Investigation of the Formation and Aging of Albumin-Based Condensates

Abstract

Liquid–liquid phase separation is an important process in living cells allowing for partitioning the cytoplasm and triggering biochemical reactions with higher reactivity than in the dilute case. The resulting droplets are typically composed of highly concentrated components such as proteins, which condensate due to particular intermolecular interactions. Macromolecular crowding is one driving force responsible for protein condensation and can be leveraged in vitro to induce condensation of several types of molecules. Herein, the condensation of bovin serum albumin (BSA) is induced by an inert macromolecular crowder (i.e., poly(ethylene glycol), PEG), screening several conditions such as ionic strength, concentration of PEG or BSA, etc. A set of suitable conditions is identified and compared with the existing literature. BSA condensates are characterized via dynamic light scattering, native PAGE, or Fourier transform infrared spectroscopy to correlate the ability of BSA to form condensates, with structural rearrangements and oligomerization. It appears that condensation occurs immediately after PEG addition and that condensed BSA forms larger oligomers compared with conditions without PEG. BSA exhibits no major change of its secondary structure upon condensation, indicating that the protein remains in its native state. In addition, the stability of the resulting condensates is evaluated and compared with classical emulsions, showing that BSA condensates are subject to the classical law of settling droplets in the regime of low volume fractions.