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 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.
Luo
J
J
Department of Chemistry, Pennsylvania State University, University Park 16802, USA.
Iwakura
M
M
Matthews
C R
CR
D002942:Circular Dichroism; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics
D013762:Tetrahydrofolate Dehydrogenase; D003545:Cysteine