NMR reveals specific remodelling of protein folding landscapes in ionic liquids

Abstract Ionic liquids (ILs) are designer solvents with tunable properties that have emerged as powerful cosolutes in protein science and biotechnology. While ILs are known to stabilize or destabilize proteins, their molecular mechanisms remain poorly defined, particularly regarding effects on the unfolded ensemble. Here, we use the metastable SH3 domain of the Drosophila adaptor protein Drk, which coexists in a slow two-state equilibrium between folded and unfolded states, as a model to dissect how ILs modulate protein conformational equilibria. Using nuclear magnetic resonance (NMR) spectroscopy, we show that cholinium glutamate ([Ch][Glu]) stabilizes the folded state by preferential exclusion from the protein surface, raising the barrier to unfolding in a manner reminiscent of osmolytes and crowding agents. In contrast, 1-butyl-3-methylimidazolium dicyanamide ([Bmim][dca]) shifts the equilibrium toward the unfolded state, not by destabilizing the native fold, but by stabilizing a compact, non-native α-helical conformation within the unfolded ensemble. Notably, this concentration-dependent mechanism differs from traditional denaturants such as urea or guanidinium chloride, which promote random-coil unfolded states. Kinetic measurements further reveal that [Ch][Glu] slows unfolding, whereas [Bmim][dca] slows folding, indicating that the two ILs reshape the folding landscape in opposite directions. These findings challenge the prevailing view that cosolutes act primarily on the folded state and establish unfolded-state stabilization as a critical determinant of protein stability. More broadly, this work provides a molecular framework for understanding how ILs reshape protein folding landscapes and offers design principles for tailoring ILs as stabilizers or destabilizers in biotechnology and therapeutic applications. Significance Statement Proteins exist in a delicate balance between folded and unfolded states, and cosolutes such as salts are generally viewed as acting only on the folded state. Here, we show that ionic liquids (ILs) are able to reshape this balance and uniquely modulate both folded and unfolded protein states through distinct molecular mechanisms. These results challenge the conventional view of protein stabilization/destabilization and demonstrate that unfolded-state modulation is a critical determinant of protein behavior. By establishing principles for how ILs alter protein folding landscapes, this work provides a framework for designing ILs with tailored effects, with implications for biocatalysis, protein engineering, and therapeutic formulation. Competing Interest Statement The authors have declared no competing interest. Footnotes Competing Interest Statement: The authors declare no competing interest.