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. Study holds ProTherm entries: 3977, 3978, 3979, 3980, 3981 Extra Details: additive : EDTA(1 mM), van der Waals contact; disulfide bond; left-handed spiral;,thermal motion; folding/unfolding pathway

Submission Details

ID: XyY3VoPo3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:23 p.m.

Version: 1

Publication Details
Villafranca JE;Howell EE;Oatley SJ;Xuong NH;Kraut J,Biochemistry (1987) An engineered disulfide bond in dihydrofolate reductase. PMID:3304420
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Dihydrofolate reductase P0ABQ5 DYR_ECOL6
100.0 Dihydrofolate reductase P0ABQ4 DYR_ECOLI
100.0 Dihydrofolate reductase P0ABQ6 DYR_SHIFL
96.2 Dihydrofolate reductase P31073 DYR_CITFR
91.8 Dihydrofolate reductase P31074 DYR_KLEAE