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The unfolding pathway for Apo Escherichia coli aspartate aminotransferase is dependent on the choice of denaturant
- Source :
- Biochemistry. 46(19)
- Publication Year :
- 2007
-
Abstract
- The guanidine hydrochloride (GdnHCl) mediated denaturation pathway for the apo form of homodimeric Escherichia coli aspartate aminotransferase (eAATase) (molecular mass = 43.5 kDa/monomer) includes a partially folded monomeric intermediate, M* [Herold, M., and Kirschner, K. (1990) Biochemistry 29, 1907-1913; Birolo, L., Dal Piaz, F., Pucci, P., and Marino, G. (2002) J. Biol. Chem. 277, 17428-17437]. The present investigation of the urea-mediated denaturation of eAATase finds no evidence for an M* species but uncovers a partially denatured dimeric form, D*, that is unpopulated in GdnHCl. Thus, the unfolding process is a function of the employed denaturant. D* retains less than 50% of the native secondary structure (circular dichroism), conserves significant quaternary and tertiary interactions, and unfolds cooperatively (mD*==U = 3.4 +/- 0.3 kcal mol-1 M-1). Therefore, the following equilibria obtain in the denaturation of apo-eAATase: D==2M 2M*==2U in GdnHCl and D==D*==2U in urea (D = native dimer, M = folded monomer, and U = unfolded state). The free energy of unfolding of apo-eAATase (D==2U) is 36 +/- 3 kcal mol-1, while that for the D* 2U transition is 24 +/- 2 kcal mol-1, both at 1 M standard state and pH 7.5.
- Subjects :
- Protein Denaturation
Protein Folding
Molecular mass
Stereochemistry
Hydrochloride
Circular Dichroism
Temperature
medicine.disease_cause
Biochemistry
chemistry.chemical_compound
Monomer
Apoenzymes
Spectrometry, Fluorescence
chemistry
medicine
Escherichia coli
Urea
Denaturation (biochemistry)
Aspartate Aminotransferases
Guanidine
Protein Structure, Quaternary
Subjects
Details
- ISSN :
- 00062960
- Volume :
- 46
- Issue :
- 19
- Database :
- OpenAIRE
- Journal :
- Biochemistry
- Accession number :
- edsair.doi.dedup.....5628771968ad6b8576489e68f88dff9c