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 [C8mim][Cl], and 1-decyl-3-methylimidazolium chloride [C10mim][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 [C8mim]+ and [C10mim]+ cations promote the refolding of alkali-denatured h-cyt c. The IL having the [C10mim]+ cation efficiently refolded the alkali-denatured h-cyt c with the formation of the MG state, whereas the IL having the [C8mim]+ 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 [C10mim][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 [C10mim][Cl] was also confirmed, as some regular structure exists far below the CMC of IL. The overall results suggested that the [C10mim]+ cation bound to the unfolded h-cyt c triggers its refolding by electrostatic and hydrophobic interactions that stabilize the MG state.