Protéines amphitropiques (diversité conformationnelle et versatilité des interactions protéines-lipides. Cas d'étude de l'a-lactalbumine et de l'apo-myoglobine)

Certaines protéines, appelées protéines amphitropiques, sont solubles en milieux aqueux et capables de se lier aux membranes pour leurs activités biologiques. Ces protéines apparaissent comme d'excellents modèles pour la compréhension des principes généraux de stabilité et de repliement des protéines membranaires intrinsèques. L'a-lactalbumine et l'apo-myoglobine ont été choisies comme modèles de protéines amphitropiques. Nous proposons une étude exhaustive des facteurs qui influencent la liaison aux membranes de ces deux protéines telles que le pH, la présence de cofacteurs, la force ionique, la charge et la courbure des membranes. Une description macroscopique des mécanismes de liaison a été entreprise par des expériences de centrifugation et de fluorescence. Enfin, la structure et la stabilité des états liés aux membranes ont été sondées par des expériences d'échanges hydrogène/deutérium (H/D). L'état confonnationnel de l'a-lactalbumine liée aux membranes s'est révélé très dépendant de la courbure membranaire. Nous avons identifié deux hélices amphiphiles qui sont directement impliquées dans les interactions a-lactalbumine-lipides. D'autre part, nos expériences d'échanges H/D pennettent une description thennodynamique de la liaison à la membrane. Dans le cas de l'apomyoglobine, nos résultats suggèrent un mécanisme commun d'insertion membranaire pour les protéines qui possèdent une topologie de type globine. Par ailleurs, la liaison de l'apo-myoglobine aux membranes ne semble pas nécessaire pour la fonction biologique de cette protéine. Une description des structures membranaires de l'apo-myoglobine a été entreprise par des expériences d'échanges H/D suivies par spectrométrie de masse.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Réalisation de nanofils de protéines

Ce travail de thèse propose de réaliser un nanofil électrique auto-assemblé constitué de protéines. L'unité de base de ce nanofil est une protéine chimère comprenant un domaine capable de former des fibres amyloïdes (Het-s 218-289) et un domaine capable d'effectuer des transferts d'électrons (une rubrédoxine). Le premier domaine permet la réalisation d'une fibre par auto-assemblage tandis que le deuxième est exposé à la surface de cette structure. Les caractéristiques redox du domaine exposé permettent aux électrons de se déplacer d'un bout à l'autre de la fibre par sauts successifs. Un tel nanofil a été créé et caractérisé par différentes techniques biophysiques. Ensuite, la preuve de la conduction des nanofils a été apportée sur des ensembles d'objets, de manière indirecte par électrochimie, et de manière directe par des mesures tension/courant. Ces travaux ouvrent la voie à la réalisation d'objets biocompatibles, biodégradables, possédant des propriétés électroniques exploitables dans des dispositifs technologiques.The research described in this thesis aims at creating a self-assembled nanowire only made of proteins. The building block of this wire is a chimeric protein that comprises an amyloid fibril forming domain (Het-s 218-289) and an electron transfer domain (rubredoxin). The first one self-assembles in amyloid fibrils which display the second at their surface. Redox characteristics of the exposed domain allow electrons to move from one extremity of the fibril to the other by successive jumps. Such a nanowire has been created and characterized by various biophysical experiments. Then, the conductivity of the nanowires has been demonstrated on sets of wires by electrochemistry and by direct current measurements. These experiments pave the way for future design of biocompatible and biodegradable objects that possess usable electronic properties.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Approche biophysique de l'étude de l'insertion du domaine de translocation de la toxine botulique dans les membranes

