Effect of a Good buffer on the fate of metastable protein-rich droplets near physiological composition

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Metastable protein-rich microdroplets are produced from liquid-liquid phase separation (LLPS) of protein aqueous solutions. These globules can be intermediates for the formation of other protein-rich phases. Lysozyme aqueous solutions undergo LLPS around 0 °C in the presence of NaCl near physiological conditions. Here, it is shown that insertion of small amounts of 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (HEPES, 0.1 M) as a second additive to lysozyme-NaCl-water solutions near physiological ionic strength (0.2 M) is an essential step for triggering conversion of protein-rich droplets into another phase. Specifically, LLPS induced by cooling reproducibly leads to a rapid and high-yield formation of compact tetragonal crystalline microparticles only in the presence of HEPES. These microcrystals exhibit small size (1-3 μm), narrow size distribution and guest-binding properties. The temperature-concentration phase diagram shows a characteristic topology with LLPS boundary metastable with respect to tetragonal microcrystals, which in turn become less stable than rod-shaped orthorhombic crystals above 40 °C. Interestingly, dynamic light scattering, hydrogen-ion titrations and isothermal titration calorimetry reveal that lysozyme-HEPES interactions were found to be weakly attractive and exothermic. Our findings indicate that additives of salting-in type can represent an important factor controlling the fate of metastable protein-rich microdroplets relevant to drug formulations, femtosecond crystallography, and potential implications in protein-driven cytoplasmic compartmentalization.