualité satisfaisante des paramètres RMN mesurés. Dans ce travail de thèse nous avons développé une nouvelle méthode qui récupère la forme théorique des spectres de poudre : l’expérience RODEO (ROtor-Directed Exchange of Orientations). La méthode a été appliquée avec succès à des peptides insérés dans des membranes models. Enfin nous avons appliquée l’expérience RODEO à un mélange de lipides extraits de membranes naturelles, et, pour la première fois, in vivo, dans des bactéries Escherichia coli. Dans la deuxième partie de ce travail, nous rapportons des résultats inattendus sur le mécanisme d'insertion du peptide antimicrobien LAH4. Il a été précédemment montré que l'hélice LAH4 est orientée parallèlement à la surface de la membrane dans des conditions acides, tandis que, à pH neutre, le peptide adopte une orientation trans-membranaire. En revanche, nous avons constaté que lorsque le tampon citrate est ajouté pour réguler le pH à 5, le peptide s’insère de manière transmembranaire. Quelques explications possibles sont proposées.Solid-state NMR (SS-NMR) spectroscopy is a powerful tool to investigate structural details of membrane-associated peptides. In particular, the tilt angle of helical peptides reconstituted in non-oriented membranes can be derived by line-shape analysis of ^{15}N powder patterns. This approach allow a better control on several parameters with respect oriented SS-NMR spectroscopy.
The spectra of peptides undergoing to fast uniaxial rotation diffusion around the membrane normal are characterized by distortions close to the isotropic chemical shift (Magic Angle Hole, MAH) that prevent to reach a satisfying fit quality. We developed a method that recovers the theoretical powder pattern line-shape based on ROtor-Directed Exchange of Orientations Cross-Polarization (RODEO). The method was successfully applied to designed peptides in unoriented membranes resulting in tilt angles values in agreement with published data. RODEO was applied also to complex membrane systems, such as lipid mixture extracted from natural membranes, and, for the first time, we obtained an estimation of the alignment of an antimicrobial peptide in vivo.
In the second part of this work, we report some unexpected results of biophysical investigations conducted to elucidate the insertion mechanism of the antimicrobial peptide LAH4 in zwitterionic membranes. It was previously shown that the LAH4-helix adopts an in-plane orientation in acidic conditions, while, at neutral pH, the peptide adopts a trans-membrane orientation. In contrast, we found that when citrate buffer is added to regulate the pH at 5, the peptide inserts in a transmembrane manner. Some possible explanations are suggested
Solid-state NMR investigations of interaction contributions that determine the alignment of helical polypeptides in biological membranes
No full textAbstractHelical peptides reconstituted into oriented phospholipid bilayers were studied by proton-decoupled 15N solid-state NMR spectroscopy. Whereas hydrophobic channel peptides, such as the N-terminal region of Vpu of HIV-1, adopt transmembrane orientations, amphipathic peptide antibiotics are oriented parallel to the bilayer surface. The interaction contributions that determine the alignment of helical peptides in lipid membranes were analysed using model sequences, and peptides that change their topology in a pH-dependent manner have been designed. The energy contributions of histidines, lysines, leucines and alanines as well as the alignment of peptides and phospholipids under conditions of hydrophobic mismatch have been investigated in considerable detail
The structure, dynamics and orientation of antimicrobial peptides in membranes by multidimensional solid-state NMR spectroscopy
No full textAbstractLinear peptide antibiotics have been isolated from amphibians, insects and humans and used as templates to design cheaper and more potent analogues for medical applications. Peptides such as cecropins or magainins are ≤40 amino acids in length. Many of them have been prepared by solid-phase peptide synthesis with isotopic labels incorporated at selected sites. Structural analysis by solid-state NMR spectroscopy and other biophysical techniques indicates that these peptide antibiotics strongly interact with lipid membranes. In bilayer environments they exhibit amphipathic α-helical conformations and alignments of the helix axis parallel to the membrane surface. This contrasts the transmembrane orientations observed for alamethicin or gramicidin A. Models that have been proposed to explain the antibiotic and pore-forming activities of membrane-associated peptides, as well as other experimental results, include transmembrane helical bundles, wormholes, carpets, detergent-like effects or the in-plane diffusion of peptide-induced bilayer instabilities
