Approches biomimétiques de l'assemblage de protéines de réserve de blé

L’assemblage des protéines de réserve de blé (prolamines) sous forme de corpuscules protéiques lors du développement du grain reste aujourd’hui un mécanisme peu compris. Ce travail de thèse a consisté à utiliser plusieurs approches biomimétiques dans le but de déterminer les paramètres physico-chimiques pouvant entrer en jeu dans l’assemblage in vivo de ces protéines. Les γ-gliadines et ω-gliadines, ont été choisies comme prolamines modèles, et leurs modes d’assemblages ont été étudiés in vitro dans des solutions aqueuses. Une attention particulière s’est portée sur leur comportement aux interfaces. Pour mimer le contexte biologique, ces protéines ont été étudiées en présence de membranes lipidiques. Les résultats de ces travaux indiquent que, du fait de leur insolubilité, les gliadines présentent une forte capacité à s’assembler en milieu aqueux, en particulier aux interfaces. Les études spectroscopiques montrent que ces protéines présentent des structures secondaires très labiles susceptibles de s’orienter aux interfaces. Que ce soit à l’interface air-eau, ou sous des monocouches lipidiques, les gliadines s’adsorbent sous forme de monocouches. Pour de fortes concentrations en protéines, l’épaisseur des couches adsorbées de γ-gliadines croît pour former des domaines protéiques denses, phénomène non observé dans le cas des ω-gliadines. Un modèle d’auto-assemblage, basé sur la structure primaire de ces gliadines est ainsi proposé, et suggéré comme mécanisme initial de formation des corpuscules protéiques.Wheat storage proteins (prolamins) assembly in protein bodies during grain development still remains an ill-understood mechanism. This work displays different biomimetic strategies in order to elucidate physical chemistry parameters important for these proteins assembly in vivo. The γ-gliadins and ω-gliadins were chosen as model prolamins, and their assembly in aqueous solution was studied in vitro. This work focused on their interfacial behaviour. To mimic the biological context, these proteins were studied in the vicinity of lipidic membranes. Results indicate that, due to their low solubility, gliadins display an important ability to self-assemble in aqueous media, preferentially to the interfaces. Spectroscopic studies show that these proteins display labile secondary structures able to change their orientation at interfaces. In addition, at the air-water interface or under lipidic monolayers, gliadins adsorb to form protein monolayers. At high γ-gliadins concentrations, the thickness of adsorbed protein layer increases to form dense protein domains, result not observed in the case of ω-gliadins. A self assembly model, based on the primary structure of gliadins is thus proposed, and suggested as an initial mechanism of protein bodies formation

Tuning Structure and Rheology of Silica-Latex Nanocomposites with the Molecular Weight of Matrix Chains: A Coupled SAXS-TEM-Simulation Approach

The structure of silica-latex nanocomposites of three matrix chain masses (20, 50, and 160 kg/mol of poly(ethyl methacrylate)) are studied using a SAXS/TEM approach, coupled via Monte Carlo simulations of scattering of fully polydisperse silica nanoparticle aggregates. At low silica concentrations (1 vol. %), the impact of the matrix chain mass on the structure is quantified in terms of the aggregation number distribution function, highest mass leading to individual dispersion, whereas the lower masses favor the formation of small aggregates. Both simulations for SAXS and TEM give compatible aggregate compacities around 10 vol. %, indicating that the construction algorithm for aggregates is realistic. Our results on structure are rationalized in terms of the critical collision time between nanoparticles due to diffusion in viscous matrices. At higher concentrations, aggregates overlap and form a percolated network, with a smaller and lighter mesh in the presence of high mass polymers. The linear rheology is investigated with oscillatory shear experiments. It shows a feature related to the silica structure at low frequencies, the amplitude of which can be described by two power laws separated by the percolation threshold of aggregates

Spontaneous gelation of wheat gluten proteins in a food grade solvent

Structuring wheat gluten proteins into gels with tunable mechanical properties would provide more versatility for the production of plant protein-rich food products. Gluten, a strongly elastic protein material insoluble in water, is hardly processable. We use a novel fractionation procedure allowing the isolation from gluten of a water/ethanol soluble protein blend, enriched in glutenin polymers at an unprecedented high ratio (50%). We investigate here the viscoelasticity of suspensions of the protein blend in a water/ethanol (50/50 v/v) solvent, and show that, over a wide range of concentrations, they undergo a spontaneous gelation driven by hydrogen bonding. We successfully rationalize our data using percolation models and relate the viscoelasticity of the gels to their fractal dimension measured by scattering techniques. The gluten gels display self-healing properties and their elastic plateaus cover several decades, from 0.01 to 10000 Pa. In particular very soft gels as compared to standard hydrated gluten can be produced.Comment: Food Hydrocolloids, in pres

