Tam GREAVES1, Qi(Hank) HAN1, Kate SMITH2,3, Calum DRUMMOND1, Connie DARMANIN4
1RMIT University, Melbourne, Australia
2Australian Synchrotron, Australian Nuclear Science and Technology Organisation, Clayton, Australia
3Swiss Light Source, Villigen, Switzerland
4La Trobe University, Bundoora, Australia
Buffered aqueous salt solutions are used as solvents for proteins, but these do not sufficiently control protein solubility and stability, which adversely affects protein activity, folding-unfolding transitions, aggregation and crystallisation. Therefore, there is a need for new solvents which can control protein and biomolecule solubility and stability. Protic ionic liquids (PILs) are cost efficient “designer” solvents which can be tailored to have properties suitable for a broad range of applications. Certain aqueous PIL solutions have beneficial properties, including stabilising biomolecules, suppressing aggregation and enhancing protein crystal growth. However, there is a lack of understanding about the interactions present, which prevents solvent design for specific protein applications.
This work presents the solvent effect of ILs on different globular proteins including lysozyme, green fluorescent protein, β-lactoglobulin, myoglobin, trypsin and pepsin. We employ small angle X-ray scattering (SAXS) to investigate the effect of a range of IL systems on the proteins using the SAXS/WAXS beamline at the Australian Synchrotron.[2, 3] The protein properties such as size, shape, conformational changes, and aggregation are discussed. The results show that the acetate anion can retain protein folding more than the mesylate anion. In addition, we use size exclusion chromatography (SEC)-SAXS to explore the protein aggregation and conformational changes to a high level of detail. We further employ modelling tools including CRYSOL, GASBOR and SREFLEX to reconstruct and model protein structure and understand the solvent effect on protein behaviours at an atomic level.
Furthermore, we have used Protein Crystallography at the Australian Synchrotron MX2 beamline to obtain a deeper understanding of ionic liquid-protein interactions.2 Specifically, we have identified conformational changes of the protein in solution due to changes in the ionic liquid chemical structure and/or concentrations. We have also identified the ion-binding sites of the ionic liquid solvated cations and anions. From these results we have clearly shown that the anion has significantly more interactions with the protein, and preferentially binds to positively charged and aromatic side chains, whereas few of the cations were identified in the solvation layer.
We have been able to relate properties of ionic liquid solutions to their ability to stabilise and maintain protein structure. This is significant progress towards being able to design ionic liquid solutions for specific proteins.
 Greaves, T. L.; Drummond, C. J., Protic ionic liquids: properties and applications. Chem. Rev. 2008, 108 (1), 206-237.
 Han, Q.; Smith, K. M.; Darmanin, C.; Ryan, T. M.; Drummond, C. J.; Greaves, T. L., Lysozyme conformational changes with ionic liquids: Spectroscopic, small angle x-ray scattering and crystallographic study. J. Colloid Interface Sci. 2021, 585, 433-443.
 Han, Q.; Binns, J.; Zhai, J.; Guo, X.; Ryan, T. M.; Drummond, C. J.; Greaves, T. L., Insights on lysozyme aggregation in protic ionic liquid solvents by using small angle x-ray scattering and high throughput screening. J. Mol. Liq. 2022, 15, 2430-2454.
 Han, Q.; Brown, S.; Drummond, C. J.; Greaves, T. L., Protein aggregation and crystallization with ionic liquids: insights into the influence of solvent properties. J. Colloid Interface Sci. 2022, 608, 1173-1190.