Interactions et assemblages entre l'alpha lactalbumine et le lysozyme: mécanismes, structures et stabilité

Understanding the mechanisms of protein-protein interactions and assemblies is of relevant interest in many research areas (food, medical, pharmaceutical) that allows a better control of overall processes and properties of resulting supramolecular structures. The aim of my thesis work was to acquire knowledges at different scales from protein assemblies at molecular level to the resulting supramolecular structures. This work was performed using a protein binary system including two oppositely charged globular proteins at neutral pH: lysozyme (LYS) and alpha-lactalbumin (alpha-LA). Fluorescence technics allow us to characterise molecular interaction and formation of heterodimers between LYS and the two forms of alpha-LA i.e. calcium loaded (holo) or calcium free (apo) alpha-LA. The formation of these heterodimers was driven by electrostatic forces. Assembly properties of these heterodimers are distinct and strongly linked to the stability of alpha-lactalbumin. LYS – holo alpha-LA heterodimers do not seem to self-assemble further into supramolecular structures, while LYS – apo alpha-LA heterodimers self-assemble into aggregates or microspheres according to the conformation of apo alpha-LA. A “molten globule” conformation of apo alpha-LA induces the formation of microspheres. Their formation follows an original nucleation and reorganisation mechanism. These microspheres contain equimolar ratio of both proteins that are perfectly co-localised within the overall microstructure. This work highlights the key role of protein conformation and flexibility in the formation and modulation of food protein assemblies.L'assemblage des protéines est une problématique fondamentale d'intérêt pour différents secteurs (alimentaire, médical, pharmaceutique, etc.). La compréhension des mécanismes à l'origine des interactions initiales et des assemblages protéiques offre la possibilité de contrôler et d'orienter le processus de formation ainsi que la nature et les propriétés fonctionnelles des structures supramoléculaires résultantes. L'objectif de la thèse était d'acquérir de nouvelles connaissances à différentes échelles d'étude sur les mécanismes d'assemblages protéiques et les structures supramoléculaires dans un mélange protéique binaire incluant deux protéines globulaires de charge globale opposée à pH neutre : le lysozyme (LYS) et l'alpha-lactalbumine (alpha-LA). L'utilisation de techniques de fluorescence a permis de caractériser l'interaction moléculaire et la formation d'hétérodimères entre ces deux protéines aussi bien avec les formes chargée (holo alpha-LA) et déplétée (apo alpha-LA) en calcium de l'alpha-LA. La formation de ces hétérodimères s'effectue par la mise en œuvre d'interactions électrostatiques. Les propriétés d'assemblage de ces hétérodimères sont différentes et intimement liées à la stabilité de l'alpha-LA. Les hétérodimères LYS – holo alpha-LA ne semblent pas former de structures supramoléculaires, alors que les hétérodimères LYS – apo alpha-LA s'assemblent en agrégats ou en structures sphériques selon la conformation de l'apo alpha-LA. Une conformation de type « molten globule » de l'apo alpha-LA favorise la formation de microsphères. Ces dernières sont constituées de LYS et d'apo alpha-LA en quantité équimolaire qui sont parfaitement co-localisés au sein de la microstructure. Ce travail souligne le rôle clé joué par la conformation et la flexibilité des protéines dans la formation et l'orientation des assemblages entre protéines alimentaires

Interactions et assemblages entre l &-Lactalbumine et le lysozyme : mécanismes, structures et stabilité

L assemblage des protéines est une problématique fondamentale d intérêt pour différents secteurs (alimentaire, médical, pharmaceutique, etc.). La compréhension des mécanismes à l origine des interactions initiales et des assemblages protéiques offre la possibilité de contrôler et d orienter le processus de formation ainsi que la nature et les propriétés fonctionnelles des structures supramoléculaires résultantes. L objectif de la thèse était d acquérir de nouvelles connaissances à différentes échelles d étude sur les mécanismes d assemblages protéiques et les structures supramoléculaires dans un mélange protéique binaire incluant deux protéines globulaires de charge globale opposée à pH neutre :: le lysozyme (LYS) et l &-lactalbumine (&-La). L utilisation de techniques de fluorescence a permis de caractériser l interaction moléculaire et la formation d hétérodimère entre ces deux protéines aussi bien avec les formes chargée (holo &-LA) et déplétée (apo &-LA) en calcium de l &-LA. La formation de ces hétérodimères s effectue par la mise en œuvre d interactions électrostatiques. Les propriétés d assemblage de ces hétérodimères sont différentes et intimement liées à la stabilité de l &-LA. Les hétérodimères LYS holo &-LA s assemblent en agrégats ou en structures sphériques selon la conformation de l apo &-LA. Une conformation de type molten globule de l apo &-LA en quantité équimolaire qui sont parfaitement co-localisés au sein de la microstructure. Ce travail souligne le rôle clé joué par la conformation et la flexibilité des protéines dans la formation et l orientation des assemblages entre protéines alimentaires.Understanding the mechanisms of protein-protein interactions and assemblies is of relevant interest in many research areas (food, medical, pharmaceutical) that allows a better control of overall processes and properties of resulting supramolecular structures. The aim of my thesis work was to acquire knowledges at different scales from protein assemblies at molecular level to the resulting supramolecular structures. This work was performed using a protein binary system including two oppositely charged globular protein at neutral pH : lysozyme (LYS) and &-lactalbumin (&-LA). The formation of these heterodimers was driven by eletrosatic forces. Assembly properties of these heterodimers are distinct and strongly linked to the stability of &-lactoalbumin. LYS holo &-LA &-LA heterodimers selsf-assemble into aggregates or microsphesrs according to the conformationof apo &-LA. A molten globule conformation of apo &-LA induces the formatinof microspheres. Their formation follows an original nucleationand reorganisation mechanims. These microspheres contain equimolar ratio of both proteins that are perfectly co-localised within the overal microstructure. This work highlights the key role of protein conformation and flexibility in the formation and modulation of food protein assemblies.RENNES-Agrocampus-CRD (352382323) / SudocSudocFranceF

