Charge-controlled metastable liquid–liquid phase separation in protein solutions as a universal pathway towards crystallization

TLDR

The study demonstrates that multivalent metal ions can induce metastable liquid–liquid phase separation in protein solutions at room temperature and explores how this mechanism can guide protein crystallization. The authors induce LLPS with multivalent metal ions and quantify salt and protein partitioning between the two coexisting phases. Small‑angle X‑ray scattering reveals a short‑range attraction responsible for LLPS metastability, and an extended phase diagram with temperature, protein, and salt concentration aligns with a second‑virial‑coefficient criterion, offering an isothermal control framework for understanding and guiding protein crystallization.

Abstract

We demonstrate that a metastable liquid–liquid phase separation (LLPS) in protein aqueous solutions can be induced by multivalent metal ions at room temperature. We determine the salt and protein partitioning in the two coexisting phases. The structure factor obtained by small angle X-ray scattering provides direct evidence for a short-ranged attraction, which leads to the metastability of the LLPS. An extended phase diagram with three control parameters (temperature, protein and salt concentration) provides a conclusive physical picture consistent with a criterion for the second virial coefficient. The presented isothermal control mechanism of the phase behavior opens new perspectives for the understanding of controlled phase behavior in nature. Furthermore, we discuss the application of this framework in predicting and optimizing conditions for protein crystallization.