Regulation on liquid-liquid phase separation of Bovine γ-globulin by polyphenols

Liquid-liquid phase separation (LLPS) for proteins can provide specific local physiological environments for some crucial biological activities.Elucidating its regulation has a potential implication for curing the related diseases of LLPS.Our results show that epicatechin gallate (GCG), a polyphenol, can promote LLPS of bovine -globulin in the binary solution.Specifically, we found that a small amount of GCG (1 mM) induces LLPS in a homogeneous solution of 4% W/V PEG-1000 and 20 mgmL-1 -globulin below the critical concentration for the phase transition.This suggests that adding polyphenols reduces the critical concentrations of PEG and proteins for LLPS, thereby expanding the LLPS region in the phase diagram.However, we observed that the polyphenol itself only induces -globulin solution to precipitate, rather than LLPS in the absence of PEG.Furthermore, when we fixed the concentration of polyphenol and protein, we observed the reentrant of liquid-liquid phase separation in the mixed solution with increasing PEG concentration.We concluded that the reentrant of phase separation is the result of the combined effects of the crowding effect of polyethylene glycol and the hydrophobic effect provided by GCG through the hydrophobic disruption regulation.We investigated the interaction between protein and polyphenol using fluorescence spectroscopy.We constructed a schematic model to elucidate the regulation mechanism of LLPS of polyphenol and PEG.