Reentrant Liquid-Liquid Phase Separation in Protein Solutions at Elevated Hydrostatic Pressures

We present results from small-angle x-ray scattering data on the effect of high pressure on the phase behavior of dense lysozyme solutions in the liquid-liquid phase separation region, and characterize the underlying intermolecular protein-protein interactions as a function of temperature and pressure in this region of phase space. A reentrant liquid-liquid phase separation region has been discovered at elevated pressures, which originates in the pressure dependence of the solvent-mediated protein-protein interactions.

Figure 1

Schematic temperature-concentration phase diagram of dense protein solutions. A transition to a (metastable) liquid-liquid phase separation region (LLPS) occurs at lower temperatures than the separation between the solution and solid (crystal) phase.

Figure 2

Measured effective structure factor $S_{\mathrm{eff}}(q)$ of a 20 wt.% lysozyme solution as a function of pressure at $T=16°\text{C}$. Black lines display the refinement of the data using the sticky hard sphere model, the red line shows the scattering intensity at $q=0.2{\text{nm}}^{-1}$ (see also Fig. 3(b)).

Figure 3

(a) Pressure dependence of the correlation length, $\xi$, for the 20.0 wt.% lysozyme solution at $p\mathrm{H}$ 7 and $T=16°\text{C}$. (b) Pressure dependence of the effective structure factor $S_{\mathrm{eff}}(q)$ at $q=0.2{\text{nm}}^{-1}$ for 8, 16, 20, and 26 °C (top to bottom) (see also Fig. 2). (c) Pressure dependence of the reduced second virial coefficient $b_2$ obtained from refinement of the SAXS data measured at 8, 16, 20, and $26°\text{C}$ (bottom to top) for a protein concentration of 20 wt.%. The gray area corresponds to solution conditions that exhibit LLPS.

Figure 4

Temperature dependence of the reduced second virial coefficient $b_2$ at pressures of 1 bar, 1 kbar, and 2.25 kbar. The protein concentration is 21.5 wt.%. Black lines indicate a linear fit to assess the LL phase boundary (circles in Fig. 5).

Figure 5

$p-T$ phase diagram of the LLPS of lysozyme for concentrations of 21.5, 20.0, and 18.5 wt.%. Low and high pressure (HP) areas of the LLPS are marked in gray. The phase boundaries are refined using a Gaussian function with a center at 1.45 kbar.