Adsorption Behavior of Arabinogalactan-Proteins (AGPs) from Acacia senegal Gum at a Solid–Liquid Interface

International audienceAdsorption of five different hyperbranched arabinogalactan-protein (AGP) fractions from Acacia senegal gum was thoroughly studied at the solid-liquid interface using a quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR), and atomic force microscopy (AFM). The impact of the protein/sugar ratio, molecular weight, and aggregation state on the adsorption capacity was investigated by studying AGP fractions with different structural and biochemical features. Adsorption on a solid surface would be primarily driven by the protein moiety of the AGPs through hydrophobic forces and electrostatic interactions. Increasing ionic strength allows the decrease in electrostatic repulsions and, therefore, the formation of high-coverage films with aggregates on the surface. However, the maximum adsorption capacity was not reached by fractions with a higher protein content but by a fraction that contains an average protein quantity and presents a high content of high-molecular-weight AGPs. The results of this thorough study highlighted that the AGP surface adsorption process would depend not only on the protein moiety and high-molecular-weight AGP content but also on other parameters such as the structural accessibility of proteins, the molecular weight distribution, and the AGP flexibility, allowing structural rearrangements on the surface and spreading to form a viscoelastic fil

Adsorption of Hyperbranched Arabinogalactan-Proteins from Plant Exudate at the Solid–Liquid Interface

Adsorption of hyperbranched arabinogalactan-proteins (AGPs) from two plant exudates, A. senegal and A. seyal, was thoroughly studied at the solid−liquid interface using quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR), and atomic force microscopy (AFM). Isotherms of the adsorption reveal that 3.3 fold more AGPs from A. seyal (500 ppm) are needed to cover the gold surface compared to A. senegal (150 ppm). The pH and salt concentration of the environment greatly affected the adsorption behavior of both gums, with the surface density ranging from 0.92 to 3.83 mg m−2 using SPR (i.e., “dry” mass) and from 1.16 to 19.07 mg m−2 using QCM-D (wet mass). Surprisingly, the mass adsorbed was the highest in conditions of strong electrostatic repulsions between the gold substrate and AGPs, i.e., pH 7.0, highlighting the contribution of other interactions involved in the adsorption process. Structural changes of AGPs induced by pH would result in swelling of the polysaccharide blocks and conformational changes of the polypeptide backbone, therefore increasing the protein accessibility and hydrophobic interactions and/or hydrogen bonds with the gold substrate

Acacia senegal vs. Acacia seyal gums - Part 1: Composition and structure of hyperbranched plant exudates

Acacia gum is a natural arabinogalactan-protein type polysaccharide widely used in industrial applications. The two varieties of Acacia gum, A. senegal and A. seyal, are hyperbranched polysaccharides rich in arabinose and galactose (PRAG) mainly formed by chains of 3,6-linked beta-D-Galp substituted in position 6 by side chains of 3-linked alpha-L-Araf. Beyond this common structure, A. senegal presented the highest degree of branching (78.2% vs. 59.2%) with more branched galactopyranoses, shorter arabinosyl side branches, and more rhamnopyranoses in terminal position. Circular dichroism experiments evidenced that both Acacia gums were partially structured into polyproline type II helices but in a lesser extent for A. senegal gum, suggesting its less structured organisation. The fingerprint of both Acacia gums and their spectral differences determined by FTIR spectroscopy were described. Analysis of HPSEC-MALLS data suggested that macromolecules of both Acacia gums adopted an ellipsoid-like conformation in solution. The average molecular weight of A. seyal macromolecules was larger than that of A. senegal ones (8.2 x 10(5) g mol(-1) vs. 6.8 x 10(5) g mol(-1)). Nevertheless, A. seyal macromolecules appeared more compact and less viscous (R-g: 17.1 nm; [eta]: 16.5 mL g(-1)) than A. senegal ones (R-g: 30.8 nm; [eta]: 22.8 mL g(-1)). The most compact structure of A. seyal gum can be partly explained by the lowest concentration of charged sugars that induces less electrostatic repulsion, and the highest content of long arabinose side chains that may self-organize and interact between them (e.g. hydrogen bonding, steric effect, etc.). For both Acacia gums, the anisotropy of macromolecules increased with the molecular weight, however, A. senegal macromolecules were the most anisotropic one

