Fluorescence quenching by ionic liquid as a potent tool to study protein unfolding intermediates

Abstract

This study aimed to utilize the fluorescence quenching property of imidazolium-based ionic liquid to characterize the unfolding intermediates of proteins along the denaturation pathway. Low concentrations (<10 mM) of imidazolium-based ionic liquid are known to quench tryptophan fluorescence without affecting the protein structure; we have successfully utilized this property to evaluate the tertiary structure of intermediates in the GdmCl-induced denaturation pathway of bovine serum albumin (BSA). BSA is a globular protein with 583 amino acids and two intrinsic tryptophans. The Trp-fluorescence quenching experiments have been supplemented by measurements of Trp fluorescence lifetime, energy transfer efficiency between tryptophan and a nonpolar acceptor, 1,6-diphenyl-1,3,5-hexatriene (DPH), near- and far-UV circular dichroism measurements. Overall, our results revealed the denaturation of BSA to proceed via two parallel pathways in presence of low concentrations of GdmCl (≤1.5 M), wherein a dry molten globule was in equilibrium with the native conformation. At higher GdmCl concentrations, both the conformations entered the unfolding pathway, assuming a partially unfolded intermediate at 1.85 M GdmCl, which subsequently unfolded to form the denatured protein at yet higher GdmCl concentrations. Based on the fluorescence quenching property of imidazolium-based ionic liquid, we proposed a plausible mechanism of GdmCl-induced unfolding of BSA, which is in conformance with previous reports, although with more details. Therefore, the study suggested the potential of imidazolium-based ionic liquid as a reliable tool to investigate the intermediate conformations of BSA in the GdmCl-induced denaturation pathway, and proposed a mechanism of unfolding of BSA.