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.

中文翻译：

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在水合深共晶溶剂中引入蛋白质结晶

众所周知，深共熔溶剂 (DESs) 的超分子结构和性质受添加水的影响很大，并且正在积极研究将其用作水溶性差的大分子的溶剂。我们报告了DESs中蛋白质结晶的首次实验研究。不同的亲水性和疏水性共晶混合物，以不同的水平水合，已被筛选为结晶介质。将 DES 添加到含有沉淀剂和结晶三种测试蛋白质所需的缓冲液的溶液中，我们观察到 DES 与相应溶液之间的体积比是结晶过程的关键参数。当使用氯化胆碱：尿素、氯化胆碱：甘油和氯化胆碱时，鸡蛋清溶菌酶成功结晶：摩尔比为 1:2 的谷氨酸共晶混合物。高分辨率 X 射线衍射实验揭示了在水分子存在下研究蛋白质和 DES 之间形成的分子复合物的有趣超分子网络的可能性。已发现单个 DES 组分系统地占据由暴露于溶剂的芳香族残基填充的特定蛋白质位点。DES 组分之间的弱相互作用，可能由水分子介导，导致在晶格中蛋白质单元周围的有序溶剂中冻结，以原子分辨率重建。发现 DES 对蛋白质构象及其在固态下的灵活性的影响可以忽略不计。另一方面，DESs 大大减少了结晶滴中的溶剂蒸发，从而增加蛋白质晶体的溶解时间。最后，发现 DES 作为有序溶剂的局部调节剂，这导致蛋白质溶解度的显着变化。此外，我们发现通过调节 DES 水合作用加速了蛋白质结晶。这使得使用这些对环境负责的溶剂能够在工业水平上改进生物技术过程。