Role of protein conformation and weak interactions on \u03b3-gliadin liquid-liquid phase separation

Line Sahli,Denis Renard,Adeline Boire,Véronique Solé-Jamault,Alexandre Giuliani

doi:10.1038/s41598-019-49745-2

Line Sahli, Denis Renard + Show 3 more

Open Access

https://doi.org/10.1038/s41598-019-49745-2

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Abstract

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.

Highlights

Liquid-liquid phase separation (LLPS) of disordered or partially disordered proteins emerges as a widespread phenomenon with broad implications for cell physiology[1,2,3]
We investigate the behavior of a purified storage model protein, γ-gliadin, starting from a mixed solvent where the protein is soluble to an aqueous solvent in order to be closer to physiological conditions found in PBs30. γ-gliadins comprise various isoforms denominated from their electrophoretic mobility[31,32]
We show the ability of γ44-gliadin to phase separates into dynamic liquid-like droplets or condensates even in the absence of ethanol

Summary

Introduction

Liquid-liquid phase separation (LLPS) of disordered or partially disordered proteins emerges as a widespread phenomenon with broad implications for cell physiology[1,2,3]. Wheat seed contains storage proteins, prolamins, which are synthetized and accumulated into the rough endoplasmic reticulum before being deposited into PBs18–20 These PBs correspond to highly dense spherical organelles, surrounded by a limiting membrane, able to fuse subsequently by coalescence[19,21,22]. 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 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]. We discuss the importance of gliadins condensates in the formation and regulation of PBs

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