Molecular basis of structural stability of Irisin: A combined molecular dynamics simulation and in vitro studies for Urea-induced denaturation

Abstract

Irisin is a recently discovered myokine having important role in adipose tissue metabolism, neuronal functioning as well as bone metabolism. In this study, urea-induced denaturation was performed to determine the conformational stability and equilibrium folding intermediate of irisin. Understanding of the folding/unfolding pathway is fundamental to study the structure and stability of proteins. Herein, the urea-induced conformational changes in irisin were monitored employing spectroscopic techniques as well as all-atom molecular dynamics (MD) simulations. It was revealed that process of irisin unfolding is biphasic, i.e., native state (N) ↔ intermediate state (I) ↔ denatured state (D) with I state populated around 2.5 M urea. The stabilized intermediate state at 2.5 M urea was confirmed through binding of the fluorescence probe, 1-anilino-8-naphthalene sulfonate (ANS). Further, 250 ns MD simulation of irisin in urea was carried out to have detailed mechanistic insight. The assessment of structural parameters, free energy landscapes and native contacts indicated the presence of intermediate state during unfolding of irisin, which was characterised as molten globule state. The results of MD simulation showed a strong correlation with the in vitro observations establishing the occurrence of the intermediate population during the unfolding of irisin induced by urea.