Detection of a Stable Intermediate in the Thermal Unfolding of a Cysteine-Free Form of Dihydrofolate Reductase from Escherichia Coli

• Published in: Biochemistry (Easton) 1995-08, Vol.34 (33), p.10669-10675
• Main Authors: Luo, J, Iwakura, M, Matthews, C. R
• Format: Article
• Language: English
• Subjects: Circular Dichroism; Cysteine; Escherichia coli; Escherichia coli - enzymology; Hot Temperature; Hydrogen-Ion Concentration; Mutagenesis, Site-Directed; Protein Folding; Structure-Activity Relationship; Tetrahydrofolate Dehydrogenase - chemistry; Tetrahydrofolate Dehydrogenase - genetics; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00033a043

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.