An engineered disulfide bond in dihydrofolate reductase.
Substitution of cysteine for proline-39 in Escherichia coli dihydrofolate reductase by oligonucleotide-directed mutagenesis positions the new cysteine adjacent to already existing cysteine-85. When the mutant protein is expressed in the E. coli cytosol, the cysteine sulfur atoms are found, by X-ray crystallographic analysis, to be in van der Waals contact but not covalently bonded to one another. In vitro oxidation by dithionitrobenzoate results in formation of a disulfide bond between residues 39 and 85 with a geometry close to that of the commonly observed left-handed spiral. Comparison of 2.0-A-refined crystal structures of the oxidized (cross-linked) and reduced (un-cross-linked) forms of the mutant enzyme shows that the conformation of the enzyme molecule was not appreciably affected by formation of the disulfide bond but that details of the molecule's thermal motion were altered. The disulfide-cross-linked enzyme is at least 1.8 kcal/mol more stable with respect to unfolding, as measured by guanidine hydrochloride denaturation, than either the wild-type or the reduced (un-cross-linked) mutant enzyme. Nevertheless, the cross-linked form is not more resistant to thermal denaturation. Moreover, the appearance of intermediates in the guanidine hydrochloride denaturation profile and urea-gradient polyacrylamide gels indicates that the folding/unfolding pathway of the disulfide-cross-linked enzyme has changed significantly.
Villafranca
J E
JE
Howell
E E
EE
Oatley
S J
SJ
Xuong
N H
NH
Kraut
J
J
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003545:Cysteine; D004220:Disulfides / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D014961:X-Ray Diffraction
D004220:Disulfides; D006146:Guanidines; D013762:Tetrahydrofolate Dehydrogenase; D019791:Guanidine; D003545:Cysteine