Membrane topologies of the PGLa antimicrobial peptide and a transmembrane anchor sequence by Dynamic Nuclear Polarization/ solid-state NMR spectroscopy OPEN

International audienceDynamic Nuclear Polarization (DNP) has been introduced to overcome the sensitivity limitations of nuclear magnetic resonance (NMR) spectroscopy also of supported lipid bilayers. When investigated by solid-state NMR techniques the approach typically involves doping the samples with biradicals and their investigation at cryo-temperatures. Here we investigated the effects of temperature and membrane hydration on the topology of amphipathic and hydrophobic membrane polypeptides. Although the antimicrobial PGLa peptide in dimyristoyl phospholipids is particularly sensitive to topological alterations, the DNP conditions represent well its membrane alignment also found in bacterial lipids at ambient temperature. With a novel membrane-anchored biradical and purpose-built hardware a 17-fold enhancement in NMR signal intensity is obtained by DNP which is one of the best obtained for a truly static matrix-free system. Furthermore, a membrane anchor sequence encompassing 19 hydrophobic amino acid residues was investigated. Although at cryotemperatures the transmembrane domain adjusts it membrane tilt angle by about 10 degrees, the temperature dependence of two-dimensional separated field spectra show that freezing the motions can have beneficial effects for the structural analysis of this sequence

Helicobacter pylori Adapts to Chronic Infection and Gastric Disease via pH-Responsive BabA-Mediated Adherence

International audienceThe BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive-binding is reduced at low pH and restored by acid neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions and evolves during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA's extraordinary reversible acid responsiveness enables tight mucosal bacterial adherence while also allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen and that bio-selection and changes in BabA binding properties through mutation and recombination with babA-related genes are selected by differences among individuals and by changes in gastric acidity over time. These processes generate diverse H. pylori subpopulations, in which BabA's adaptive evolution contributes to H. pylori persistence and overt gastric disease

Loss of Metal Ions, Disulfide Reduction and Mutations Related to Familial ALS Promote Formation of Amyloid-Like Aggregates from Superoxide Dismutase

Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) are one of the causes of familial amyotrophic lateral sclerosis (FALS). Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1) formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1

Etude topologique de polypeptides membranaires par RMN du solide et spectroscopie infrarouge par réflexion totale atténuée (ATR-FTIR)

Cette thèse est divisée en trois parties qui suivent des approches différentes. Dans la première partie, l'hydrophobicité d'acides amines, propriété bien connue, est déterminée. Un modèle thermodynamique détaillé de la topologie de polypeptides dérivés du LAH4 en fonction du pH a été développé. Ces modifications dans la topologie ont été mises en évidence par la technique de spectroscopie infrarouge de réflexion totale atténuée. (abrégée en anglais ATR-FTIR). Grâce à ces résultats et à un modèle théoretique, nous avons pu déterminer l'enthalpie nécessaire à l'insertion d'acides aminés de la phase aqueuse vers l'intérieur d'une bicouche lipidique.La seconde partie donne une vue d'ensemble (avantages et limites) de l'utilisation de RMN du deutérium sur la 2H3C-alanine. Il est en effet prouvé que cette méthode peut fournir des informations sur la structure et la dynamique des polypeptides membranaires. Bien que la RMN du deutérium ait déjà été utilisée auparavant sur des peptides marqués à l'aide de 2H3C-alanine, l'étude systematique de tels peptides par cette technique éclaire de nouveaux aspects du sujet.Dans la troisième partie, la RMN à l'état solide est appliquée à différents polypeptides et protéines d'origine naturelle. Ainsi, pour la bacteriorhodopsine et la gramicidine A, certains détails de la structure déjà connue du protéine / polypeptide ont été néanmoins élucidés. La structure du ß-peptide amyloide dans sa forme membranaire est quant à elle largement inconnue : nous avons essayé de savoir si la RMN du solide pouvait fournir de plus amples informations. Enfin, pour les protéines colicine E1 et BCL-XL , nous avons testé la validité de modèles disponibles dans la littérature. Dans le cas du domaine formant le canal de la colicine E1, une protéine marquée uniquement à une seule position a été utilisée : les spectres 15N RMN à l'état solide sont parmi les premier mesurés pour une protéine exprimée par bactérie et marquée sur une seule position.The thesis consists of three different sections, which follow slightly different approaches. In the first part the hydrophobicity a very basic property, of amino acids is determined. For the series of LAH4-like polypeptides a detailed thermodynamic model for the pH dependent topology of the peptides was developed. The topological changes were measured by attenuated total reflection infrared spectroscopy (ATR-FTIR). The results of the measurements and the theoretical model were used to determine the enthalpy for the insertion of some amino acid from water phase into the lipid bilayer interior. The second part gives a detailed overview of the advantages and problems of the application of deuterium NMR using 2H3C-Alanine. It is shown that the method can give valuable information of structure and dynamics of membrane polypeptides. Even thought solid-state NMR on 2H3C-Alanine labelled peptides was used before the systematic investigation of the properties of deuterium NMR on 2H3C-Alanine labelled peptides gives new and important aspects into the topic.In the third part the solid-state NMR method is applied to different natural polypeptides and proteins. For bacteriorhodopsin and gramicidine A solid-state NMR was used to investigate some details of the known structure of the protein/polypeptide. The structure of the membrane associated form of the amyloid b-peptide is widely unknown. It was tested, if solid-state NMR can provide further information of the membrane associated form of the amyloid b-peptide. For the proteins colicin E1 and BCL-XL detailed models are published and the solid-state NMR experiments are used to test these models. For the measurement of the channel-forming domain of colicin E1 a single-side labelled protein was used. The measured spectra belong to the first 15N solid-state NMR spectra of a single-side labelled protein (expressed in bacteria).STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

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

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Membrane Interactions Accelerate the Self-Aggregation of Huntingtin Exon 1 Fragments in a Polyglutamine Length-Dependent Manner

International audienceThe accumulation of aggregated protein is a typical hallmark of many human neurodegenerative disorders, including polyglutamine-related diseases such as chorea Huntington. Misfolding of the amyloidogenic proteins gives rise to self-assembled complexes and fibres. The huntingtin protein is characterised by a segment of consecutive glutamines which, when exceeding ~ 37 residues, results in the occurrence of the disease. Furthermore, it has also been demonstrated that the 17-residue amino-terminal domain of the protein (htt17), located upstream of this polyglutamine tract, strongly correlates with aggregate formation and pathology. Here, we demonstrate that membrane interactions strongly accelerate the oligomerisation and β-amyloid fibril formation of htt17-polyglutamine segments. By using a combination of biophysical approaches, the kinetics of fibre formation is investigated and found to be strongly dependent on the presence of lipids, the length of the polyQ expansion, and the polypeptide-to-lipid ratio. Finally, the implications for therapeutic approaches are discussed