Influence of pH and lipid membrane on the liquid-liquid phase separation of wheat γ-gliadin in aqueous conditions.

Protein body (PB) formation in wheat seeds is a critical process influencing seed content and nutritional quality. In this study, we investigate the potential mechanisms governing PB formation through an in vitro approach, focusing on γ-gliadin, a key wheat storage protein. We used a microfluidic technique to encapsulate γ-gliadin within giant unilamellar vesicles (GUVs) and tune the physicochemical conditions in a controlled and rapid way. We examined the influence of pH and protein concentration on LLPS and protein-membrane interactions using various microscopy and spectroscopy techniques. We showed that γ-gliadin encapsulated in GUVs can undergo a pH-triggered liquid-liquid phase separation (LLPS) by two distinct mechanisms depending on the γ-gliadin concentration. At low protein concentrations, γ-gliadins phase separate by a nucleation and growth-like process, while, at higher protein concentration and pH above 6.0, γ-gliadin formed a bi-continuous phase suggesting a spinodal decomposition-like mechanism. Fluorescence and microscopy data suggested that γ-gliadin dense phase exhibited affinity for the GUV membrane, forming a layer at the interface and affecting the reversibility of the phase separation.

Biochemical and physical-chemical characterisation of leaf proteins extracted from Cichorium endivia leaves.

This study provides a detailed characterisation of a leaf protein concentrate (LPC) extracted from Cichorium endivia leaves using a pilot scale process. This concentrate contains 74.1% protein and is mainly composed of Ribulose-1,5-BISphosphate Carboxylase/Oxygenase (RuBisCO). We show that the experimentally determined extinction coefficient (around 5.0 cm-1 g-1 L depending on the pH) and refractive index increment (between 0.27 and 0.39 mL g-1) are higher than the predicted ones (about 1.6 cm-1 g-1 L and 0.19 mL g-1, respectively). In addition, the UV-visible absorption spectra show a maximum at 258 nm. These data suggest the presence of non-protein UV-absorbing species. Chromatographic separation of the concentrate components in denaturing conditions suggests that RuBisCO SC may be covalently bounded to few phenolic compounds. Besides, the solubility of LPC proteins is higher than 90% above pH 6. Such high solubility could make LPC a good candidate as a functional food ingredient.

Data on agronomic traits, biochemical composition of lipids, proteins and polysaccharides and rheological measurement in a brown mustard seed collection.

The data were collected from a brown mustard seeds collection of 18 accessions during two years and in three distinct sites of production in France. The 18 accessions of mustard seeds were selected to be representative of genetic, agronomical and technological variabilities. All accessions were produced in the "Bourgogne" area. This article describes agronomical data (PMG, yield), genotyping data, global composition of mustard seeds (lipids, proteins and polysaccharides) and fine composition of the previous macronutrients potentially involved in the technological properties (fatty acids, storage proteins and osidic composition of polysaccharides). Additional data regarding the potential rheological property of each accessions were also reported. These data can be reused by food industries, breeders and geneticists in order to understand pedoclimatic effects (year and location) and the relation between mustard seed composition and the end-uses properties (paste mustard quality).

New exploration of the γ-gliadin structure through its partial hydrolysis.

The partial enzymatic hydrolysis of wheat gliadins constitutes an interesting tool to unravel their structural specificity. In this work, the structure and conformation of γ-gliadin were investigated through its limited chymotrypsic digestion. Using a combination of computational, biochemical and biophysical tools, we studied each of its N and C terminal domains. Our results reveal that γ-gliadin is a partially disordered protein with an unfolded N-terminal domain surprisingly resistant to chymotrypsin and a folded C-terminal domain. Using spectroscopic tools, we showed that structural transitions occured over the disordered N-terminal domain for decreasing ethanol/water ratios. Using SAXS measurements, low-resolution 3D structures of γ-gliadin were proposed. To relate the repeated motifs of the N-terminal domain of γ-gliadin to its structure, engineered peptide models PQQPY/F were also studied. Overall results demonstrated similarities between the N-terminal domain and its derived model peptides. Our findings support the use of these peptides as general templates for understanding the wheat protein assembly and dynamics.

Deamidation and Enzymatic Hydrolysis of Gliadins Alter Their Processing by Dendritic Cells in Vitro.

