New Paper Online

Reshaping the folding landscape of the N-terminal Src homology 3 domain of the Drosophila adapter protein Drk with ionic liquids

Micael S Silva, Aldino Viegas, Philip O'Toole, Sara S Felix, Ângelo Miguel Figueiredo, Eurico J Cabrita

https://doi.org/10.1016/j.ijbiomac.2026.151173

This study investigates how ionic environments modulate the conformational equilibrium and stability of the SH3 domain, combining high-resolution NMR spectroscopy with biophysical analysis. The work provides residue-level insight into folding–unfolding equilibria and ligand interactions under physiologically relevant ionic conditions.

By integrating structural, thermodynamic and kinetic data, we demonstrate how specific ionic liquid components influence conformational exchange and binding behavior. These findings contribute to a more detailed molecular understanding of protein stability and modulation in complex environments.

Highlights

• ILs modulate protein folding via distinct, ion-specific molecular mechanisms.

• NMR reveals [Ch][Glu] stabilizes proteins through entropic excluded-volume effects.

• [Bmim][dca] stabilizes a compact non-native helix within the unfolded ensemble.

• Cation–anion synergy exceeds effects of individual ions on protein stability.

• ILs reshape protein energy landscapes beyond classical Hofmeister trends. 

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.

By identifying distinct pathways through which ILs stabilize or destabilize proteins, our study provides conceptual and mechanistic foundations for the rational design of ILs tailored to control protein behavior. Such principles hold relevance not only for biocatalysis and therapeutic protein formulation but also for understanding how non-native environments shape protein misfolding, aggregation, and disordered-state function.

Read more:

https://doi.org/10.1016/j.ijbiomac.2026.151173