Enhanced structural stability of insulin aspart in cholinium aminoate ionic liquids.

Cholinium aminoates [Ch][AA] have gained tremendous interest as a promising ionic liquid medium for the synthesis and storage of proteins. However, high alkalinity of [Ch][AA] limits its usage with pH-sensitive proteins. Here, we probed the structure, stability, and interactions of a highly unstable therapeutic protein, insulin aspart (IA), in a range of buffered [Ch][AA] (b-[Ch][AA]) using a combination of biophysical tools and in silico pipeline including ultraviolet-visible, fluorescence, and circular dichroism spectroscopies, dynamic light scattering measurements and molecular docking. b-[Ch][AA] used in the study differed in concentrations and their anionic counterparts. We reveal information on ion and residue specific solvent-protein interactions, demonstrating that the structural stability of IA was enhanced by a buffered cholinium prolinate. In comparison to the glycinate and alaninate anions, the hydrophilic prolinate anions established more hydrogen bonds with the residues of IA and provided a less polar environment that favours the preservation of IA in its active monomeric form, opening new opportunities for utilizing [Ch][AA] as storage medium.