Découvrir le mode d'action des peptides mimétiques antimicrobiens dans des environnements membranaires modèles : une étude biophysique
No full textFace à la résistance croissante aux antimicrobiens, les peptides antimicrobiens et diverses imitations synthétiques sont au centre de l'attention. Dans cette thèse, trois types d'imitations de PAM sont étudiés : Imitations de petites molécules (à base d'aryl-alkyl-lysine, inspirées des détergents, à base de cyclam), Peptides cycliques (riches de 3 arginines et de 3 tryptophanes), Peptoïdes (peptoïdes cationiques triazolium), ainsi que les PAM (SFV33 et GVR28) dérivés du collagène VI. Ces mimétiques synthétiques sont conçus d’après le modèle PAM (nature amphipathique, charge cationique).L'objectif de cette thèse est de réaliser des études biophysiques de l'interaction peptide/composé-membrane afin de comprendre la force motrice des activités antibactériennes présentées par ces mimétiques et peptides ciblés. Des études de RMN à l'état solide non orientée, fluorescence et dichroïsme circulaire réalisées avec diverses membranes modèles montrent que ces peptides/composés non seulement interagissent avec les membranes bactériennes, mais aussi qu'ils présentent une nette sélectivité pour la membrane bactérienne comparé à la membrane eucaryote.Due to the rise of antimicrobial resistance, the antimicrobial peptide (AMP) and its various synthetic mimics are much in focus. In this thesis, three branches of AMP mimics; Small molecule mimics (aryl-alkyl-lysine based, detergent inspired, and cyclam-based), Cyclic peptides (rich with three arginine and three tryptophan units), Peptoids (triazolium cationic peptoids), and also AMPs (SFV33 and GVR28) derived from collagen VI are investigated. These synthetic mimics are designed using the template of AMP (amphipathic nature and cationic charge). The aim of this thesis is to do biophysical studies of the peptide/compound-membrane interaction to understand the driving force of the antibacterial activities exhibited by these mimics and peptides discussed. Non-oriented solid-state NMR, fluorescence, and circular dichroism studies done with various model membranes show that these peptides/compounds not only undergo interaction with the bacterial membranes but also show clear selectivity towards the bacterial membrane over the eukaryotic membrane
Production of polypeptides derived from the envelope glycoproteins of HIV-1 for biophysical and structural studies by NMR and CD
No full textQuelques régions stables ont été découvertes sur les gp d’env du VIH-1 contre lesquelles des patients produisent des anticorps neutralisants. Les épitopes les plus prometteurs se trouvent dans la MPER et sont probablement exposés durant la fusion. Alors que les peptides isolés à partir de cette région ne sont pas parvenus à induire une réaction immunogène neutralisante, des études antérieures suggèrent que la membrane lipidique joue un rôle dans la structuration des antigènes et dans la réponse immunogénique.C’est pourquoi nous étudions la structure de ces épitopes. Cela nécessite leur surexpression, leur purification et leur reconstitution dans des liposomes. Une étude de CD montre qu’ils pourraient changer de conformation, cela sera confirmé par RMN. En outre, leur immunogénicité sera vérifiée par vaccination des souris. En plus, nous trouvons que le cholestérol peut modifier l’orientation des peptides englobant le motif CRAC de la gp41.A few stable regions have been discovered on the HIV-1 env gp against which some patients produce neutralizing antibodies. The most promising ones are located in the MPER and are probably exposed transiently during the fusion. Whereas the peptides isolated from this region failed to induce immunogenic response, previous studies suggest the lipid membrane plays a role in antigens structure and in the immunogenic response.That is why we investigate the structure of these épitopes in membrane models. This requires the production of these épitopes by bacterial overexpression, their purification and their reconstitution in liposomes. A CD study shows that they could undergo a conformational change; this will be confirmed by NMR. Also their immunogenicity will be checked by mice immunization. In addition, we find that cholesterol could change the orientation of peptides encompassing a gp41 CRAC motif
