Effect of sequential deletion of extra N-terminal residues on the structure and stability of yeast iso-1-cytochrome-c

Abstract

A sequence alignment of yeast cytochrome-c (y-cyt-c) with mammalian cyts-c shows that the yeast protein has a five residue long N-terminal extension. A question arises: Does this N-terminal extension play any roles in the stability, structure, and folding of the yeast protein? To answer this question, in silico and in vitro studies were carried out on the wild type (WT) protein and its five deletants (Δ(−5/−5), Δ(−5/−4), Δ(−5/−3), Δ(−5/−2), and Δ(−5/−1) where Δ denotes the deletion and the numbers refer to the residues deleted, e.g. Δ(−5/−1) denotes the deletion of residues numbered from −5 to −1 (TEFKA), while Δ(−5/−2) denotes the deletion of resides numbered from −5 to −2 (TEFK) and so on). The main conclusion of the in silico study is that the order of stability of deletants and WT protein is Δ(−5/−4) > WT > Δ(−5/−3) > Δ(−5/−5) > Δ(−5/−1) ~ Δ(−5/−2). In vitro studies involved (i) measurements of thermodynamic stability of all proteins by differential scanning calorimetry and from sigmoidal curves of two different structural properties ([θ]₂₂₂, a probe for detecting change in secondary structure, and Δε₄₀₅, a probe for detecting alteration in the heme environment), and (ii) characterization of all proteins by various spectral properties. The main conclusions of the in vitro studies are as follows: (i) The order of thermodynamic stability of all proteins is in excellent agreement with that predicted by in silico studies, and (ii) A sequential deletion of the N-terminal extension has no effects on protein structure and folding.

Keywords:

• yeast iso-1-cytochrome-c
• protein stability
• two-state denaturation
• differential scanning calorimetry

Acknowledgments

SU, MAH, and SZ are thankful to DBT, UGC, and ICMR, respectively, for their fellowships. We thank Professor Bruce E. Bowler, University of Montana (USA) for providing us the plasmid pBTR1(WT). We also thank Professor Rajiv Bhat, Jawaharlal Nehru University (India) for his help with the DSC measurements.

Supplementary material

The supplementary material for this paper is available online at http://dx.doi.10.1080/07391102.2013.848826.