Acetylation degree, a key parameter modulating chitosan rheological, thermal and film-forming properties

International audienceThe effect of blue crab chitosan acetylation degree (AD) on its rheological, thermal and film-forming properties was investigated. Determination of the dynamic viscoelastic properties (elastic modulus G' and viscous modulus G") of chitosan solutions revealed a typical Newtonian viscous-like behavior, with higher viscosity values at lower acetylation degrees. Moreover, the gelation temperature of blue crab chitosan was strongly dependent on its acetylation degree, with lower values at lower acetylation degrees. Regarding chitosan thermal stability, thermogravimetric profiles revealed greater thermal resistance for chitosans with lower acetylation degrees, showing significantly higher degradation temperature. Likewise, the values of glass transition temperature decreased with the increase of the acetylation degree. In another side, chitosan-based films, with lower acetylation degrees, are found to be more flexible, resistant and transparent. Therefore, blue crab chitosan acetylation degree modulated toughly its viscosity, elongation at break, tensile strength and thermal resistance. Further, chitosans with ADs less than 20% seemed to have potential industrial applications

The role of active site flexible loops in catalysis and of zinc in conformational stability of Bacillus cereus 569/H/9 beta-lactamase.

Metallo-beta-lactamases catalyse the hydrolysis of most beta-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the diversity and flexibility of their substrate binding sites, motivating research into their structure and function. In this study, we examined the conformational properties of the Bacillus cereus beta-lactamase II in the presence of chemical denaturants using a variety of biochemical and biophysical techniques. The apoenzyme was found to unfold cooperatively, with a Gibbs free energy of stabilization (DeltaG degrees ) of 32 +/- 2 kJ.mol11. For holoBcII, a first non-cooperative transition leads to multiple interconverting native-like states, in which both zinc atoms remain bound in an apparently unaltered active site and the protein displays a well-organized compact hydrophobic core with structural changes confined to the enzyme surface, but with no catalytic activity. 2D NMR data revealed that the loss of activity occurs concomitantly with perturbations in two loops that border the enzyme active site. A second cooperative transition, corresponding to global unfolding, is observed at higher denaturant concentrations, with DeltaG degrees value of 65 +/- 1.4 kJ.mol11. These combined data highlight the importance of the two zinc ions in maintaining structure as well as a relatively well-defined conformation for both active site loops in order to maintain enzymatic activity

Acacia gums new fractions and sparkling base wines: How their biochemical and structural properties impact foamability?

International audienceFoam is the first attribute observed when sparkling wine is served. Bentonite is essentially used to flocculate particles in sparkling base wines but can impair their foamability. Gums from Acacia senegal and Acacia seyal improved the foamability of different bentonite-treated base wines. Our main goal was to see how the supplementation with new fractions separated from Acacia gums by Ion Exchange Chromatography affected foamability of sparkling base wines, deepening the relation between foam behavior and characteristics of wine and gums. High molar mass fractions increased the maximal foam height and the foam height during the stability period in, respectively, 11 out and 8 out of 16 cases (69% and 50%, respectively). The properties of the supplementing gums fractions obtained by IEC and, although to a minor extent, the wine characteristics, affected positively and/ or negatively the foam behavior. Wine foamability also depended on the relationship between wine and gums fractions properties

Improvement of the foamability of sparkling base wines by the addition of Acacia gums

International audienceIn sparkling wine, foam characteristics are one of the major attributes. The foam quality depends on wine components. Bentonite is added to the base wine to facilitate the riddling process, but causes a loss of foamability. Acacia gum can be used as additive in wine. We have studied if the addition of Acacia senegal gum (AsenG), Acacia seyal gum (AseyG) and different AsenG fractions could improve the foamability of different base wines treated with bentonite. The foamability differs depending on the gum or the gum fraction treatment but also on the wine, being these differences linked to some aspects of their respective compositions and molecular parameters. AsenG and AseyG increase the foamability (by Mosalux - sparging procedure), respectively, in five and seven out of eight base wines treated with bentonite. Therefore, AsenG and AseyG are potential treatments increasing the foamability of these wines

Effect of mono or co-culture of EPS-producing <em>Streptococcus thermophilus</em> strains on the formation of acid milk gel and the appearance of texture defects

International audienceOne acidifying (ST1) and two texturing strains (ST2 and ST3) of Streptococcus thermophilus were used as pure or co-cultures to identify and understand their effects on the structuring of acid milk gels and on the appearance of texture defects, i.e., syneresis and graininess. Symbiosis between specific texturing and acidifying strains reduced acidification time and increased exocellular polysaccharide (EPS) content. The texturing strain could simultaneously produce low and high molar mass EPS and their distribution in mass and/or in number were influenced by the proportion of acidifying to texturing strain used. The results of this study suggest that the high molar mass EPS contributes to acid gel firmness, but less so compared with the acidification rate. The ability of strain ST3 to prevent texture defects, specifically graininess, did not depend on the acidification kinetics or final EPS content, but rather on the structural properties of EPS and/or the bacterial chain morphology