Dynamic trafficking of wheat γ-gliadin and of its structural domains in tobacco cells, studied with fluorescent protein fusions

Prolamins, the main storage proteins of wheat seeds, are synthesized and retained in the endoplasmic reticulum (ER) of the endosperm cells, where they accumulate in protein bodies (PBs) and are then exported to the storage vacuole. The mechanisms leading to these events are unresolved. To investigate this unconventional trafficking pathway, wheat γ-gliadin and its isolated repeated N-terminal and cysteine-rich C-terminal domains were fused to fluorescent proteins and expressed in tobacco leaf epidermal cells. The results indicated that γ-gliadin and both isolated domains were able to be retained and accumulated as protein body-like structures (PBLS) in the ER, suggesting that tandem repeats are not the only sequence involved in γ-gliadin ER retention and PBLS formation. The high actin-dependent mobility of γ-gliadin PBLS is also reported, and it is demonstrated that most of them do not co-localize with Golgi body or pre-vacuolar compartment markers. Both γ-gliadin domains are found in the same PBLS when co-expressed, which is most probably due to their ability to interact with each other, as indicated by the yeast two-hybrid and FRET-FLIM experiments. Moreover, when stably expressed in BY-2 cells, green fluorescent protein (GFP) fusions to γ-gliadin and its isolated domains were retained in the ER for several days before being exported to the vacuole in a Golgi-dependent manner, and degraded, leading to the release of the GFP ‘core’. Taken together, the results show that tobacco cells are a convenient model to study the atypical wheat prolamin trafficking with fluorescent protein fusions

Le gluten vu par diffusions de rayonnements

National audience-

Biomimetical approaches of the wheat storage protein assembly

L'assemblage des protéines de réserve de blé(prolamines) sous forme de corpuscules protéiques lors du développement du grain reste aujourd'hui un mécanisme peu compris. Ce travail de thèse a consisté à utiliser plusieurs approches biomimétiques dans le but de déterminer les paramètres physico-chimiques pouvant entrer en jeu dans l'assemblage in vivo de ces protéines. Les gamma-gliadines et omega-gliadines, ont été choisies comme prolamines modèles, et leurs modes d'assemblages ont été étudiés in vitro dans des solutions aqueuses. Une attention particulière s'est portée sur leur comportement aux interfaces. Pour mimer le contexte biologique, ces protéines ont été étudiées en présence de membranes lipidiques. Les résultats de ces travaux indiquent que, du fait de leur insolubilité, les gliadines présentent une forte capacité à s'assembler en milieu aqueux, en particulier aux interfaces. Les études spectroscopiques montrent que ces protéines présentent des structures secondaires très labiles susceptibles de s'orienter aux interfaces. Que ce soit à l'interface air-eau, ou sous des monocouches lipidiques, les gliadines s'adsorbent sous forme de monocouches. Pour de fortes concentrations en protéines, l'épaisseur des couches adsorbées de gamma-gliadines croît pour former des domaines protéiques denses, phénomène non observé dans le cas des omega-gliadines. Un modèle d'auto-assemblage, basé sur la structure primaire de ces gliadines est ainsi proposé, et suggéré comme mécanisme initial de formation des corpuscules protéiques.Wheat storage proteins (prolamins) assembly in protein bodies during grain development still remains an ill-understood mechanism. This work displays different biomimetic strategies in order to elucidate physical chemistry parameters important for these proteins assembly in vivo. The g-gliadins and w-gliadins were chosen as model prolamins, and their assembly in aqueous solution was studied in vitro. This work focused on their interfacial behaviour. To mimic the biological context, these proteins were studied in the vicinity of lipidic membranes. Results indicate that, due to their low solubility, gliadins display an important ability to selfassemble in aqueous media, preferentially to the interfaces. Spectroscopic studies show that these proteins display labile secondary structures able to change their orientation at interfaces. In addition, at the air-water interface or under lipidic monolayers, gliadins adsorb to form protein monolayers. At high g-gliadins concentrations, the thickness of adsorbed protein layer increases to form dense protein domains, result not observed in the case of w-gliadins. A self assembly model, based on the primary structure of gliadins is thus proposed, and suggested as an initial mechanism of protein bodies formation

