Dynamics of liquid-liquid phase separation of wheat gliadins

Abstract

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.

Insight into gluten structure in a mild chaotropic solvent by asymmetrical flow field-flow fractionation (AsFlFFF) and evidence of non-covalent assemblies between glutenin and ω-gliadin

“…Our results are in agreement with these findings. By contrast, the phase diagram measured for gliadin by Boire et al (Boire et al, 2018) displays a critical temperature of 12 °C and a protein concentration dependence that is reminiscent of what we observe for samples with Glu ≥ 29 %. This could be explained by the presence of assemblies evidenced by DLS that would pilot the cloud point temperature observed, although the gliadin extract contained only 7.6 % glutenin.…”

“…By definition gliadins are monomeric proteins, but several authors have nevertheless identified the presence of high molar mass proteins (up to 700 000 g/mol) solubilized in this type of solvent (Bietz and Wall, 1980;Schmid, 2016;Shewry et al, 1983). Objects with hydrodynamic radius of about 100 nm co-existing with monomers were observed but disregarded (Boire et al, 2018). Exploiting the low temperature liquid-liquid phase separation of solutions of wheat gluten in ethanol/water 50/50 v/v (Boire et al, 2013), we developed extraction procedures to obtain ethanol/water soluble gluten protein extracts enriched in glutenin.…”

Glutenin and Gliadin, a Piece in the Puzzle of their Structural Properties in the Cell Described through Monte Carlo Simulations

“…The formation of protein bodies is most probably a result of similar hydrophobic interactions and crosslinking, as described above ( Figure 9 g). An increasing concentration and amount of hydrophobic groups contribute to proteins more prone to self-associating [ 77 , 92 ], which is speculated to be an initial stage of protein body formation for gliadins [ 93 , 94 ]. Folded and aggregated proteins are then transported via different routes to the assembly in PBs at various places in the cell, depending on the stages of grain development.…”

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

“…Pyrenoid 12–14 and photobodies 15 , respectively located in chloroplast and nucleus, are best-known plant-specific membrane-less compartments, containing proteins with high disorder profiles. Recent in vitro studies suggest that LLPS could also drive membrane-enclosed organelles formation in wheat seed 16,17 . It has been hypothesized that condensates of wheat storage proteins might play a role as precursor in the formation of their storage organelles, also called protein bodies (PBs).…”

“…Remarkably, all prolamins are known for their low complexity sequence containing rich interspersed repeats which can lead to disordered structures 24–26 . Previous works in aqueous buffer/ethanol solution (45/55) (v/v) have shown the ability of whole gliadins extract, comprising a mixture of α, β, γ and ω-gliadins, to self-assemble through LLPS by decreasing temperature 16,27 . It also has been demonstrated the ability of dense gliadins phases to remain in the liquid-like state, even at high protein concentrations (up to 500 g/L) and at low temperatures (from 2 to 20 °C) 16,28 .…”