title: The partially folded homodimeric intermediate of Escherichia coli aspartate aminotransferase contains a "molten interface" structure.
abstract: The role of intersubunit side chain-side chain interactions in the stability of the Escherichia coli aspartate aminotransferase (eAATase) homodimer was investigated by directed mutagenesis at 10 different interface contacts. The urea-mediated unfolding pathway of this enzyme proceeds through the formation of a dimeric intermediate, D*, that retains only 40% of the native enzyme secondary structure as judged by circular dichroism. Disruption of any single intersubunit interaction results in a >2.6 kcal mol(-1) decrease in native state stability, independent of its location or nature. However, the stability of D* with respect to U, the unfolded monomer, is the same for all mutants. The stability of the eAATase interface cannot be ascribed to the contribution of a few hot spots, or to the accumulation of a large number of weak interactions, but only to the presence of multiple important and interconnected interactions. It is proposed that a "molten interface" structure, flexible enough to accommodate point mutations, accounts for the stability of D*. Nuclei of tertiary structure, which are not involved in native intersubunit contacts, likely provide a scaffold for the unstructured interface of D*. Such a scaffold would account for the cooperative unfolding of the intermediate.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3206, USA.
authors.forename: Edgar
authors.initials: E
authors.lastname: Deu
authors.forename: Jashdeep
authors.initials: J
authors.lastname: Dhoot
authors.forename: Jack F
authors.initials: JF
authors.lastname: Kirsch
mesh_terms: D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001219:Aspartate Aminotransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D055672:Static Electricity; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
chemical_list: D029968:Escherichia coli Proteins; D014508:Urea; D001219:Aspartate Aminotransferases; D000409:Alanine