Cholinium glutamate stabilizes the folded SH3 domain by slowing unfolding, while 1-butyl-3-methylimidazolium dicyanamide stabilizes unfolded non-native α-helices and slows folding.
Ionic liquids can reshape the protein folding landscape in opposite directions, providing molecular design principles for tuning protein behavior.
Ionic liquids (ILs) are tunable designer solvents that have emerged as powerful cosolutes in protein science and biotechnology. Although ILs are known to stabilize or destabilize proteins, their molecular mechanisms, particularly their effects on unfolded ensembles, remain poorly understood. Here, we use the metastable N-terminal Src homology 3 (SH3) domain of the Drosophila adapter protein Drk, which exists in a slow two-state equilibrium between folded and unfolded conformations, to investigate how aqueous IL solutions modulate protein conformational equilibria. Using site-resolved nuclear magnetic resonance spectroscopy, complemented by thermodynamic and kinetic analyses, we show that cholinium glutamate (ChGlu) stabilizes the folded state through preferential exclusion from the protein surface, raising the barrier to unfolding via entropy-dominated, crowding-like effects. In contrast, 1-butyl-3-methylimidazolium dicyanamide (Bmimdca) shifts the equilibrium toward the unfolded state primarily by stabilization of a non-native α-helical conformation within the unfolded ensemble, with additional contributions from perturbation of the folded state. Comparisons with the corresponding simple salts reveal that for ChGlu the effect is primarily anion driven, while for Bmimdca there is a pronounced synergistic destabilization, indicating cooperative cation-anion interactions under aqueous conditions. This mechanism differs fundamentally from that of classical denaturants such as urea or guanidinium chloride, which promote random-coil unfolded states. Kinetic measurements show that ChGlu slows unfolding, whereas Bmimdca slows folding, demonstrating that these ILs reshape the protein folding landscape in opposite directions. Together, these findings establish unfolded-state stabilization as a critical determinant of protein stability and provide molecular design principles for tuning protein behavior using ILs.
Silva et al. (2026) studied this question. Cholinium glutamate stabilizes the folded SH3 domain by slowing unfolding, while 1-butyl-3-methylimidazolium dicyanamide stabilizes unfolded non-native α-helices and slows folding.