Abstract

The effects of ionic liquids (ILs) on proteins are well known and widely studied for various applications [1]. Compared to conventional solvents, ILs exhibit several advantageous properties, including negligible volatility, moderate conductivity and viscosity, liquid state over a wide temperature range, excellent solvation ability, and high thermal stability. A diverse range of interactions promotes the self-organization of ILs into complex structures. Biocompatible ionic liquids retain these properties while maintaining manageable toxicity levels, making them attractive for pharmaceutical applications, such as reducing the need for additives in vaccine formulations and simplifying handling logistics [2]. In this work, Hamiltonian Replica Exchange Molecular Dynamics simulations were employed to investigate the effect of concentration changes in choline-derived ionic liquids on the stability and structure of Plasmodium falciparum proteins. Additionally, solvation structures were characterized using Kirkwood–Buff solution theory [3]. The proteins analyzed—AMA1, MSP1, and MSP2, associated with P. falciparum, the primary causative agent of malaria—were studied in aqueous solutions of choline geranate (CAGE), choline lactate (CAL), and choline chloride (CACL) at concentrations of 0.125 M, 0.250 M, 0.5 M, 1.0 M, and 2.0 M. Kirkwood–Buff integrals (KBIs) revealed distinct behaviors among the three ionic liquids. Choline chloride behaves similarly to a conventional salt, showing minimal variation in solvation effects with concentration and acting as a preferentially excluded, protein-stabilizing cosolvent. In contrast, CAGE and CAL exhibit behavior typical of biocompatible ionic liquids: their affinities, as quantified by KBIs, vary with concentration. Although they are not completely excluded from the protein surface, their electrostatic interactions—modulated by the nature of both cations and anions—contribute to protein stabilization at optimal concentrations. Furthermore, the KBIs of the ions forming CAGE converge to different values, indicating distinct local structuring. The choline cation also exhibits complex behavior, with variations in accumulation at different distances from the protein surface. Structural analyses based on RMSD and RMSF indicate that lower IL concentrations are sufficient to maintain protein structures in their functional conformations. This suggests the existence of an optimal concentration range in which epitope structures remain preserved in the presence of cosolvents. In AMA1, key residues in disordered regions—K489, R503, K508, and R512—were stabilized. The spatial distributions of relevant functional groups of each cosolvent were further characterized using Minimum Distance Distribution Functions and KBIs. These approaches enabled a molecular-level understanding of protein–cosolvent interactions and their impact on protein stability.

References

[1] V. Piccoli, L. Martínez, J Phys Chem B, 129, 6765-6776 (2025).

[2] Y-L. Wang, et al, Chem Rev, 120, 5798–5877 (2020).

[3] IL. Shulgin, E. Ruckenstein, J Phys Chem B, 110, 12707–12713 (2006).