Functional and structural study of Vectofusin-1 peptide supramolecular assembly
No full textLes peptides de la famille LAH4 sont des peptides cationiques qui se replient en hélices alpha et in- teragissent avec les membranes phospholipidiques. Ces peptides peuvent s’assembler en nano-fibrilles. Dans cette thèse, nous avons étudié le dérivé LAH4-A4 breveté sous le nom Vectofusin-1. Une étude fonctionnelle a révélé que ses propriétés d’auto-assemblage influencent son activité biologique en tant qu’adjuvant améliorant la transduction lentivirale. Lors d’une collaboration avec le laboratoire ART- TG, nous avons montré que les formes fibrillaires et monomériques de Vectofusin-1 diffèrent en activité biologique, notamment selon le type cellulaire. Un protocole de production et purification du peptide Vectofusin-1 basé sur l’expression en système bactérien avec marquage isotopique a été mis en place. Une étude biophysique, par microscopie électronique, a démontré que la formation de fibrilles dépend de paramètres précis comme le pH, le solvant et la température. Une étude structurale par RMN du solide a permis de caractériser en partie les interactions impliquées dans la formation et la stabilisation des fibrilles.LAH4 peptides are cationic peptides that fold into alpha-helices and interact with phospholipid mem- branes. These peptides can assemble into nanofibrils.In this thesis, we studied the LAH4-A4 derivative patented under the name Vectofusin-1. A functional study revealed that its self-assembly properties influence its biological activity as an adjuvant enhancing lentiviral transduction. In collaboration with the ART-TG laboratory, we demonstrated that the fibrillar and monomeric forms of Vectofusin-1 differ in biological activity, particularly depending on the cell type. A protocol for producing and purifying Vectofusin-1 peptide based on isotopically labeled bacterial ex- pression was established.A biophysical study using electron microscopy showed that fibril formation depends on specific pa- rameters such as pH, solvent, and temperature. A structural study using solid-state NMR partially characterized the interactions involved in the formation and stabilization of fibrils
Des interactions moléculaires médiées par les lipides à une activité antimicrobienne synergique : une étude des peptides amphipathiques PGLa et Mag2 par spectroscopie RMN et simulations de dynamique moléculaire in silico
No full textPGLa et magainin 2 sont deux peptides amphiphiles, avec une activité antimicrobienne synergique. Nous avons étudié la structure et la dynamique de ces peptides, ainsi que leurs interactions avec les membranes. Par spectroscopie RMN en phase solide et liquide nous avons obtenu des informations sur la structure de PGLa dans un environnement hydrophobe.Un modèle 3D de PGLa en micelles a été construit, et différentes populations de PGLa en bicouche lipidique on été identifiées. Des simulations de MD ont permis d'étudier l'interaction de PGLa et magainin 2 entre eux et avec avec la membrane. Nous en avons déduis un mode de recrutement de ces peptides, et une forme d’interaction avec elle, en « clusters ». Nous avons aussi trouvé un modèle d’insertion de ces peptides dans la membrane : le « flip », et montré le rôle des lysines de PGLa pour toutes ces actions, et nous avons muté ces résidus en arginine pour comparer d’un point de vue structural, dynamique et antimicrobien.PGLa and magainin 2 are two amphiphilic peptides that exhibit synergistic antimicrobial activity. The structure, dynamics, and interactions of these peptides with membranes were studied. Solid-phase and liquid-phase NMR spectroscopy provided information on the structure of PGLa in a hydrophobic environment. A 3D model of PGLa in micelles was constructed, and different populations of PGLa in lipid bilayers were identified. MD simulations were used to investigate the interaction of PGLa and magainin 2 with each other and with the membrane. A mode of recruitment of these peptides, as well as a form of interaction with it, in 'clusters' was deduced. Additionally, a model for the insertion of these peptides into the membrane, known as the 'flip', was discovered. The role of PGLa lysines in these actions was also demonstrated, notably by mutating these residues to arginine for structural, dynamic, and antimicrobial comparison
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