The significance of denaturant titrations of protein stability: a comparison of rat and baker's yeast cytochrome c and their site-directed asparagine-52-to-isoleucine mutants

Biochem J. 1994 Apr 15;299 ( Pt 2)(Pt 2):347-50. doi: 10.1042/bj2990347.

Abstract

The residue asparagine-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was mutated to isoleucine by site-directed mutagenesis, and the unfolding of the wild-type and mutant proteins in urea or guanidinium chloride solutions was studied. Whereas the yeast mutant cytochrome unfolded in 4-7 M urea with a rate constant (k) of 1.7 x 10(-2) s-1, the rat mutant protein unfolded with k = 5.0 x 10(-2) s-1, followed by a slow partial refolding with k = 5.0 x 10(-4) s-1. Denaturant titrations indicated that the mutation increased the stability of the yeast cytochrome by 6.3 kJ (1.5 kcal)/mol, while it decreased that of the rat protein by 11.7 kJ (2.8 kcal)/mol. These results probably reflect structural differences between yeast iso-1 and vertebrate cytochromes c in the vicinity of the Asn-52 side chain.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Asparagine*
  • Calorimetry
  • Cytochrome c Group / chemistry*
  • Cytochrome c Group / metabolism
  • Drug Stability
  • Isoleucine*
  • Kinetics
  • Mutagenesis, Site-Directed
  • Protein Denaturation
  • Protein Folding*
  • Rats
  • Saccharomyces cerevisiae / metabolism*
  • Urea

Substances

  • Cytochrome c Group
  • Isoleucine
  • Asparagine
  • Urea