ACID AND THERMAL UNFOLDING OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE

The acid and thermal unfolding of Escherichia coli dihydrofolate reduc tase (DHFR) were studied by means of circular dichroism (CD) and fluor escence spectroscopy, There existed at least one intermediate around p H 4 in the acid unfolding process at 15 degrees C, in which the tertia ry structure was disrupted before unfolding of the secondary structure , The fluorescence energy transfer from intrinsic tryptophan residues to 1-anilinonaphthalene-8-sulfonate suggested the disruption of the te rtiary structure around some tryptophan residues of the intermediate, The thermal unfolding process at pH 7.0 also involved at least one int ermediate having a disrupted tertiary structure and a folded secondary structure, The three-state thermodynamic analysis showed that the int ermediate in thermal unfolding was less stable by 1.8 kcal/mol than th e native state, The similarity of the far-ultraviolet CD spectra of ac id and thermally unfolded forms suggests that both types of unfolding produce the same structure, which may be a molten globule intermediate such as that in the folding kinetics of DHFR, The acid and thermal un folding were depressed in the presence of KCI due to stabilization of the native form.