Boosting protein crystallization from liquid-liquid phase separation by increasing metastability gap.

• HEPES acts as a salting-out agent for lysozyme crystallization and a salting-in agent for liquid–liquid phase separation (LLPS) of lysozyme-NaCl aqueous solutions. • HEPES increases metastability gap between crystal solubility and LLPS. This explains observed enhancement of LLPS-induced protein crystallization. • The opposite effects of HEPES on LLPS and protein crystallization are consistent with ability of HEPES to stabilize lysozyme crystal lattice. • Changing HEPES protonation state has no qualitative effect on observed LLPS-induced protein crystallization. • Changing HEPES with other similar organic molecules significantly reduce LLPS-induced protein crystallization. • Changing NaCl with phosphate buffer as LLPS inducer significantly reduce LLPS-induced protein crystallization. Aqueous solutions of lysozyme undergo LLPS by cooling in the presence of NaCl (0.1–0.2 M) around neutral pH. This phase transition is metastable with respect to protein crystallization. Addition of 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (HEPES, 0.1 M) to these solutions triggers a rapid, reproducible and high-yield formation of tetragonal lysozyme microcrystals through LLPS even if NaCl concentration is quite low. In this work, we demonstrate that HEPES causes opposite thermodynamic effects on solubility and LLPS boundaries in the protein phase diagram, leading to broadening of metastability gap. This increases thermodynamic driving force for crystal nucleation inside protein-rich liquid phase. The observed enhancement of LLPS-based crystallization is independent of HEPES protonation state. However, crystallization output is dramatically reduced when HEPES is replaced by other organic molecules sharing same functional groups. We attribute HEPES effect on crystallization to its ability to bind lysozyme inside crystal. Replacing NaCl with phosphate buffer as LLPS inducer radically reduces crystallization, consistent with NaCl being a more effective agent for lysozyme crystallization. This work shows that protein crystallization from metastable LLPS can be successfully enhanced by combining an additive that induces LLPS with another additive that increases metastability gap. [ABSTRACT FROM AUTHOR]