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X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050
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X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

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The partially folded homodimeric intermediate of Escherichia coli aspartate aminotransferase contains a "molten

Edgar Deu1, Jashdeep Dhoot, Jack F Kirsch

  • 1Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3206, USA.

Biochemistry
|December 23, 2008
PubMed

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.

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