Interactions between yeast iso-1-cytochrome c and its peroxidase.
Isothermal titration calorimetry was used to study the formation of 19 complexes involving yeast iso-1-ferricytochrome c (Cc) and ferricytochrome c peroxidase (CcP). The complexes comprised combinations of the wild-type proteins, six CcP variants, and three Cc variants. Sixteen protein combinations were designed to probe the crystallographically defined interface between Cc and CcP. The data show that the high-affinity sites on Cc and CcP coincide with the crystallographically defined sites. Changing charged residues to alanine increases the enthalpy of complex formation by a constant amount, but the decrease in stability depends on the location of the amino acid substitution. Deleting methyl groups has a small effect on the binding enthalpy and a larger deleterious effect on the binding free energy, consistent with model studies of the hydrophobic effect, and showing that nonpolar interactions also stabilize the complex. Double-mutant cycles were used to determine the coupling energies for nine Cc-CcP residue pairs. Comparing these energies to the crystal structure of the complex leads to the conclusion that many of the substitutions induce a rearrangement of the complex.
Pielak
G J
GJ
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, USA. gary_pielak@unc.edu
Wang
X
X
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002151:Calorimetry; D018360:Crystallography, X-Ray; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D003578:Cytochrome-c Peroxidase / Q000737:chemistry*; D045304:Cytochromes c; D018698:Glutamic Acid / Q000235:genetics; D008239:Lysine / Q000235:genetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions; D013816:Thermodynamics; D014633:Valine / Q000235:genetics
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions; D001224:Aspartic Acid; D018698:Glutamic Acid; D045304:Cytochromes c; D003578:Cytochrome-c Peroxidase; D014633:Valine; D008239:Lysine; D000409:Alanine