Thermodynamic forces from protein and water govern condensate formation of an intrinsically disordered protein domain

Abstract

Liquid-liquid phase separation (LLPS) can drive a multitude of cellular processes by compartmentalizing biological cells via the formation of dense liquid biomolecular condensates, which can function as membraneless organelles. Despite its importance, the molecular-level understanding of the underlying thermodynamics of this process remains incomplete. In this study, we use atomistic molecular dynamics simulations of the low complexity domain (LCD) of human fused in sarcoma (FUS) protein to investigate the contributions of water and protein molecules to the free energy changes that govern LLPS. Both protein and water components are found to have comparably sizeable thermodynamic contributions to the formation of FUS condensates. Moreover, we quantify the counteracting effects of water molecules that are released into the bulk upon condensate formation and the waters retained within the protein droplets. Among the various factors considered, solvation entropy and protein interaction enthalpy are identified as the most important contributions, while solvation enthalpy and protein entropy changes are smaller. These results provide detailed molecular insights on the intricate thermodynamic interplay between protein- and solvation-related forces underlying the formation of biomolecular condensates.

Fig. 1. Illustration of the process of protein condensate formation via liquid–liquid phase separation.

The zoomed-in views highlight the water molecules that are released into a bulk-like environment (top) outside the protein condensate and the ones that are retained inside the condensate (bottom).

Fig. 2. Snapshots of simulated systems.

Each of the simulated systems contains eight FUS-LCD molecules (residues 1–163 of human FUS).

Fig. 3. Hydration properties at different FUS-LCD concentrations.

a The number of water molecules in the protein hydration layer (defined here as water molecules within 0.3 nm of the protein surface). ${\mathrm{N}}_{\mathrm{W}}^{\mathrm{PHL}}$ is plotted against the protein concentration ρ. The asterisk (*) denotes that the numbers are normalized with respect to the number of hydration waters found for a single FUS-LCD protein in the high-dilution limit. b The number of water-water hydrogen bonds per water molecule is plotted as a function of ρ. c Tetrahedral order parameter distribution of water in 350 mg mL⁻¹ FUS-LCD solution (orange) compared to bulk water (blue). Data are presented as mean values ± standard deviation (SD) over three repeat simulations (the statistical errors in (b) are smaller than the size of the dots). Source data are provided as a Source Data file.

Fig. 4. Number of released and retained water molecules per protein.

The number of released molecules decreases (red dashed line) as the FUS-LCD concentration ρ approaches the condensate concentration of 350 mg mL⁻¹, where every protein is solvated (on average) by 2064 water molecules (blue dashed line), which are referred to as the retained waters. Source data are provided as a Source Data file.

Fig. 5. Entropy of water as a function of protein concentration (ρ).

a Total entropy. The horizontal dashed line represents the bulk water value. b Decomposition of the total entropy (black curve) into translational (cyan) and rotational (magenta) contributions. The values in (b) were normalized with respect to bulk water. Data in (a, b) are presented as mean values ± standard deviation (SD) over three repeat simulations. Source data are provided as a Source Data file.

Fig. 6. Interaction energies between the different components as a function of protein concentration.

a Protein–protein interaction energy, including intra- and inter-protein contributions. b Protein-water interaction energy. c Water-water interaction energy (per water molecule). Data in (a–c) are presented as mean values ± standard deviation (SD) over three repeat simulations (the statistical errors in (b, c) are smaller than the size of the dots). Source data are provided as a Source Data file.

Fig. 7. Changes of the solvation-related thermodynamic quantities.

The quantities plotted in (a–f) are indicated at the top of each panel. The dashed red, blue, gray lines denote the released, retained, and total water contributions, respectively. Data are presented as mean values ± standard deviation (SD) over three repeat simulations. Source data are provided as a Source Data file.

Fig. 8. Thermodynamic driving forces of FUS-LCD condensate formation.

The contributions from total solvation-free energy (ΔG_solv, brown dashed line), protein–protein interaction energy (ΔE_PP, blue dashed line), and protein conformational entropy (ΔS_P, orange dashed line) are plotted together with the resulting total free energy change upon formation of a FUS-LCD condensate (ΔG = ΔG_solv + ΔE_PP − TΔS_P, black dashed line), starting from a (hypothetical) homogeneous solution with protein concentration ρ. Data are presented as mean values ± standard deviation (SD) over three repeat simulations. Source data are provided as a Source Data file.