THE SIGNIFICANCE OF DENATURANT TITRATIONS OF PROTEIN STABILITY - A COMPARISON OF RAT AND BAKERS-YEAST CYTOCHROME-C AND THEIR SITE-DIRECTED ASPARAGINE-52-TO-ISOLEUCINE MUTANTS

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.