Deu Edgar, Dhoot Jashdeep, Kirsch Jack F
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3206, USA.
Biochemistry. 2009 Jan 20;48(2):433-41. doi: 10.1021/bi801431x.
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.
通过对10个不同界面接触点进行定向诱变，研究了亚基间侧链 - 侧链相互作用在大肠杆菌天冬氨酸转氨酶（eAATase）同型二聚体稳定性中的作用。该酶由尿素介导的去折叠途径通过形成二聚体中间体D进行，通过圆二色性判断，该中间体仅保留天然酶二级结构的40%。破坏任何单个亚基间相互作用都会导致天然状态稳定性降低>2.6千卡摩尔⁻¹，且与相互作用的位置或性质无关。然而，对于所有突变体，D相对于未折叠单体U的稳定性是相同的。eAATase界面的稳定性不能归因于少数热点的贡献，也不能归因于大量弱相互作用的积累，而只能归因于存在多个重要且相互关联的相互作用。有人提出，一种足够灵活以适应点突变的“熔融界面”结构解释了D的稳定性。不参与天然亚基间接触的三级结构核心可能为D的无结构界面提供了支架。这样的支架可以解释中间体的协同去折叠。
