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.