Probing the molecular interactions between cholinium-based ionic liquids and insulin aspart: A combined computational...

Despite extensive studies revealing the potential of cholinium-based ionic liquids (ILs) in protein stabilization, the nature of interaction between ILs' constituents and protein residues is not well understood. In this work, we used a combined computational and experimental approach to investigate the structural stability of a peptide hormone, insulin aspart (IA), in ILs containing a choline cation [Ch]⁺ and either dihydrogen phosphate ([Dhp]^-) or acetate ([Ace]^-) as anions. Although IA remained stable in both 1 M [Ch][Dhp] and 1 M [Ch][Ace], [Dhp]^- exhibited a much stronger stabilization effect than [Ace]^-. Both the hydrophilic ILs intensely hydrated IA and increased the number of water molecules in IA's solvation shell. Undeterred by the increased number of water molecules, the native state of IA's hydrophobic core was maintained in the presence of ILs. Importantly, our results reveal the importance of IL concentration in the medium which was critical to maintain a steady population of ions in the microenvironment of IA and to counteract the denaturing effect of water molecules. Through molecular docking, we confirm that the anions exert the dominant effect on the structure of IA, while [Ch]⁺ have the secondary influence. The computational results were validated using spectroscopic analyses (ultra-violet, fluorescence, and circular dichroism) along with dynamic light scattering measurements. The extended stability of IA at 30 °C for 28 days in 1 M [Ch][Dhp] and [Ch][Ace] demonstrated in this study reveals the possibility of stabilizing IA using cholinium-based ILs.