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

Effects of trace elements and calcium on diabetes and obesity, and their complications: Protective effect of dairy products – A mini review

An error occurred in the address of an author of the article Bouglé D., Bouhallab S., Bureau F. and Zunquin G., Effects of trace elements and calcium on diabetes and obesity, and their complications: Protective effect of dairy products – A mini review, Dairy Sci. Technol. 89 (2009) 213–218. Therefore the address of the author Saïd Bouhallab was lacking in the article: $^{2}$INRA, UMR1253 Science et Technologie du Lait et de l'Œuf, 35042 Rennes, France $^{3}$AGROCAMPUS OUEST, UMR1253 Science et Technologie du Lait et de l'Œuf, 35042 Rennes, Franc

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

Selective coacervation between lactoferrin and the two isoforms of β-lactoglobulin

This work reports on the impact of subtle change of protein charge on coacervation and subsequent liquid–liquid phase separation between two oppositely charged globular proteins. For this purpose, a comparative study was conducted on the coacervation of lactoferrin (LF) with the two β-lactoglobulin (β-LG) isoforms. Upon mixing LF with an excess of β-LG, microspheres were formed throughout coacervation under narrow pH range (5.4–6.0). At the optimal pH of coacervation, LF being the limiting partner under tested concentration ranges. The β-LG/LF molar ratio recovered in the formed coacervates varied from 4 to 8 depending on the total protein concentration. Remarkably, LF showed a selective coacervation with isoform A of β-LG as judged by a larger concentration domain for coacervation and a high yield of LF recovered once mixed with β-LG A i.e. 80% against a maximum of 42% with β-LG B. At thermodynamic level, the interaction of LF with both β-LG isoforms exhibited complex exothermic binding isotherms with both enthalpic and entropic contributions

How the presence of a small molecule affects the complex coacervation between lactoferrin and ␤-lactoglobulin

Heteroprotein complex coacervation corresponds to the formation of two liquid phases in equilibrium induced by the interaction of two oppositely charged proteins. The more concentrated phase known as coacervate phase, has attracted interest from several fields of science due to its potential applications for example for encapsulation and delivery of bioactives. Prior such application, it is necessary to understand how the presence of small ligands affects the complex coacervation. In this work, we report on the interaction of small ligand with individual proteins β-lactoglobulin (β-LG) and lactoferrin (LF) and consequences on their complex coacervation. ANS (8-Anilinonaphthalene-1-sulfonic acid), a fluorescent probe, was used as model ligand. While ANS did not interact with β-LG, it presented two sets of binding sites with LF inducing its self-aggregation. Depending on its concentration, ANS modulated the shape of β-LG-LF macromolecular assembly. Coacervates were observed for ANS/LF molar ratio <25 against amorphous aggregates for higher ANS/LF molar ratios. A maximum loading capacity of around 40 mg of ANS per gram of LF in the formed heteroprotein coacervates was reached

Interaction of natively unfolded proteins with tannins

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