No abstract

We investigated the liquid-liquid phase separation of wheat storage proteins with molecular weight (MW) ranging from 25 kg mol(-1) to 300 kg mol(-1). We characterized the onset of the phase separation upon a temperature decrease by turbidity. The protein compositions at equilibrium after temperature quenches were additionally determined by size-exclusion-high performance liquid chromatography. We found that wheat proteins can be classified into two classes according to their phase behaviour. The partition of the proteins between the two phases depends on their MW above a critical size of 45 kg mol(-1). For those high MW proteins, we observed that the behaviour is similar to the one of neutral, linear polymers. Their partition and critical parameters are well described by Flory-Huggins theory. Proteins below 45 kg mol(-1), namely alpha-, beta- and gamma-gliadins, have similar interaction potentials under the physicochemical conditions tested. Their phase diagrams, characterized by a low value of critical volume fraction, are characteristic of long-range interactions. Wheat protein behaviour can resultantly be rationalized in a much simpler way than expected from their complex composition

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.

During wheat seeds development, storage proteins are synthetized and subsequently form dense protein phases, also called Protein Bodies (PBs). The mechanisms of PBs formation and the supramolecular assembly of storage proteins in PBs remain unclear. In particular, there is an apparent contradiction between the low solubility in water of storage proteins and their high local dynamics in dense PBs. Here, we probe the interplay between short-range attraction and long-range repulsion of a wheat gliadin isolate by investigating the dynamics of liquid-liquid phase separation after temperature quench. We do so using time-resolved small angle light scattering, phase contrast microscopy and rheology. We show that gliadins undergo liquid-liquid phase separation through Nucleation and Growth or Spinodal Decomposition depending on the quench depth. They assemble into dense phases but remain in a liquid-like state over an extended range of temperatures and concentrations. The analysis of phase separation kinetics reveals that the attraction strength of gliadins is in the same order of magnitude as other proteins. We discuss the respective role of competing interactions, protein intrinsic disorder, hydration and polydispersity in promoting local dynamics and providing this liquid-like behavior despite attractive forces.