The paper, "Revealing the complexity of ionic liquid-protein interactions through a multi-technique investigation" can be found here: https://www.nature.com/articles/s42004-020-0302-5

Nature’s catalysts, enzymes, are proteins which perform a vast number of chemical reactions with incredible efficiency, and the capacity to generate enantiomerically pure products. These biological molecules can perform many industrial relevant reactions; however, due to their low stability and solubility in non-aqueous environments, enzymes are used at significantly milder conditions than their synthesised counterparts. There is an evident trade-off between aqueous low-temperature and highly selective bioprocessing, where product purification can be costly, versus the use of organic solvents or high temperatures for high substrate solubility and reaction kinetics. If the limitations of enzymes and proteins can be removed and utilised in organic solvents than the tremendous potential of enzymes as catalysts can be realised.

Ionic liquids, organic salts with a melting temperature below 100 °C, are an emerging class of solvents due to their highly tunable nature allowing them to have extreme capability solubilising biopolymers.  In particular, the high thermal stability and negligible vapour pressure of ionic liquids make them extremely attractive for energy-efficient bioprocessing. Attempts have been made to increase enzyme thermal stability in non-aqueous environments through immobilisation on a solid support; however, this comes at a great expense of low biocatalytic activity.

Our group have had success in stabilising and solubilising proteins and enzymes in ionic liquids through surface engineering while maintaining protein architecture and function with extraordinary temperature stability (up to 137 °C)^1,2. Also, we have shown that proteins and enzymes can be dissolved in ionic liquids at concentrations as high as 50 wt%. However, ionic liquid-protein interactions are still not well comprehended due to the extensive selection of ionic liquids and protein structure heterogeneity.  The Hofmeister series is often invoked to clarify ionic liquid-protein interactions^3,4, which tend to be protein-dependent⁵ and often contradictory^6,7. This contradictory nature is manifested acutely in the activity of enzymes, where different behaviours may be observed depending on whether the biocatalyst is free, crosslinked, or immobilised. Thus, we set out to provide a framework to establish the best approach for the progress of the biocatalysis field.

Using GFP as an archetype, we investigate the interactions, structure, and stability in a range (acetate, chloride, triflate) of pyrrolidinium and imidazolium salts using small-angle X-ray scattering and multiple spectroscopic (UV/Vis, fluorescence, circular dichroism, and NMR) techniques. Using this holistic framework, we discover unprecedented information on site-specific ionic liquid-protein interactions and reveal that triflate (the least interacting anion) induces a contraction in the protein size that reduces the barrier to unfolding.

                 Figure 1: Ionic liquid-protein interactions probed in this study

In this paper, we demonstrate that protein stability requires a similar robust multi-technique analytical framework to avoid the pitfalls of single-technique approaches. This work provides detailed structural analysis between ionic liquids and proteins for the advancement of non-aqueous biocatalysis and the possibility for the removal of the ubiquitous nature of water in industrial biocatalysis.

 (1)         Brogan, A. P. S., Bui-Le, L., Hallett, J. P., Non-aqueous homogenous biocatalytic conversion of polysaccharides in ionic liquids using chemically modified glucosidase. Nat. Chem. 10, 859-865 (2018).

(2)         Brogan, A. P. S., Hallett, J. P., Solubilizing and stabilizing proteins in anhydrous ionic liquids through formation of protein- polymer surfactant nanoconstructs. J. Am. Chem. Soc. 138, 4494 (2016).

(3)         Yang, Z., Hofmeister effects: an explanation for the impact of ionic liquids on biocatalysis. J. Biotechnol. 144, 12-22 (2019).

(4)         Constatinescu, D., Herrmann, C., Weingartner, H., Patterns of protein unfolding and protein aggregation in ionic liquids. Phys. Chem. Chem. Phys. 12, 1756-1763 (2010).

(5)         Zhang, Y., Cremer, P. S., The inverse and direct Hofmeister series for lysozyme. Proc. Natl. Acad. Sci. U. S. A. 106, 15249-15253 (2009).

(6)         Naushad, M., ALOthman, Z. A., Khan, A. B., Ali, M., Effect of ionic liquid on activity, stability, and structure of enzymes: a review. Int. J. Biol. Macromol. 51, 555-560 (2012).

(7)         Gao, W.-W., Zhang, F.-X., Zhang, G.-X., Zhou, C.-H., Key factors affecting the activity and stability of enzymes in ionic liquids and novel applications in biocatalysis. Biochem. Eng. J. 99, 67-84 (2015).