Aqueous BSA-PEG solutions under repeated temperature cycles: Liquid-liquid phase separation and properties of the...

Liquid-liquid phase separation (LLPS) and biomolecular condensates formation in crowded bio-milieus are of paramount importance for cell compartmentalization. A topic of biophysics relevance is the responsiveness of the condensates to environmental stimuli. Here, we investigated solutions of bovine serum albumin (BSA) and polyethylene glycol of average molecular weight of 5000 Da (PEG5k) subject up to n_c = 7 consecutive cooling cycles. The LLPS and condensate formation were assessed by temperature-dependent optical density measurements at 400 nm (OD₄₀₀) and by microscopy observations, respectively. Insight into the dynamics of the homogeneous and condensed phases of BSA/PEG5k/buffer solutions was obtained by electron paramagnetic resonance spectroscopy. Freshly prepared ternary solutions are characterized by high OD₄₀₀ over the whole temperature range and protein droplets with diameters from few to tens of micrometers. BSA in PEG5k containing solutions is more densely packed and exhibits restricted dynamics with respect to the PEG5k-free protein. Well-defined LLPS with upper critical solution temperature profiles are recorded in BSA/PEG5k/buffer solutions for n_c ≥ 2. Homogeneous solutions, with nearly zero optical density and absence of droplets, convert into the condensed state, with high optical density and presence of droplets, on decreasing the temperature. The LLPS temperature progressively decreases from approximately 35 °C at n_c = 2 to approximately 21 °C at n_c = 7, whereas the morphology, packing and dynamics of the droplets are slightly affected by n_c. Moreover, coacervates in the dense and lean phases show similar microscopic and dynamic features. The overall findings highlight the effectiveness of temperature stimuli in modulating the LLPS in protein solutions.