Introducing Protein Crystallization in Hydrated Deep Eutectic Solvents

Supramolecular structure and properties of deep eutecticsolvents (DESs) are known to be highly affected by the addition of water,and their use as solvents for poorly water-soluble macromolecules is beingactively investigated. We report the first experimental investigation ofprotein crystallization in DESs. Different hydrophilic and hydrophobiceutectic mixtures, hydrated at different levels, have been screened ascrystallization media. DESs were added to the solution containing theprecipitant and the buffer required to crystallize three test proteins, andwe observed that the volume ratio between DES and the correspondingsolution is a key parameter for the crystallization process. Successfulcrystallization was achieved for the hen-egg white lysozyme when usingcholine chloride:urea, choline chloride:glycerol, and choline chloride:-glutamic acid eutectic mixtures at a 1:2 molar ratio. High-resolution X-ray diffraction experiments disclosed the possibility to studythe intriguing supramolecular network of the molecular complexes formed between protein and DES in the presence of watermolecules. Individual DES components have been found to systematically occupy specific protein sites populated by solvent-exposedaromatic residues. Weak interactions between DES components, possibly mediated by water molecules, which resulted in beingfrozen in the ordered solvent surrounding the protein units in the crystal lattice, were reconstructed at atomic resolution. DESs werefound to have a negligible effect on the protein conformation and its flexibility in the solid state. On the other hand, DESs greatlyreduced solvent evaporation from the crystallization drop, thereby increasing the dissolution time of the protein crystals. Finally,DESs were found to serve as local modulators of the ordered solvent, and this resulted in a significant change of the proteinsolubility. In addition, we found that protein crystallization was sped up by tuning DES hydration. This enables the employment ofthese environmentally responsible solvents to improve biotechnological processes at the industrial level.