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Introducing protein crystallization in hydrated deep eutectic solvents

DOI: 10.1021/acssuschemeng.1c01230 DOI Help

Authors: Benny Danilo Belviso (Consiglio Nazionale delle Ricerche) , Filippo Maria Perna (Università degli Studi di Bari “Aldo Moro”) , Benedetta Carrozzini (Consiglio Nazionale delle Ricerche) , Massimo Trotta (Consiglio Nazionale delle Ricerche) , Vito Capriati (Università degli Studi di Bari “Aldo Moro”,) , Rocco Caliandro (Consiglio Nazionale delle Ricerche)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Sustainable Chemistry & Engineering

State: Published (Approved)
Published: June 2021
Diamond Proposal Number(s): 15832 , 21741

Abstract: 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.

Journal Keywords: deep eutectic solvents; lysozyme; protein crystallization; crystal structure; protein−DES interactions; crystal dissolution

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Engineering, Chemistry


Instruments: I03-Macromolecular Crystallography , I04-Macromolecular Crystallography

Added On: 17/06/2021 14:36

Discipline Tags:

Biotechnology Physical Chemistry Chemistry Structural biology Engineering & Technology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)