Implications of Imidazolium-Based Ionic Liquids as Refolding Additives for Urea-Induced Denatured Serum Albumins

Abstract

One of the major challenges in protein stability is that proteins can easily unfold in the presence of denaturants like urea, which alters the native structure of proteins. There are numerous studies in which ionic liquids (ILs) act as promising biocompatible solvents (Bio-IL) for biomolecules. In this context, we present the refolding ability of biocompatible imidazolium-based ILs, 1-ethyl 3-methyl imidazolium ethyl sulfate [Emim][ESO₄] (IL-1) and 1-ethyl 3-methyl imidazolium chloride [Emim][Cl] (IL-2) against the chemically induced structural changes in bovine and human serum albumin (BSA and HSA). The work is substantiated with several spectroscopic, thermal and docking studies. In steady-state fluorescence spectroscopy, we observe that the emission intensity quenches for the protein in urea, which is reversible with the addition of ILs. Circular dichroism spectral studies reflect the reappearance of α helical content, which is a good indicator of the refolding ability of ILs. Further, thermal fluorescence studies showed that ILs have the ability to refold the urea-induced denatured protein at a higher temperature range only up to 7 M urea concentration; however, above 7 M urea concentration, IL somehow fails to refold the protein. The work is also supported by dynamic light scattering measurements, and the degree of BSA/HSA aggregation was reduced with the introduction of Bio-IL to the urea–BSA/urea–HSA system, ensuring the aggregate-free refolding. Furthermore, molecular docking studies were employed to probe the binding sites, and the results are well corroborated with the spectroscopic and thermal folding results. Therefore, through this paper, we aim to unravel the mechanistic intricacy of ILs using experimental and docking approaches. Overall, ILs act as recoiling medium for both native and unfolded (denatured by urea) BSA/HSA native structures.