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[Display omitted] Ions are important to modulate protein properties, including solubility and stability, through specific ion effects. Ionic liquids (ILs) are designer salts with versatile ion combinations with great potential to control protein properties. Although protein-ion binding of common metals is well-known, the IL effect on proteins is not well understood. Here, we employ the model protein lysozyme in dilute and concentrated IL solutions to determine the specific ion binding effect on protein phase behaviour, activity, size and conformational change, aggregation and intermolecular interactions. A combination of spectroscopic techniques, activity assays, small-angle X-ray scattering, and crystallography highlights that ILs, particularly their anions, bind to specific sites in the protein hydration layer via polar contacts on charged, polar and aromatic residues. The specific ion binding can induce more flexible loop regions in lysozyme, while the ion binding in the bulk phase can be more dynamic in solution. Overall, the protein behaviour in ILs depends on the net effect of nonspecific interactions and specific ion binding. Compared to formate, the nitrate anion induced high protein solubility, low activity, elongated shape and aggregation, which is largely owing to its higher propensity for ion binding. These findings provide new insights into protein-IL binding interactions and using ILs to modulate protein properties. [ABSTRACT FROM AUTHOR]

[Display omitted] Globular proteins are well-folded model proteins, where ions can substantially influence their structure and aggregation. Ionic liquids (ILs) are salts in the liquid state with versatile ion combinations. Understanding the IL effect on protein behavior remains a major challenge. Here, we employed small angle X-ray scattering to investigate the effect of aqueous ILs on the structure and aggregation of globular proteins, namely, hen egg white lysozyme (Lys), human lysozyme (HLys), myoglobin (Mb), β-lactoglobulin (βLg), trypsin (Tryp) and superfolder green fluorescent protein (sfGFP). The ILs contain ammonium-based cations paired with the mesylate, acetate or nitrate anion. Results showed that only Lys was monomeric, whereas the other proteins formed small or large aggregates in buffer. Solutions with over 17 mol% IL resulted in significant changes in the protein structure and aggregation. The Lys structure was expanded at 1 mol% but compact at 17 mol% with structural changes in loop regions. HLys formed small aggregates, with the IL effect similar to Lys. Mb and βLg mostly had distinct monomer and dimer distributions depending on IL type and IL concentration. Complex aggregation was noted for Tryp and sfGFP. While the anion had the largest ion effect, changing the cation also induced the structural expansion and protein aggregation. [ABSTRACT FROM AUTHOR]

Solvents that support protein functionality are important for biochemical applications, and new solvents are required. Here we employ FTIR and fluorescence spectroscopies, small angle X-ray scattering (SAXS) and X-ray crystallography to understand conformational changes of lysozyme with ionic liquids (ILs) added. Spectroscopic techniques identified that the secondary structure of lysozyme was maintained at the lower IL concentrations of 1 and 5 mol%, though the Tryptophan environment was significantly altered with nitrate-based ILs present. SAXS experiments indicated that the radius of gyration of lysozyme increased with 1 mol% IL present, and then decreased with increasing IL concentrations. The tertiary structure, particularly the loop regions, changed as a function of IL concentration, and this depended on the IL type. The crystallographic structure of lysozyme with the IL of ethylammonium nitrate present confirmed the loop region was extended, and identified three specific binding sites with nitrate ions, and that the positively charged areas were IL sensitive regions. This work provides a detailed understanding of lysozyme conformational changes in the presence of ILs. This approach can be extended to other functionally-important proteins. [ABSTRACT FROM AUTHOR]