A HISTIDINE VARIANT OF YEAST ISO-1-CYTOCHROME-C THAT STRONGLY AFFECTSTHE ENERGETICS OF THE DENATURED STATE

Iso-1-cytochrome c has been engineered to remove all histidine residue s not involved in heme ligation in the native state to produce a varia nt designated TM. Single histidine residues were then introduced at po sitions 26, TM + His26, and 54, TM + His54. Since histidine residues n ot involved in native state heme ligation are known to replace the met hionine 80 heme ligand in denatured cytochrome c, these variants were expected to affect the structure of the denatured state. Guanidine hyd rochloride denaturations were performed to assess the stability of tho se proteins relative to the wild-type protein. The free energy differe nce far heme ligation in the denatured state was assessed by FH titrat ion, The experimentally observed mutation-induced change (Delta Delta G(D-state)) in the free energy of heme ligation for unfolded TM + His5 4 versus TM His26 is -0.4 kcal/mol. The expected mutation-induced chan ge in Delta Delta G(D-state) calculated for a random coil unfolded sta te is +2 kcal/mol. Thus, unfolded TM + His54 has residual structure st abilizing its denatured state by -2.4 kcal/mol relative to TM His26, T he results imply that the denatured state can contribute significantly to mutation-induced changes in the free energy of unfolding of a prot ein. (C) 1997 Academic Press Limited.