Unravelling the unfolding mechanism of human integrin linked kinase by GdmCl-induced denaturation.

Abstract Integrin-linked kinase (ILK) is a ubiquitously expressed Ser/Thr kinase which plays significant role in the cell-matrix interactions and growth factor signalling. In this study, guanidinium chloride (GdmCl)-induced unfolding of kinase domain of ILK (ILK 193–446 ) was carried out at pH 7.5 and 25 °C. Eventually, denaturation curves of mean residue ellipticity at 222 nm ([ θ ] 222 ) and fluorescence emission spectrum were analysed to estimate stability parameters. The optical properties maximum emission (λ max ) and difference absorption coefficient at 292 nm (Δ e 292 ) were analysed. The denaturation curve was measured only in the GdmCl molar concentration ranging 3.0–4.2 M because protein was aggregating below 3.0 M of GdmCl concentrations. The denaturation process of ILK 193–446 was found as reversible at [GdmCl] ≥ 3.0 M. Moreover, a coincidence of normalized denaturation curves of optical properties ([ θ ] 222 , Δ e 292 and λ max ) suggesting that GdmCl-induced denaturation of ILK 193–446 is a two-state process. In addition, 100 ns molecular dynamics simulations were performed to see the effects of GdmCl on the structure and stability of ILK 193–446 . Both the spectroscopic and molecular dynamics approaches provided clear insights into the stability and conformational properties of ILK 193–446.