Unveiling water ordering in liquid–liquid phase separation using bovine serum albumin-polyethylene glycol systems

Abstract

Liquid–liquid phase separation (LLPS) is a fundamental physicochemical process where a homogeneous liquid solution spontaneously separates into two distinct liquid phases. Initially studied in polymer science, LLPS has emerged as a crucial mechanism in various biological processes, particularly in the formation of membrane-less organelles within cells. These organelles are biomolecular condensates that compartmentalise biochemical reactions without relying on traditional lipid membranes. LLPS is driven by a balance of enthalpic and entropic contributions, with protein–protein and protein-solvent interactions playing a pivotal role. Environmental factors, including temperature, pH, and solute concentrations, critically influence these interactions and thus the phase separation process. In this study, we investigate the role of water dynamics in the regulation of LLPS processes using a binary system of Bovine Serum Albumin (BSA) and Polyethylene Glycol (PEG). Classical spectroscopic methods and fluorescence lifetime imaging microscopy (FLIM), combined with phasor plot analysis, are employed to probe the local environment within protein condensates. Central to this approach is the use of 6-acetyl-2-dimethylaminonaphthalene (ACDAN), a fluorescent dye renowned for its sensitivity to water dipolar relaxation and changes in solvent polarity. This research aims to deepen our understanding of LLPS, particularly the role of water, offering insights into cellular processes and potential therapeutic strategies for LLPS-related diseases.