Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

Designer Solvents for Protein Stability and Solubility (#351)

Stuart J Brown 1 , QI (Hank) Han 1 , Calum J Drummond 1 , Tamar L Greaves 1
  1. School of Science, RMIT University, Melbourne, VIC, Australia

Many proteins have low stability and solubility in aqueous buffer solutions. To remedy this, alternative solvents have been investigated. One class of solvents showing potential are ionic liquids (ILs) which due to their tailorable properties have been reported to increase protein activity1-2, solubility3 and long term and thermal stability4. However, the relationship between IL structure and maintaining protein solution structure is unknown. In this study, a plethora of techniques has been used to characterise protein stability, solubility and IL physicochemical properties.

A library of 48 ammonium based ILs have been synthesised for use with proteins. Neat ILs were extensively characterised and then used at 4 different concentrations. The model proteins of hen egg white lysozyme and human lysozyme were then studied across select IL and protein concentrations. Analysis of the data obtained at the Small Angle X-ray Scattering beamline at the Australian Synchrotron was used to investigate the specific ion effects, flexibility, aggregation and quantify the radius of gyration in IL-water mixtures. This non-trivial multi parameter analysis through the ATSAS5 platform enabled the visualisation of changes in the structure of both proteins. The resulting trends in protein variance showed that the structure of the ILs, particularly the anion and its functional groups significantly contributed to the stability of the proteins. Nitrate based ILs were seen to encourage unfolding of the protein at concentrations of 1-10 mol% while ILs with hydroxyl groups on either cation or anion enhanced protein stability, and were tolerated up to concentrations of 50 mol%. This study shows that individual ion species are not enough to dictate protein stability and so the ion pairing must be considered. By relating the observed changes in protein stability to solvent properties we hope to enable future studies into the development of specifically optimised solvents for protein stability and solubility.

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  2. 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. Journal of Colloid and Interface Science 2021, 585, 433-443.
  3. Byrne, N.; Angell, C. A., The solubility of hen lysozyme in ethylammonium nitrate/H2O mixtures and a novel approach to protein crystallization. Molecules 2010, 15 (2), 793-803.
  4. Fujita, K.; MacFarlane, D. R.; Forsyth, M.; Yoshizawa-Fujita, M.; Murata, K.; Nakamura, N.; Ohno, H., Solubility and Stability of Cytochrome c in Hydrated Ionic Liquids:  Effect of Oxo Acid Residues and Kosmotropicity. Biomacromolecules 2007, 8 (7), 2080-2086.
  5. Manalastas-Cantos, K.; Konarev, P. V.; Hajizadeh, N. R.; Kikhney, A. G.; Petoukhov, M. V.; Molodenskiy, D. S.; Panjkovich, A.; Mertens, H. D. T.; Gruzinov, A.; Borges, C.; Jeffries, C. M.; Svergun, D. I.; Franke, D., ATSAS 3.0: expanded functionality and new tools for small-angle scattering data analysis. Journal of Applied Crystallography 2021, 54 (1).