Abstract

The reversible temperature-induced unfolding of a cysteine-free mutant (C85S/C152E, des-Cys) of dihydrofolate reductase from Escherichia coli has been studied by absorbance and by both far- and near-ultraviolet circular dichroism spectroscopies. The non-coincidence of all three transition curves demonstrated the existence of a highly populated partially-folded form near 39 degrees C at pH 7.8. This intermediate retains substantial secondary structure and partially excludes one or more of the five tryptophans from solvent; however, the intermediate has lost specific tertiary packing around its aromatic residues. Increases in enthalpy, entropy, and heat capacity are observed for both the native/intermediate and intermediate/unfolded transitions; the majority of the changes in these parameters occurs in the first transition. These results suggest that the thermal unfolding reaction of des-Cys dihydrofolate reductase involves a stable intermediate whose properties resemble those of a molten globule. Study holds ProTherm entries: 2468, 2469 Extra Details: additive : K2EDTA(0.2 mM),cysteine free pseudo wild type; the transition is from native,to intermediate Escherichia coli dihydrofolate reductase; cysteine-free;,thermal unfolding; intermediate; secondary structure

Submission Details

ID: PMtUqFB64

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:19 p.m.

Version: 1

Publication Details

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