Approches biomimétiques de l'assemblage de protéines de réserve de blé

Wheat storage proteins (prolamins) assembly in protein bodies during grain development still remains an ill-understood mechanism. This work displays different biomimetic strategies in order to elucidate physical chemistry parameters important for these proteins assembly in vivo. The g-gliadins and w-gliadins were chosen as model prolamins, and their assembly in aqueous solution was studied in vitro. This work focused on their interfacial behaviour. To mimic the biological context, these proteins were studied in the vicinity of lipidic membranes. Results indicate that, due to their low solubility, gliadins display an important ability to selfassemble in aqueous media, preferentially to the interfaces. Spectroscopic studies show that these proteins display labile secondary structures able to change their orientation at interfaces. In addition, at the air-water interface or under lipidic monolayers, gliadins adsorb to form protein monolayers. At high g-gliadins concentrations, the thickness of adsorbed protein layer increases to form dense protein domains, result not observed in the case of w-gliadins. A self assembly model, based on the primary structure of gliadins is thus proposed, and suggested as an initial mechanism of protein bodies formation.L'assemblage des protéines de réserve de blé(prolamines) sous forme de corpuscules protéiques lors du développement du grain reste aujourd'hui un mécanisme peu compris. Ce travail de thèse a consisté à utiliser plusieurs approches biomimétiques dans le but de déterminer les paramètres physico-chimiques pouvant entrer en jeu dans l'assemblage in vivo de ces protéines. Les gamma-gliadines et omega-gliadines, ont été choisies comme prolamines modèles, et leurs modes d'assemblages ont été étudiés in vitro dans des solutions aqueuses. Une attention particulière s'est portée sur leur comportement aux interfaces. Pour mimer le contexte biologique, ces protéines ont été étudiées en présence de membranes lipidiques. Les résultats de ces travaux indiquent que, du fait de leur insolubilité, les gliadines présentent une forte capacité à s'assembler en milieu aqueux, en particulier aux interfaces. Les études spectroscopiques montrent que ces protéines présentent des structures secondaires très labiles susceptibles de s'orienter aux interfaces. Que ce soit à l'interface air-eau, ou sous des monocouches lipidiques, les gliadines s'adsorbent sous forme de monocouches. Pour de fortes concentrations en protéines, l'épaisseur des couches adsorbées de gamma-gliadines croît pour former des domaines protéiques denses, phénomène non observé dans le cas des omega-gliadines. Un modèle d'auto-assemblage, basé sur la structure primaire de ces gliadines est ainsi proposé, et suggéré comme mécanisme initial de formation des corpuscules protéiques

Ab-Initio calculations of proline vibrations with and without water, consequences on the infrared spectra of proline-rich proteins

International audienceThe infrared spectra of proline rich proteins display a strong band in the 1450 cm-1 region. In the literature, this band was assigned either to the deformation modes of the CH2 and CH3 groups or to the CN stretching mode of proline residues. In order to establish the correct assignment of this band, the impact of proline vibrations in a polypeptide chain is studied and ab-initio calculations are performed for a model molecule (I) containing a repeat unit of polyproline. A strong band is effectively calculated in the 1450 cm-1 region and mostly assigned to CN stretching whereas, due to the absence of N-H bond, there is no amide II band. These results are in good agreement with the spectral features observed in the FTIR spectra of gliadins. Moreover, the spectral shifts calculated when a water molecule is complexed with (I) are consistent with the hydration effect observed in the experimental data

Ab-Initio calculations of proline vibrations with and without water, consequences on the infrared spectra of proline-rich proteins

The infrared spectra of proline rich proteins display a strong band in the 1450 cm-1 region. In the literature, this band was assigned either to the deformation modes of the CH2 and CH3 groups or to the CN stretching mode of proline residues. In order to establish the correct assignment of this band, the impact of proline vibrations in a polypeptide chain is studied and ab-initio calculations are performed for a model molecule (I) containing a repeat unit of polyproline. A strong band is effectively calculated in the 1450 cm-1 region and mostly assigned to CN stretching whereas, due to the absence of N-H bond, there is no amide II band. These results are in good agreement with the spectral features observed in the FTIR spectra of gliadins. Moreover, the spectral shifts calculated when a water molecule is complexed with (I) are consistent with the hydration effect observed in the experimental data

Tracking an ion complexing agent within bilayers

In this Letter, we show the benefit of using a lyotropic lamellar phase as a self assembled model system to investigate the distribution of a lipophilic molecule at a hydrophilic/hydrophobic interface. For complexing agents used for ion transfer in liquid/liquid extraction it is essential to determine their interfacial activity. Coupling scattering and spectrometry techniques, we show that it is possible to determine accurately the amount of this type of molecule at the interface.Actinide reCycling by SEParation and Transmutatio