Effects of Ionic Liquids on Aqueous Urea Solutions: Insights into the Ionic Liquid-Assisted Protein Renaturation

Abstract

Ionic liquids (ILs) are designer solvents that find wide applications in various areas. Recently, ILs have been shown to induce the refolding of certain proteins that were previously denatured under the treatment of urea. A molecular-level understanding of the counteracting mechanism of ILs on urea-induced protein denaturation remains elusive. In this study, we employ atomistic molecular dynamics simulations to investigate the ternary urea–water–IL solution in comparison to the aqueous urea solution to understand how the presence of ILs can modulate the structure, energetics, and dynamics of urea–water solutions. Our results show that the ions of the IL used, ethylammonium nitrate (EAN), interact strongly with urea and disrupt the urea aggregates that were known to stabilize the unfolded state of the proteins. Results also suggest a disruption in urea–water interaction that releases more free water molecules in solution. We subsequently strengthened these findings by simulating a model peptide in the absence and presence of EAN, which showed broken versus intact secondary structure in urea solution. Analyses show that these changes were accomplished by the added IL, which enforced a gradual displacement of urea from the peptide surface by water. We propose that the ILs facilitate protein renaturation by breaking down the urea aggregates and increasing the amount of free water molecules around the protein.

Counteraction Effects of Ammonium-Based Ionic Liquids on Urea-Induced Denaturation of α-Lactalbumin: A Comprehensive Molecular Simulation Study

“…Such a counteraction is so significant that at relatively higher IL concentration (beyond 2 M), the protein conformations in the ternary solution (S5 and S6) become even more rigid than that in pure aqueous medium (S1). Our result corroborates well with several experimental and MD simulation studies, which demonstrated similar counteraction effects with other ILs. − …”

“…94,95 Urea molecules can also form protein−urea (PU) hydrogen bonds with a protein. 52 Importantly, nonuniform preferential binding propensities of urea and the IL (EAA) components with the protein as discussed in the present study are likely to modify the hydrogen bonding environment at the interface in binary urea−water and ternary urea−IL−water solutions. This in turn may shed further light on the microscopic origin behind the counteraction effects of the IL on urea-induced partial unfolding of a protein.…”

“…In this section, we attempt to explore the effects of binary urea–water and ternary urea–IL–water solutions on the conformational properties of the protein α-lactalbumin. This is done by computing the root-mean-square deviations (RMSD) of the protein non-hydrogen atoms in different solutions with respect to the reference crystal structure. , The RMSD data provide a critical parameter for first-hand information on the deviation of the simulated protein configurations with respect to its initial reference structure under different conditions. It also provides vital information on possible conformational oscillations or jumps exhibited by the protein among different states or sub-states.…”

“…It is known that the presence of a protein in an aqueous solution perturbs the regular water–water (WW) hydrogen bond network at the interface with the formation of protein–water (PW) hydrogen bonds. , It is reported in earlier studies that the presence of an IL in aqueous solution alters the PW hydrogen bond network at the interface with the formation of relatively stronger protein–IL (PIL) hydrogen bonds. , Urea molecules can also form protein–urea (PU) hydrogen bonds with a protein . Importantly, nonuniform preferential binding propensities of urea and the IL (EAA) components with the protein as discussed in the present study are likely to modify the hydrogen bonding environment at the interface in binary urea–water and ternary urea–IL–water solutions.…”

Virtual Issue on Protein Crowding and Stability

“…Closely related are studies of peptides and proteins in the presence of different osmolytes such as TMAO, − trehalose, ethylene glycol, and ethanol, as they capture different aspects of the stabilizing and destabilizing molecules that may be encountered in cellular environments. Equally relevant are a number of mostly simulation studies that explore peptide and protein conformations in the presence of ionic liquids, − an area of increased interest in itself, as the organic ions present in these solvents also have parallels in biological environments.…”