title: Evaluating the effects of a single amino acid substitution on both the native and denatured states of a protein.
abstract: For proteins that contain a disulfide bond, stability is linked thermodynamically to thiol-disulfide exchange. We use this relationship to obtain unfolding free energies for both the reduced and oxidized forms of Escherichia coli thioredoxin from measurements of the effective concentrations of protein thiols. We then evaluate the effect of an amino acid substitution on disulfide bond formation in both the native and denatured states of the protein. Although the Pro-34----Ser substitution in thioredoxin results in a decrease of the effective concentration of protein thiols in the native state, the effective concentration increases in the denatured state. The net effect of the amino acid substitution is to increase the stability of reduced thioredoxin by approximately 2.4 kcal/mol, whereas the stability of the oxidized protein remains the same. By assuming a two-state unfolding equilibrium and a mutation free energy of -7.7 kcal/mol for the Pro-34----Ser substitution in the reduced, urea-unfolded state (based on estimates of solvation and entropic changes), we obtained relative free energies for the native and denatured states of the mutant and wild-type proteins, in both the reduced and oxidized forms.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Nine Cambridge Center 02142.
authors.forename: T Y
authors.initials: TY
authors.lastname: Lin
authors.forename: P S
authors.initials: PS
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D002151:Calorimetry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011392:Proline; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011506:Proteins; D012694:Serine; D013879:Thioredoxins; D011392:Proline; D005978:Glutathione