Investigation of guanidinium chloride-induced unfolding pathway of sphingosine kinase 1
Preeti Gupta, Faez Iqbal Khan, Dilkash Ambreen, Dakun Lai, Mohamed F. Alajmi, Afzal Hussain, Asimul Islam, Faizan Ahmad, Md. Imtaiyaz Hassan
International Journal of Biological Macromolecules
Sphingosine kinase 1 (SphK1) is a lipid kinase which plays vital role in the regulation of varieties of biological processes including, cell growth, apoptosis and mitogenesis. In the present study, we investigated the guanidinium chloride (GdmCl)-induced denaturation of SphK1 at pH 8.0 and 25 °C using two different spectroscopic probes, i.e., mean residue ellipticity at 222 nm ([θ]222) and fluorescence emission maxima (λmax). A significant overlap between the transition curves obtained from both the spectral properties indicate that GdmCl-induced unfolding of SphK1 follows two-state process i.e., Native (N) ⇌ Denatured (D) state. Interestingly, a visible protein aggregation was observed at low concentrations of GdmCl ([GdmCl] ≤ 1.5 M). The analysis of transition curves was done to estimate the thermodynamic parameters associated with the stability of SphK1. To complement our experimental findings, 100 ns molecular dynamics (MD) simulations were performed. Spectroscopic studies together with MD simulations provided mechanistic insights of unfolding pathway of SphK1 along with its stability parameters.
V-660, FP-6200, J-1500
Absorption, Quantitation, Circular dichroism, Protein folding, Secondary structure, Chemical stability, Thermodynamics, Protein denaturation, Aggregation, Fluorescence, Protein structure, Ligand binding, Biochemistry