Abstract
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 ([Ch][Glu]) 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 ([Bmim][dca]) 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 [Ch][Glu] the effect is primarily anion driven, while for [Bmim][dca] 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 [Ch][Glu] slows unfolding, whereas [Bmim][dca] 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.
