Mechanistic insights into the urea-induced denaturation of kinase domain of human integrin linked kinase
- Authors: Syed, Sunayana B , Khan, Faez I , Khan, Sabab H , Srivastava, Saurabha , Hasan, Gulam M , Lobb, Kevin A , Islam, Asimul , Ahmad, Faizan , Hassan, M Imtaiyaz
- Date: 2018
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/448013 , vital:74691 , xlink:href="https://doi.org/10.1016/j.ijbiomac.2017.12.164"
- Description: Integrin-linked kinase (ILK), a ubiquitously expressed intracellular Ser/Thr protein kinase, plays a major role in the oncogenesis and tumour progression. The conformational stability and unfolding of kinase domain of ILK (ILK193–446) was examined in the presence of increasing concentrations of urea. The stability parameters of the urea-induced denaturation were measured by monitoring changes in [θ]222 (mean residue ellipticity at 222 nm), difference absorption coefficient at 292 nm (Δε292) and intrinsic fluorescence emission intensity at pH 7.5 and 25 ± 0.1 °C. The urea-induced denaturation was found to be reversible. The protein unfolding transition occurred in the urea concentration range 3.0–7.0 M. A coincidence of normalized denaturation curves of optical properties ([θ]222, Δε292 and λmax, the wavelength of maximum emission intensity) suggested that ureainduced denaturation of kinase domain of ILK is a two-state process. We further performed molecular dynamics simulation for 100 ns to see the effect of urea on structural stability of kinase domain of ILK at atomic level. Structural changes with increasing concentrations of urea were analysed, and we observed a significant increase in the root mean square deviation, root mean square fluctuations, solvent accessible surface area and radius of gyration. A correlation was observed between in vitro and in silico studies.
- Full Text:
- Date Issued: 2018
Unravelling the unfolding mechanism of human integrin linked kinase by GdmCl-induced denaturation
- Authors: Syed, Sunayana B , Khan, Faez I , Khan, Sabab H , Srivastava, Saurabha , Hasan, Gulam M , Lobb, Kevin A , Islam, Asimul , Hassan, M Imtaiyaz , Ahmad, Faizan
- Date: 2018
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/447220 , vital:74593 , xlink:href="https://doi.org/10.1016/j.ijbiomac.2018.06.025"
- Description: 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 ap proaches provided clear insights into the stability and conformational properties of ILK.
- Full Text:
- Date Issued: 2018