Alginate-ionic liquid injectable hydrogels supporting protein crystallization

Protein crystallization is essential for determining 3D structures, yet innovative media are needed to enhance crystallization efficiency and optimize crystallography experiments. Injectable hydrogels offer a dynamic, tunable environment for protein crystallization while enabling continuous sample delivery for serial crystallography. In this study, we combined two emerging media and developed novel injectable alginate-ionic liquid hydrogels to support protein crystallization. Our results demonstrate that the ionic liquid ethylammonium nitrate promoted the formation of stable, continuous injection streams and slightly decreased the viscoelastic properties and pore size of the alginate hydrogels. In-situ lysozyme crystallization was successfully conducted in hydrogels containing 10 wt% alginate and 5 wt% ethylammonium nitrate. Small-angle X-ray scattering revealed that the Bragg peaks of the lysozyme crystals in hydrogels were consistent with those in solutions, indicating stable crystal formation within the hydrogels and minimal interaction between alginate and protein. Furthermore, analysis of lysozyme crystallized in different ionic liquids and salts showed that nitrate ions favor the monoclinic lattice, with cation variation altering the crystal packing. These findings offer new insights into developing hydrogel-based media for protein crystallization by integrating ionic liquids and alginate, advancing our understanding of crystal polymorphism and the protein crystallization process.