In vitro and in silico studies of urea-induced denaturation of yeast iso-1-cytochromecand its deletants at pH 6.0 and 25 °C

Abstract

Yeast iso-1-cytochrome c (y-cyt-c) has five extra residues at N-terminus in comparison to the horse cytochrome c. These residues are numbered as -5 to -1. Here, these extra residues are sequentially removed from y-cyt-c to establish their role in folding and stability of the protein. We performed urea-induced denaturation of wild-type (WT) y-cyt-c and its deletants. Denaturation was followed by observing change in Δε405 (probe for measuring change in the heme environment within the protein), [θ]405 (probe for measuring the change in Phe82 and Met80 axial bonding), [θ]222 (probe for measuring change in secondary structure) and [θ]416 (probe for measuring change in the heme-methionine environment). The urea-induced reversible denaturation curves were used to estimate Δ[Formula: see text], the value of Gibbs free energy change (ΔGD) in the absence of urea; Cm, the midpoint of the denaturation curve, i.e. molar urea concentration ([urea]) at which ΔGD = 0; and m, the slope (=∂ΔGD/∂[urea]). Our in vitro results clearly show that except Δ(-5/-4) all deletants are less stable than WT protein. Coincidence of normalized transition curves of all physical properties suggests that unfolding/refolding of WT protein and its deletants is a two-state process. To confirm our in vitro observations, we performed 40 ns MD simulation of both WT y-cyt-c and its deletants. MD simulation results clearly show that extra N-terminal residues play a role in stability but not in folding of the protein.

Chitinase from Thermomyces lanuginosus SSBP and its biotechnological applications

“…Molecular dynamic (MD) simulations of chitinase provided a detailed information on the fluctuations and conformational changes . MD simulations can track system behavior and understanding of protein folding (Ubaid-Ullah et al 2014;Haque et al 2015;Naiyer et al 2015). These methods are used to investigate the structure, dynamics and thermodynamics of biological molecules (Anwer et al 2015;Hoda et al 2015;Naz et al 2015).…”

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 ).…”

Spectroscopic and MD simulation studies on unfolding processes of mitochondrial carbonic anhydrase VA induced by urea

“…The coordinates of CAVA were energy minimized with steepest descent, up to a tolerance of 100 kJ mol -1 to remove bad contacts. CHARMM topology for urea is generated and molecule is equilibrated with spc216 water model in a cubic box with the size of 6 nm (Zoete, Cuendet, Grosdidier & Michielin 2011) Three independent MD simulations were carried with standard protocol (Smith, Jones & van Gunsteren 2005;Haque et al 2014) in water in water and the co-solvent (3.0 and 5.0 M urea) at 300K to evaluate the structural changes and stability in course of simulation.…”

“…The coordinates of CAVA was energy minimized with steepest descent, up to a tolerance of 100 kJ mol -1 to remove bad contacts. Three independent MD Journal of Biomolecular Structure and Dynamics simulations were carried with standard protocol Haque et al 2014) in water and co-solvent (urea 3.0 M and 5.0 M) at 300K to evaluate the structural changes and stability in course of simulation.…”