Role of protein conformation and weak interactions on gamma-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 gamma-gliadin as a model of wheat proteins to probe gliadins behavior in conditions near physiological conditions. Bioinformatic analyses suggest that gamma-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 gamma-gliadin.We developed an in vitro approach consisting to first solubilize gamma-gliadin in 55% ethanol (v/v) and to progressively decrease ethanol ratio in favor of increased aqueous solution. Our results show the ability of gamma-gliadin to self-assemble into dynamic droplets through LLPS, with saturation concentrations ranging from 25.9 mu M +/- 0.85 mu M (35% ethanol (v/v)) to 3.8 mu M +/- 0.1 mu M (0% ethanol (v/v)). We demonstrate the importance of the predicted ordered C-terminal domain of gamma-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