Recovery of enzyme structure and activity following rehydration from ionic liquid

Long-term preservation of proteins at room temperature continues to be a major challenge. Towards using ionic liquids (ILs) to address this challenge, here we present a combination of experiments and simulations to investigate changes in lysozyme upon rehydration from IL mixtures using two imidazolium-based ILs (1-ethyl-3-methylimidazolium ethylsulfate, [EMIM][EtSO₄] and 1-ethyl-3-methylimidazolium diethylphosphate, [EMIM][Et₂PO₄]). Various spectroscopic experiments and molecular dynamics simulations are performed to ascertain the structure and activity of lysozyme. Circular dichroism spectroscopy confirms that lysozyme maintains its secondary structure upon rehydration, even after 295 days. Increasing the IL concentration decreases the activity of lysozyme and is ultimately quenched at sufficiently high IL concentrations, but the rehydration of lysozyme from high IL concentrations completely restores its activity. Such rehydration occurs in the most common lysozyme activity assay, but without careful attention, this effect on the IL concentration can be overlooked. From simulations we observe occupation of [EMIM⁺] ions near the vicinity of the active site and the ligand-lysozyme complex is less stable in the presence of ILs, which results in the reduction of lysozyme activity. Upon rehydration, fast leaving of [EMIM⁺] is observed and the availability of active site is restored. In addition, suppression of structural fluctuations is also observed when in high IL concentrations, which also explains the decrease of activity. This structure suppression is recovered after undergoing rehydration. The return of native protein structure and activity indicates that after rehydration lysozyme returns to its original state. Our results also suggest a simple route to protein recovery following extended storage.