PMID:8257692
| Citation |
Iwakura, M, Jones, BE, Falzone, CJ and Matthews, CR (1993) Collapse of parallel folding channels in dihydrofolate reductase from Escherichia coli by site-directed mutagenesis. Biochemistry 32:13566-74 |
|---|---|
| Abstract |
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. |
| Links | |
| Keywords |
Circular Dichroism; Cysteine/chemistry; Escherichia coli/enzymology; Flow Cytometry; Folic Acid Antagonists; Kinetics; Magnetic Resonance Spectroscopy; Methotrexate/pharmacology; Mutagenesis, Site-Directed; NADP/pharmacology; Protein Conformation; Protein Folding; Tetrahydrofolate Dehydrogenase/chemistry; Tetrahydrofolate Dehydrogenase/genetics; Thermodynamics; Urea/pharmacology |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<annotationlinks/>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
References
See Help:References for how to manage references in EcoliWiki.