title: A histidine variant of yeast iso-1-cytochrome c that strongly affects the energetics of the denatured state.
abstract: Iso-1-cytochrome c has been engineered to remove all histidine residues not involved in heme ligation in the native state to produce a variant designated TM. Single histidine residues were then introduced at positions 26, TM + His26, and 54, TM + His54. Since histidine residues not involved in native state heme ligation are known to replace the methionine 80 heme ligand in denatured cytochrome c, these variants were expected to affect the structure of the denatured state. Guanidine hydrochloride denaturations were performed to assess the stability of these proteins relative to the wild-type protein. The free energy difference for heme ligation in the denatured state was assessed by pH titration. The experimentally observed mutation-induced change (delta deltaG(D-state)) in the free energy of heme ligation for unfolded TM + His54 versus TM + His26 is -0.4 kcal/mol. The expected mutation-induced change in delta deltaG(D-state) calculated for a random coil unfolded state is +2 kcal/mol. Thus, unfolded TM + His54 has residual structure stabilizing its denatured state by -2.4 kcal/mol relative to TM + His26. The results imply that the denatured state can contribute significantly to mutation-induced changes in the free energy of unfolding of a protein.
authors.affiliation: Department of Chemistry and Biochemistry, University of Denver, CO 80208-2436, USA.
authors.forename: S
authors.initials: S
authors.lastname: Godbole
authors.forename: B E
authors.initials: BE
authors.lastname: Bowler
mesh_terms: D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D045304:Cytochromes c; D004734:Energy Metabolism; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D016296:Mutagenesis; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013816:Thermodynamics
chemical_list: C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D045304:Cytochromes c