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

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, the value of Gibbs free energy change (ΔG_D⁰) in the absence of urea; C_m, the midpoint of the denaturation curve, i.e. molar urea concentration ([urea]) at which ΔG_D = 0; and m, the slope (=∂ΔG_D/∂[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.