Les neurotoxines botuliques (BoNTs), toxines les plus efficaces connues, sont responsables du botulisme. Elles inhibent la libération d acétylcholine aux jonctions neuromusculaires causant des paralysies flasques. Lors de l intoxication, la BoNT se lie à ses récepteurs à la surface des neurones, puis est internalisée par endocytose. L interaction de son domaine de translocation avec la membrane à l'intérieur de compartiments cellulaires acides permet le passage du domaine catalytique dans le cytosol. Le domaine de translocation de BoNT/A (Tm) a été exprimé à partir d un gène de synthèse. L insertion de Tm dans la membrane et son activité sont dépendantes du pH et apparaissent dès pH 5,5. Aucun changement de structure n'est détecté par spectroscopie. Cependant, la formation d'une structure quaternaire n'est pas exclue. L'activité de Tm est aussi sensible à la courbure de la membrane, ce qui pourrait permettre une régulation supplémentaire de sa fonction. L apomyoglobine appartient à la famille des protéines à repliement de type globine, comme certaines toxines bactériennes. Son interaction avec la membrane présente de fortes homologies avec celle du domaine de translocation de la toxine diphtérique. Ce processus à deux étapes (liaison puis insertion) est dépendant du pH et requiert le passage par un état partiellement replié. Pour chaque étape, les régions impliquées ont été localisées par des expériences d échanges Hydrogène/Deuterium analysées par spectrométrie de masse. La liaison à la bicouche se fait par une hélice amphiphile, puis une hélice hydrophobe intervient pour l insertion. Cette dernière n est accessible qu après formation de l état partiellement replié.Botulinum neurotoxins (BoNTs), the most potent known toxins, are responsible for botulism. They inhibit acetylcholine release at the neuromuscular junction, inducing a flaccid paralysis. Upon intoxication, BoNT binds to its receptor at the plasma membrane of neurons and is then internalized by endocytosis. Inside acidic compartments, the interaction of its translocation domain with the membrane drives the translocation of the catalytic domain into the cytosol. The translocation domain of BoNT/A (Tm) was expressed and produced using a synthetic gene. Its insertion and activity have been shown to be pH dependent and to occur below pH 5.5. No structural change could be detected by spectroscopy. However, the formation of a quaternary structure is still possible. The sensitivity of Tm activity to membrane curvature has been observed. This could be an additional control to its function. Apomyoglobin belongs to the globin fold family which counts several bacterial toxin domains as members. Its interaction with membranes shares some characteristics with that of the translocation domain of diphtheria toxin. It is a pH-dependent two-step process (binding and insertion) which requires the accumulation of a partially unfolded state. The protein parts involved in each step have been identified using Hydrogen/Deuterium exchanges analyzed by mass spectrometry. An amphipathic helix allows the membrane binding, and a hydrophobic helix is involved in the insertion step. The last helix becomes available upon formation of the partially folded state.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy

International audienceAtom-resolved real-time studies of kinetic processes in proteins have been hampered in the past by the lack of experimental techniques that yield sufficient temporal and atomic resolution. Here we present band-selective optimized flip-angle short transient (SOFAST) real-time 2D NMR spectroscopy, a method that allows simultaneous observation of reaction kinetics for a large number of nuclear sites along the polypeptide chain of a protein with an unprecedented time resolution of a few seconds. SOFAST real-time 2D NMR spectroscopy combines fast NMR data acquisition techniques with rapid sample mixing inside the NMR magnet to initiate the kinetic event. We demonstrate the use of SOFAST real-time 2D NMR to monitor the conformational transition of α-lactalbumin from a molten globular to the native state for a large number of amide sites along the polypeptide chain. The kinetic behavior observed for the disappearance of the molten globule and the appearance of the native state is monoexponential and uniform along the polypeptide chain. This observation confirms previous findings that a single transition state ensemble controls folding of α-lactalbumin from the molten globule to the native state. In a second application, the spontaneous unfolding of native ubiquitin under nondenaturing conditions is characterized by amide hydrogen exchange rate constants measured at high pH by using SOFAST real-time 2D NMR. Our data reveal that ubiquitin unfolds in a gradual manner with distinct unfolding regimes

Is folding of β-lactoglobulin non-hierarchic? intermediate with native-like β-sheet and non-native α-helix

The refolding of β-lactoglobulin, a β-barrel protein consisting of β strands βA-βI and one major helix, is unusual because non-native α-helices are formed at the beginning of the process. We studied the refolding kinetics of bovine β-lactoglobulin A at pH 3 using the stopped-flow circular dichroism and manual H/2H exchange pulse labeling coupled with heteronuclear NMR. The protection pattern from the H/2H exchange of the native state indicated the presence of a stable hydrophobic core consisting of βF, βG and βH strands. The protection pattern of the kinetic intermediate obtained about one second after initiating the reaction was compared with that of the native state. In this relatively late kinetic intermediate, which still contains some non-native helical structure, the disulfide-bonded β-hairpin made up of βG and βH strands was formed, but the rest of the molecule was fluctuating, where the non-native α-helices may reside. Subsequently, the core β-sheet extends, accompanied by a further α-helix to β-sheet transition. Thus, the refolding of β-lactoglobulin exhibits two elements: the critical role of the core β-sheet is consistent with the hierarchic mechanism, whereas the α-helix to β-sheet transition suggests the non-hierarchic mechanism

