Thermodynamic effects of reduction of the active-site disulfide of Escherichia coli thioredoxin explored by differential scanning calorimetry.
Intramolecular disulfide bonds in protein molecules, whether present in the wild-type protein or engineered via site-directed mutagenesis, are capable of significantly increasing the stability. Establishing thermodynamic parameters associated with the redox formation of this linkage is often encumbered by other global structural changes within the protein molecule. The active site of Escherichia coli thioredoxin possesses a disulfide/dithiol in a short loop, oxidation/reduction of which is accompanied by little structural alteration of the protein. Data for the thermal denaturation of the reduced protein are presented, which on comparison to the data obtained for the oxidized form [Ladbury, J.E., Wynn, R., Hellinga, H.W., & Sturtevant, J.M. (1993) Biochemistry 32, 7526-7530] are used to establish thermodynamic parameters for the redox reaction in this molecule. Data for an isosteric double mutation in the active site of thioredoxin (Cys32Ser/Cys35Ser) are also presented. Although the wild-type and mutated proteins show a similar reduction in free energy compared to the oxidized form (-3.0 +/- 0.4 and -3.1 +/- 0.3 kcal mol-1, respectively), the enthalpic and entropic contributions to this destabilization are different for the two proteins.
Ladbury
J E
JE
Department of Chemistry, Yale University, New Haven, Connecticut 06520.
Kishore
N
N
Hellinga
H W
HW
Wynn
R
R
Sturtevant
J M
JM
D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D013816:Thermodynamics; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004220:Disulfides; D046911:Macromolecular Substances; D013879:Thioredoxins