Gliadins are major wheat allergens. Their treatment by acid or enzymatic hydrolysis has been shown to modify their allergenic potential. As the interaction of food proteins with dendritic cells (DCs) is a key event in allergic sensitization, we wished to investigate whether deamidation and enzymatic hydrolysis influence gliadin processing by DC and to examine the capacity of gliadins to activate DCs. We compared the uptake and degradation of native and modified gliadins by DCs using mouse bone marrow-derived DCs. We also analyzed the effects of these interactions on the phenotypes of DCs and T helper (Th) lymphocytes. Modifying gliadins induced a change in physicochemical properties (molecular weight, hydrophobicity, and sequence) and also in the peptide size. These alterations in turn led to increased uptake and intracellular degradation of the proteins by DCs. Native gliadins (NGs) (100 µg/mL), but not modified gliadins, increased the frequency of DC expressing CD80 (15.41 ± 2.36% vs 6.81 ± 1.10%, p < 0.001), CCR7 (28.53 ± 8.17% vs 17.88 ± 2.53%, p < 0.001), CXCR4 (70.14 ± 4.63% vs 42.82 ± 1.96%, p < 0.001), and CCR7-dependent migration (2.46 ± 1.45 vs 1.00 ± 0.22, p < 0.01) compared with NGs. This was accompanied by Th lymphocyte activation (30.37 ± 3.87% vs 21.53 ± 3.14%, p < 0.1) and proliferation (16.39 ± 3.97% vs 9.31 ± 2.80%, p > 0.1). Moreover, hydrolysis decreases the peptide size and induces an increase in gliadin uptake and degradation. Deamidation and extensive enzymatic hydrolysis of gliadins modify their interaction with DCs, leading to alteration of their immunostimulatory capacity. These findings demonstrate the strong relationship between the biochemical characteristics of proteins and immune cell interactions.

Role of protein conformation and weak interactions on γ-gliadin liquid-liquid phase separation.

Wheat storage proteins, gliadins, were found to form in vitro condensates in 55% ethanol/water mixture by decreasing temperature. The possible role of this liquid-liquid phase separation (LLPS) process on the in vivo gliadins storage is elusive and remains to be explored. Here we use γ-gliadin as a model of wheat proteins to probe gliadins behavior in conditions near physiological conditions. Bioinformatic analyses suggest that γ-gliadin is a hybrid protein with N-terminal domain predicted to be disordered and C-terminal domain predicted to be ordered. Spectroscopic data highlight the disordered nature of γ-gliadin. We developed an in vitro approach consisting to first solubilize γ-gliadin in 55% ethanol (v/v) and to progressively decrease ethanol ratio in favor of increased aqueous solution. Our results show the ability of γ-gliadin to self-assemble into dynamic droplets through LLPS, with saturation concentrations ranging from 25.9 µM ± 0.85 µM (35% ethanol (v/v)) to 3.8 µM ± 0.1 µM (0% ethanol (v/v)). We demonstrate the importance of the predicted ordered C-terminal domain of γ-gliadin in the LLPS by highlighting the protein condensates transition from a liquid to a solid state under reducing conditions. We demonstrate by increasing ionic strength the role displayed by electrostatic interactions in the phase separation. We also show the importance of hydrogen bonds in this process. Finally, we discuss the importance of gliadins condensates in their accumulation and storage in the wheat seed.

Contrasting Assemblies of Oppositely Charged Proteins.

Oppositely charged proteins can form soluble assemblies that under specific physical chemical conditions lead to liquid-liquid phase separation, also called heteroprotein coacervation. Increasing evidence suggests that surface charge anisotropy plays a key role in heteroprotein complexation, and coacervation. Here, we investigated complexation of an acidic protein, ß-lactoglobulin (BLG), with two basic proteins, rapeseed napin (NAP) and lysozyme (LYS), of similar net charge and size but differing in surface charge distribution. Using turbidity measurements and isothermal titration calorimetry, we confirmed that LYS binds BLG as expected from previous studies. This interaction leads to two types of phase separation phenomena, depending on pH: liquid-solid phase separation in the case of strong electrostatic attraction and liquid-liquid phase separation for weaker attraction. More interestingly, we showed using dynamic light scattering that NAP interacts with BLG, resulting in formation of assemblies in the nanometer size range. The formation of assemblies was also evident when modeling the interactions using Brownian dynamics for both BLG + NAP and BLG + LYS. Similarly, to DLS, BLG and NAP formed smaller assemblies than BLG with LYS. The molecular details rather than the net charge of BLG and NAP may therefore play a role in their assembly. Furthermore, simulated BLG + NAP assemblies were larger than those experimentally detected by DLS. We discuss the discrepancy between experiments and simulations in relation to the limitations of modelling precisely the molecular characteristics of proteins.