Title
Unravelling the unfolding mechanism of human integrin linked kinase by GdmCl-induced denaturation
Author
Sunayana Begum Syed, Faez Iqbal Khan, Sabab Hasan Khan, Saurabha Srivastava, Gulam Mustafa Hasan, Kevin A. Lobb, Asimul Islam, Md. Imtaiyaz Hassan, Faizan Ahmad
Year
2018
Journal
International Journal of Biological Macromolecules
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 (ILK193–446) 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 (λmax) and difference absorption coefficient at 292 nm (Δε292) 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 ILK193–446 was found as reversible at [GdmCl] ≥ 3.0 M. Moreover, a coincidence of normalized denaturation curves of optical properties ([θ]222, Δε292 and λmax) suggesting that GdmCl-induced denaturation of ILK193–446 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 ILK193–446. Both the spectroscopic and molecular dynamics approaches provided clear insights into the stability and conformational properties of ILK193–446.
Instrument
J-1500
Keywords
Circular dichroism, Secondary structure, Protein denaturation, Chemical stability, Thermodynamics, Protein folding, Biochemistry