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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.

The partial enzymatic hydrolysis ofwheat gliadins constitutes an interesting tool to unravel their structural spec ificity. In this work, the structure and conformation of -y 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 -y gliadin is a partially disordered protein with an unfolded N terminal demain surprisingly resistant to chymotrypsin and a folded C terminal dornain. Using spec troscopic tools, we showed that structural transitions occured over the disordered N terminal domain for de creasing ethanol/water ratios. Using SAXS measurements, low resolution 30 structures of-y gliadin were proposed. To relate the repeated motifs of the N terminal demain of-y gliadin to its structure , engineered peptide models PQQPY/F were aise studied. Overall results demonstrated similarities between the N terminal demain and its derived model peptides. Our findings support the use of these peptides as general templates for under standing the wheat protein assembly and dynamics.

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