Interactions and self-assembly of globular proteins: Mechanism and properties of formed supramolecular structures

Sustainability in food manufacture involves a profound reasoning of the way food are produced. Reducing energy input during food processing and optimizing ingredient formulation in order to meet both sensorial acceptance and nutritional benefit through a controlled release of macro- and micronutriments constitute great challenges for food industries. Controlled self-assembly of molecules into biomaterials throughout bottom-up approach is a promising way to achieve these goals. Because of their omnipresence in food systems, proteins are the focus of many attempts for their use as building blocks for such biomaterials. For instance, the apo form of α-lactalbumin (α-LA) and β-lactoglobulin are able to self-assemble into well-defined microspheres in the presence of an oppositely charged protein such as lysozyme (LYS).1 Formation and destabilization of microspheres are inducible by changing the physicochemical conditions. Because of this reversibility, such microspheres could be used to trap, protect during processing and storage, carry and deliver bioactives. However, to complete this challenge, a perfect understanding of protein assembly and disassembly mechanisms are necessary. In this communication we address the mechanism of formation of microspheres of α-LA and LYS from the molecular scale to the micropheres. One of the first events in the mechanism of formation of microspheres is a specific interaction between a α-LA and LYS leading to a heterodimer.2 Probably throughout charge screening, α-LA/LYS heterodimers rapidly self-assembled into nanometer sized-aggregates. These small entities further aggregate into clusters following a diffusion limited mechanism (DLCA) and fuse upon physical contact into spherical microspheres. The driving force for the reorganisation of the clusters into microspheres is suggested to be the decrease of the total surface free energy. However, the reorganization of the clusters was only inducible when the temperature was increased above 30°C, temperature above which a α-LA adopt a molten globule structure. We put forward that the higher flexibility of α-LA above 30°C may facilitate clusters – microspheres transition

Synergistic pore formation by type III toxin translocators of Pseudomonas aeruginosa.

International audienceType III secretion/translocation systems are essential actors in the pathogenicity of Gram-negative bacteria. The injection of bacterial toxins across the host cell plasma membranes is presumably accomplished by a proteinaceous structure, the translocon. In vitro, Pseudomonas aeruginosa translocators PopB and PopD form ringlike structures observed by electron microscopy. We demonstrate here that PopB and PopD are functionally active and sufficient to form pores in lipid vesicles. Furthermore, the two translocators act in synergy to promote membrane permeabilization. The size-based selectivity observed for the passage of solutes indicates that the membrane permeabilization is due to the formation of size-defined pores. Our results provide also new insights into the mechanism of translocon pore formation that may occur during the passage of toxins from the bacterium into the cell. While proteins bind to lipid vesicles equally at any pH, the kinetics of membrane permeabilization accelerate progressively with decreasing pH values. Electrostatic interactions and the presence of anionic lipids were found to be crucial for pore formation whereas cholesterol did not appear to play a significant role in functional translocon formation

Kinetics and Structure during Self-Assembly of Oppositely Charged Proteins in Aqueous Solution

International audienceSelf-assembly in aqueous solution of two oppositely charged globular proteins, hen egg white lysozyme (LYS) and bovine calcium-depleted α-lactalbumin (apo α-LA), was investigated at pH 7.5. The aggregation rate of equimolar mixtures of the two proteins was determined using static and dynamic light scattering as a function of the ionic strength (15−70 mM) and protein concentration (0.28−2.8 g/L) at 25 and 45 °C. The morphology of formed supramolecular structures was observed by confocal laser scanning microscopy. When the two proteins are mixed, small aggregates were formed rapidly that subsequently grew by collision and fusion. The aggregation process led on larger length scales to irregularly shaped flocs at 25 °C, but to monodisperse homogeneous spheres at 45 °C. Both the initial rate of aggregation and the fraction of proteins that associated decreased strongly with decreasing protein concentration or increasing ionic strength but was independent of the temperature