Dehydration entropy drives liquid-liquid phase separation by molecular crowding

Abstract

Complex coacervation driven liquid-liquid phase separation (LLPS) of biopolymers has been attracting attention as a novel phase in living cells. Studies of LLPS in this context are typically of proteins harboring chemical and structural complexity, leaving unclear which properties are fundamental to complex coacervation versus protein-specific. This study focuses on the role of polyethylene glycol (PEG)-a widely used molecular crowder-in LLPS. Significantly, entropy-driven LLPS is recapitulated with charged polymers lacking hydrophobicity and sequence complexity, and its propensity dramatically enhanced by PEG. Experimental and field-theoretic simulation results are consistent with PEG driving LLPS by dehydration of polymers, and show that PEG exerts its effect without partitioning into the dense coacervate phase. It is then up to biology to impose additional variations of functional significance to the LLPS of biological systems.

Fig. 1. Schematic and general properties of εPL-HA complex coacervation.

a Schematic of εPL-HA complex coacervation (0.1 M, pH 5.0 sodium acetate, additional 40 mM NaCl) with and without PEG. The microdroplet coacervate suspension was observed by an optical microscope, and the macrophase separation occurred after centrifugation. b Turbidity (at 500 nm) of the suspension and (c) coacervate volume fraction as a function of additional NaCl in 0.1 M sodium acetate (pH 5.0).

Fig. 2. ¹H PFG NMR full and zoom-in (50-fold amplification) spectra.

The spectrum from macro-separated a dilute phase (without PEG), b dilute phase (with PEG), c dense phase (without PEG), and d dense phase (with PEG). Complex coacervation occurred in 0.1 M, pH 5.0 sodium acetate buffer, with no additional NaCl.

Fig. 3. Illustration of the εPL-HA complex coacervation with and without PEG.

PEG did not directly participate in the complex coacervation but increased the coacervate yield and density.

Fig. 4. Microscope images of the εPL-HA microdroplet suspension.

a Without PEG and b with 10% (w/v) PEG at specific temperature (under 0.1 M, pH 5.0 sodium acetate, additional 60 mM NaCl).

Fig. 5. Temperature dependences of the εPL-HA complex coacervation.

a The absorbance of the εPL-HA microdroplet suspension (0.1 M, pH 5.0 sodium acetate, additional 60 mM NaCl) with different PEG% (w/v) in a function of temperature. b Photo of macrophase separation after 2 h incubation of the micro-separated coacervate suspension with 10% PEG at 25 and 70 °C. c Time effect on the εPL-HA complex coacervation in the presence of PEG at 25, 70 and 90 °C.

Fig. 6. Water dynamics.

a Diffusion coefficients of interstitial water in the macro-separated phases (0.1 M, pH 5.0 sodium acetate, without additional NaCl) were measured by PFG-NMR. Error bars indicate the standard deviation. b Diffusion coefficients of hydration water were obtained by Overhauser-DNP. Error bars with the 5% deviation on the means have been marked to show the effect of experimental uncertainty.

Fig. 7. FTS results.

The density distribution of a polyelectrolytes and b PEG in a two-phase solution in which a dilute supernatant and dense coacervate phase of polyelectrolytes coexist. The density distribution (normalized histogram) of c polyelectrolytes and d PEG. In this system, we set the total density of polyelectrolytes and PEG at ρ_Pb³ = 1.5 and ρ_Cb³ = 15. The black, orange and blue curves show the density distribution of polyelectrolytes without PEG and with PEG at cross-excluded volume strengths v_PC = 0.034b³ and 0.068b³, respectively.

Fig. 8. Viscosity and interfacial tension.

a MSD for tracer beads in coacervate with and without PEG. Viscosity was estimated from the Stokes−Einstein relation. b Coacervate droplet coalescence event in the presence of PEG. The coalescence event was well fit by an exponential decay, and this determined the relaxation time t. c The linear fit of the relaxation time with respect to the radius of the coacervate. The interfacial tension was calculated from the slope. Coacervate with and without PEG was formed at 0.1 M sodium acetate (pH 5.0), without additional NaCl.

Fig. 9. Fluorescence images of the εPL-HA coacervate droplets formed with and without PEG (0.1 M, pH 5.0 sodium acetate, without additional NaCl).

Two different bleaching geometries were used, and the white circles indicated the bleaching area. a In partial droplet bleaching (radius ~1.5 μm), droplet images represent the droplet before bleaching and right after bleaching. b In entire droplet bleaching (radius ~16 μm), droplet images represent the droplet before bleaching, right after bleaching, and after 120 s of bleaching. c The recovery curves after entire droplet bleaching. Blue: without PEG, Red: with PEG.