Dynamics of Ionic Liquid-Assisted Refolding of Denatured Cytochrome c: A Study of Preferential Interactions toward Renaturation.

In vitro refolding of denatured protein and the influence of the alkyl chain on the refolding of a protein were tested using long chain imidazolium chloride salts, 1-methyl-3-octylimidazolium chloride [C8 mim][Cl], and 1-decyl-3-methylimidazolium chloride [C10 mim][Cl]. The horse heart cytochrome c (h-cyt c) was denatured by urea and guanidinium hydrochloride (GdnHCl), as well as by base-induced denaturation at pH 13, to provide a broad overview of the overall refolding behavior. The variation in the alkyl chain of the ionic liquids (ILs) showed a profound effect on the refolding of denatured h-cyt c. The ligand-induced refolding was correlated to understand the mechanism of the conformational stability of proteins in aqueous solutions of ILs. The results showed that the long chain ILs having the [C8 mim]+ and [C10 mim]+ cations promote the refolding of alkali-denatured h-cyt c. The IL having the [C10 mim]+ cation efficiently refolded the alkali-denatured h-cyt c with the formation of the MG state, whereas the IL having the [C8 mim]+ cation, which is known to be compatible for protein stability, shows slight refolding and forms a different transition state. The lifetime results show successful refolding of alkaline-denatured h-cyt c by both of the ILs, however, more refolding was observed in the case of [C10 mim][Cl], and this was correlated with the fast and medium lifetimes (τ1 and τ2 ) obtained, which show an increase accompanied by an increase in secondary structure. The hydrophobic interactions plays an important role in the refolding of chemically and alkali-denatured h-cyt c by long chain imidazolium ILs. The formation of the MG state by [C10 mim][Cl] was also confirmed, as some regular structure exists far below the CMC of IL. The overall results suggested that the [C10 mim]+ cation bound to the unfolded h-cyt c triggers its refolding by electrostatic and hydrophobic interactions that stabilize the MG state.