The role of a hinge region in the folding, stability, and activity of Escherichia coli dihydrofolate reductase was investigated with three site-directed mutants at valine-88, the central residue of the hinge. The three mutants, V88A and V88I and a valine-88 deletion, were created to perturb the packing of hydrophobic residues in the interior of a loose turn formed by residues 85-91. Deleting the valine-88 residue destabilized the protein by 2.93 +/- 0.6 kcal/mol as determined by equilibrium unfolding transitions in urea monitored by circular dichroism at 20 degrees C. Substitution of alanine for valine-88 stabilized the protein by -0.20 +/- 0.02 kcal/mol, and the isoleucine substitution was mildly destabilizing by 1.73 +/- 0.2 kcal/mol. Although there was no clear correlation between side-chain volume and stability, these results suggest that side-chain interactions in the interior of the turn influence the folding and stability of dihydrofolate reductase. The specific activity of the valine deletion mutant was approximately twice that of the wild-type protein while the specific activities of the V88A and V88I proteins were only slightly greater than the wild type. The full time courses of the reactions catalyzed by the mutants were almost identical with that for the wild type, indicating no major changes in the kinetic mechanism. Additionally, the rate constants associated with interconversion between various forms of the apoenzyme were identical for the mutant and wild-type enzymes. The rate constants for refolding transitions were examined by dilution of urea-inactivated protein.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 325, 326, 327 Extra Details: additive : EDTA(0.1 mM),dG and ddG were measured in the presence of [urea]50% Escherichia coli dihydrofolate reductase; enzymatic activity;,point mutations; folding; stability
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:15 p.m.
|Number of data points||14|
|Proteins||Dihydrofolate reductase ; Dihydrofolate reductase|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: ddG ; Derived Quantity: ddG_H2O|
|Libraries||Mutations for sequence MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLDKPVIMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR|