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

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

T I Koshy  1 , T L Luntz, B Plotkin, A Schejter, E Margoliash
Affiliations
Comparative Study

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

T I Koshy et al. Biochem J. .

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.

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