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.

Characterization of pre-molten globule state of yeast iso-1-cytochrome c and its deletants at pH 6.0 and 25 °C

“…The near-UV CD spectrum obtained for the native WT protein is in agreement with the one reported elsewhere [56]. The near-UV CD spectra of all deletants in the native state are, within experimental errors, comparable with that of the native WT protein as reported earlier [34]. Absence of dichroic bands in the near-UV region in urea/GdmCl denatured WT y-cyt-c (D state) has been attributed to the absence of tertiary interactions in the protein [56,57].…”

“…The complete procedure for making deletants, their expression and purification has already been reported [34]. Deletants lacking extra N-terminal residues are denoted by (−5/−5), (−5/−4), (−5/−3), (−5/−2), and (−5/−1) where denotes the deletion and the numbers refer to the residues deleted, e.g., (−5/−1) denotes the deletion of residues numbered from −5 to −1 (i.e., TEFKA), while (−5/−2) denotes the deletion of resides numbered from −5 to −2 (i.e., TEFK) and so on [37].…”

“…We have been trying to understand questions: "Is the N-terminal extension required for the stability and/or proper folding?" The answer to the first part of the question has already been provided elsewhere [34]. To answer the other part of the question, we cloned, expressed and purified the WT y-cyt-c and its deletants lacking the N-terminal residues as described earlier [34].…”

“…The answer to the first part of the question has already been provided elsewhere [34]. To answer the other part of the question, we cloned, expressed and purified the WT y-cyt-c and its deletants lacking the N-terminal residues as described earlier [34]. To find the effect of the extra five N-terminal residues on the folding of y-cyt-c, we performed LiCl-induced denaturations of WT and its each deletant.…”

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).…”