Quantifying the free energy landscapes of biomolecular condensation reveals the underlying physical mechanisms of phase separation

Abstract

Protein condensation, driven by phase separation in living cells, gives rise to the emergence of membraneless assemblies. This physiological behavior is sensitive to the temperature, pressure, concentration, and protein composition, governed by physical laws behind phase transition. Aberrant phase transitions can lead to irreversible pathological aggregates linked to aging and plenty of human diseases. Despite its significance, the intricate degrees of freedom, especially near critical transition temperature, have long hindered efforts to elucidate the underlying physical mechanisms. We reveal the underlying global and physical process of biomolecular condensation by establishing coarse-grained models and performing extensive thermodynamic calculations. Our results demonstrate that condensation proceeds via the formation of condensed droplets below the critical temperature, followed by droplet maturation accompanied by the emergence of multiple droplets. We quantify the free energy landscapes at multiple temperatures and show that biomolecular condensation is a weak first-order phase transition, consistent with classical theoretical models. Additional thermodynamic quantities, including heat capacity, titration curves, and affinities, are determined to assess the thermodynamic stability. Critically, we identify “nucleation seeds” or “hot spots” by analyzing the transition state ensemble, providing direct mechanistic insight for the rational design of biomolecules with tailored phase-separation characteristics.