Molecular basis of the structural stability of hemochromatosis factor E: A combined molecular dynamic simulation and GdmCl‐induced denaturation study

Abstract

Hemochromatosis factor E (HFE) is a member of class I MHC family and plays a significant role in the iron homeostasis. Denaturation of HFE induced by guanidinium chloride (GdmCl) was measured by monitoring changes in [θ]222 (mean residue ellipticity at 222 nm), intrinsic fluorescence emission intensity at 346 nm (F346 ) and the difference absorption coefficient at 287 nm (Δε287) at pH 8.0 and 25°C. Coincidence of denaturation curves of these optical properties suggests that GdmCl-induced denaturation (native (N) state ↔ denatured (D) state) is a two-state process. The GdmCl-induced denaturation was found reversible in the entire concentration range of the denaturant. All denaturation curves were analyzed for ΔGD0, Gibbs free energy change associated with the denaturation equilibrium (N state ↔ D state) in the absence of GdmCl, which is a measure of HFE stability. We further performed molecular dynamics simulation for 40 ns to see the effect of GdmCl on the structural stability of HFE. A well defined correlation was established between in vitro and in silico studies.

Effect of pH on the structure, function, and stability of human calcium/calmodulin-dependent protein kinase IV: combined spectroscopic and MD simulation studies

“…MD simulation in combinations with experimental studies help to get better insight into the conformation of protein in different environments (Anwer et al 2015;Idrees et al 2015;Khan et al 2016a;Khan et al 2016b;Khan et al 2016c;Naiyer et al 2015;Ubaid-Ullah et al 2014). After equilibration CAMKIV was simulated for 20 ns at different protonation state, its behavior is analyzed using the utilities of GROMACS.…”

Structural basis of urea-induced unfolding: Unraveling the folding pathway of hemochromatosis factor E

“…We previously reported the GdmCl-induced denaturation of HFE [33], in which we found that GdmCl-induced denaturation of HFE follows a classical two-state mechanism. To further confirm that, whether the stability parameters calculated from GdmCl-induced denaturation are the intrinsic property of HFE or depends upon the nature of denaturant used, we follow up this study and carried out urea-induced denaturation of HFE.…”

“…Here, we report the effect of urea on the structure and stability of HFE (residues 22-306). We previously reported the GdmCl-induced denaturation studies of HFE, and proposed that the unfolding of HFE in the presence of GdmCl is cooperative, occurring in a single step by a two-state mechanism [33]. But in the case of urea-induced denaturation of HFE, it is not a simple two-state process; the unfolding of HFE in urea shows a biphasic transition with a stable intermediate (X) state.…”

“…HFE protein was successfully cloned, expressed and purified using our well established protocol described elsewhere [33,34]. In short the whole cell-lysate was obtained by homogenization in 50 mM Tris-buffer of pH 8.0 containing 10 mM 1,4-dithiothreitol and 15 mM sodium azide.…”

Purification and characterization of RGA2, a Rho2 GTPase-activating protein from Tinospora cordifolia

“…Secondary structure measurement of protein using CD provided information that whether the purified protein is folded, and to checks its conformation or stability (Alam Khan et al 2009 , 2010 ; Greenfield 2006 ; Rehman et al 2011 ; Singh et al 2015 ). In addition, analysis of CD spectra helps to estimate the secondary structure composition of a protein (Anwer et al 2014 , 2015 ; Haque et al 2015 ; Khan et al 2016 ; Rahaman et al 2015 ). Prediction of secondary structure for the CD data measured to 178 nm for α-helix: 0.97 for β-sheet: 0.75 for β-turn: 0.50 and for other structures: 0.89 (Manavalan and Johnson 1987 ).…”