Temperature and cosolute regulate the liquid-liquid phase separation in BSA solutions

Biomolecular condensates form in vivo in the crowded cellular environment and are favoured in macromolecules containing low structural complexity or large portions of disordered regions. The liquid-liquid phase separation (LLPS) of protein solutions, in which macromolecule-rich regions are separated from the aqueous solution, can also be observed in vitro under specific experimental conditions of temperature, pH, pressure and components concentration. In this study, we investigate the formation of LLPS of bovine serum albumin (BSA) induced by polyethylene glycol (PEG-5000) and temperature. The LLPS of BSA solutions and droplets formation were assessed and characterized by temperature dependent turbidity, optical microscopy and infrared spectroscopy experiments. The results show that the lower the PEG concentration, the lower the LLPS transition temperature of BSA solution. At PEG concentration of 10 % (w/v) the average diameter of BSA droplets is about 9 μm at 10 °C, decreases to about 3 μm at 20 °C and at higher temperature the droplets dissolve and a homogeneous phase is observed. The real time formation of the BSA droplets is also followed by ATR-FTIR kinetic experiments that allow an estimate of protein concentration in the droplets giving a value 50 times higher than the initial solution (100 μM). No variation of the protein secondary structure within the condensates compared to the homogeneous phase is evidenced.