DETECTION OF A STABLE INTERMEDIATE IN THE THERMAL UNFOLDING OF A CYSTEINE-FREE FORM OF DIHYDROFOLATE-REDUCTASE FROM ESCHERICHIA-COLI

The reversible temperature-induced unfolding of a cysteine-free mutant (C85S/C152E, des-Cys) of dihydrofolate reductase from Escherichia col i has been studied by absorbance and by both far-and near-ultraviolet circular dichroism spectroscopies. The non-coincidence of all three tr ansition curves demonstrated the existence of a highly populated parti ally-folded form near 39 degrees C at pH 7.8. This intermediate retain s substantial secondary structure and partially excludes one or more o f the five tryptophans from solvent; however, the intermediate has los t 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 t he changes in these parameters occurs in the first transition. These r esults suggest that the thermal unfolding reaction of des-Cys dihydrof olate reductase involves a stable intermediate whose properties resemb le those of a molten globule.