Introducing Protein Crystallization in Hydrated Deep Eutectic Solvents

Supramolecular structure and properties of deep eutectic solvents (DESs) are known to be highly affected by the addition of water, and their use as solvents for poorly water-soluble macromolecules is being actively investigated. We report the first experimental investigation of protein crystallization in DESs. Different hydrophilic and hydrophobic eutectic mixtures, hydrated at different levels, have been screened as crystallization media. DESs were added to the solution containing the precipitant and the buffer required to crystallize three test proteins, and we observed that the volume ratio between DES and the corresponding solution is a key parameter for the crystallization process. Successful crystallization was achieved for the hen-egg white lysozyme when using choline 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 study the intriguing supramolecular network of the molecular complexes formed between protein and DES in the presence of water molecules. Individual DES components have been found to systematically occupy specific protein sites populated by solvent-exposed aromatic residues. Weak interactions between DES components, possibly mediated by water molecules, which resulted in being frozen in the ordered solvent surrounding the protein units in the crystal lattice, were reconstructed at atomic resolution. DESs were found to have a negligible effect on the protein conformation and its flexibility in the solid state. On the other hand, DESs greatly reduced 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 protein solubility. In addition, we found that protein crystallization was sped up by tuning DES hydration. This enables the employment of these environmentally responsible solvents to improve biotechnological processes at the industrial level.

Automated summary

The study revealed that the volume ratio between DES and the corresponding solution is a key parameter for protein crystallization, DES can prolong the dissolution time of protein crystals and modulate the ordered solvent, leading to a significant change in protein solubility.