Collapse of parallel folding channels in dihydrofolate reductase from Escherichia coli by site-directed mutagenesis.


The rate-limiting steps in the folding of dihydrofolate reductase from Escherichia coli have been shown to involve the conversion of a set of four intermediates to a corresponding set of native conformers via four parallel channels [Jennings et al. (1993) Biochemistry 32, 3783-3789]. Fluorescence and absorbance studies of the unfolding and refolding of the C85S/C152E double mutant at various final urea concentrations reveal two slow folding reactions, two fewer than observed in the wild-type protein. Refolding in the presence of substoichiometric levels of the inhibitor methotrexate shows that the two remaining slow reactions correspond to two parallel channels which lead to a pair of native conformers capable of binding the inhibitor. A combination of stopped-flow circular dichroism and cofactor binding studies confirms that the four parallel channels observed in the wild-type protein have collapsed into two channels in the mutant. Kinetic and equilibrium studies of the single cysteine mutants suggest that replacements of Cysteine-85 which perturb the hydrophobic core containing this side chain are responsible for the simplification of the kinetic mechanism. These results demonstrate that at least two of the parallel folding channels in dihydrofolate reductase arise when tertiary structure develops and are not dependent upon cis/trans isomerization at prolyl peptide bonds. Study holds ProTherm entries: 4643, 4644, 4645, 4646 Extra Details: additive : K2EDTA(0.2 mM),

Submission Details


Submitter: Connie Wang

Submission Date: April 24, 2018, 8:26 p.m.

Version: 1

Publication Details
Iwakura M;Jones BE;Falzone CJ;Matthews CR,Biochemistry (1993) Collapse of parallel folding channels in dihydrofolate reductase from Escherichia coli by site-directed mutagenesis. PMID:8257692
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