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, the value of Gibbs free energy change (ΔG_D) 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.

Keywords:

• yeast iso-1-cytochrome c
• protein stability
• protein folding
• molecular dynamics and simulation
• urea denaturation
• two-state unfolding

Acknowledgements

SU, MAH and SZ are thankful to DBT, UGC and ICMR, respectively, for their fellowships. FA and MIH gratefully acknowledge the financial support from the Department of Science and Technology for financial support [SB/SO/BB-71/2010(G)]. We thank Dr Bruce E. Bowler, the University of Montana (USA) for providing us the plasmid pBTR1(WT). AP is thankful to the UGC (BSR grant), Delhi, India, for providing Dr DS Kothari postdoctoral fellowship to carry out this work. Workstation for MD simulation was supported by the Indian Council of Medical Research (BIC/12(04)/2012) to MIH and FA.