title: In vitro and in silico studies of urea-induced denaturation of yeast iso-1-cytochrome c and 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.
authors.affiliation: a Centre for Interdisciplinary Research in Basic Sciences , Jamia Millia Islamia , New Delhi 110025 , India.
authors.forename: Md Anzarul
authors.initials: MA
authors.lastname: Haque
authors.forename: Sobia
authors.initials: S
authors.lastname: Zaidi
authors.forename: Shah
authors.initials: S
authors.lastname: Ubaid-Ullah
authors.forename: Amresh
authors.initials: A
authors.lastname: Prakash
authors.forename: Md Imtaiyaz
authors.initials: MI
authors.lastname: Hassan
authors.forename: Asimul
authors.initials: A
authors.lastname: Islam
authors.forename: Janendra K
authors.initials: JK
authors.lastname: Batra
authors.forename: Faizan
authors.initials: F
authors.lastname: Ahmad
mesh_terms: D000818:Animals; D045304:Cytochromes c / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins; D011489:Protein Denaturation / Q000187:drug effects; D013696:Temperature; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D005656:Fungal Proteins; D050505:Mutant Proteins; D014508:Urea; D045304:Cytochromes c
keywords: molecular dynamics and simulation; protein folding; protein stability; two-state unfolding; urea denaturation; yeast iso-1-cytochrome c