title: Acid and thermal unfolding of Escherichia coli dihydrofolate reductase.
abstract: The acid and thermal unfolding of Escherichia coli dihydrofolate reductase (DHFR) were studied by means of circular dichroism (CD) and fluorescence spectroscopy. There existed at least one intermediate around pH 4 in the acid unfolding process at 15 degrees C, in which the tertiary 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 tertiary structure around some tryptophan residues of the intermediate. The thermal unfolding process at pH 7.0 also involved at least one intermediate having a disrupted tertiary structure and a folded secondary structure. The three-state thermodynamic analysis showed that the intermediate in thermal unfolding was less stable by 1.8 kcal/mol than the native state. The similarity of the far-ultraviolet CD spectra of acid 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 unfolding were depressed in the presence of KCl due to stabilization of the native form.
authors.affiliation: Department of Materials Science, Faculty of Science, Hiroshima University.
authors.forename: E
authors.initials: E
authors.lastname: Ohmae
authors.forename: T
authors.initials: T
authors.lastname: Kurumiya
authors.forename: S
authors.initials: S
authors.lastname: Makino
authors.forename: K
authors.initials: K
authors.lastname: Gekko
mesh_terms: D000817:Anilino Naphthalenesulfonates; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D000817:Anilino Naphthalenesulfonates; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D011189:Potassium Chloride; D013762:Tetrahydrofolate Dehydrogenase