Deep Eutectic Solvents A new opportunity for protein crystallization

Deep Eutectic Solvents (DESs) are mixtures of Lewis or Brønsted acids and bases showing a melting point much lower than that of the individual components.1 Today, the interest toward DESs is related to their exploitation in several industrial applications2 and to the role that such mixtures appear to have in the transport of macromolecules of diverse polarities inside the plants.3 Ionic liquids share several physicochemical properties (e.g., thermal stability, conductivity, negligible vapor pressures, and easy recycling) with eutectic mixtures and are already exploited as additives in protein crystallization.4 This prompted us to investigate the yet unexplored opportunity to use DESs in protein crystallization.Here, we show, for the first time, that protein molecules are able to crystallize in the presence of DESs.5 Our crystallization experiments have shown that hen-egg white lysozyme crystallizes according to the DES components and the hydration level (Figure 1). Successful crystallization have been achieved by using choline chloride:urea, choline chloride:glycerol, and choline chloride:glutamic acid eutectic mixtures at a 1:2 molar ratio. X-ray diffraction experiments have provided details about the binding sites of DES components on lysozyme protein surface and precise information about the intriguing non-covalent bond network by which protein, DES components and water molecules interact each other. DESs appear to have negligible effects on protein conformation and significantly reduce solvent evaporation from the crystallization drop, a key property to increase the dissolution time of the protein crystals. Moreover, DESs could tune protein solubility because it appears to affect hydration shell of the protein.Our results prove that high-quality protein crystals can be obtained in the presence of DESs and that such crystals can be used to get precise structural information about the interaction between DESs and proteins, which is still lacking in the literature. Moreover, the ability of DESs to protect protein crystals from their rapid dissolution (which has been observed in this work) could be exploited in biotechnological applications involving enzymes in crystalline form.