Wallace L A, Burke J, Dirr H W
Protein Structure-Function Research Programme, Department of Molecular and Cell Biology, University of the Witwatersrand, 2050, Johannesburg, South Africa.
Biochim Biophys Acta. 2000 May 23;1478(2):325-32. doi: 10.1016/s0167-4838(00)00023-6.
The folding and assembly of the dimeric glutathione transferases (GST) involves the association of two structurally distinct domains per subunit. A prominent and conserved domain-domain interaction in class alpha GSTs is formed by the packing of the indole side chain of Trp-20 from domain I into a hydrophobic pocket in domain II. Stability studies have shown that partial dissociation of the domains near Trp-20 occurs as an initial fast event during the unfolding kinetics of human GSTA1-1 (Wallace et al., Biochemistry 37 (1998) 5320-5328; Wallace et al., Biochem. J. 336 (1998) 413-418). The contribution of Trp-20 toward stabilising the domain-domain interface was investigated by mutating it to either a phenylalanine (W20F) or alanine (W20A) and determining the functionality (catalysis and non-substrate ligand binding) and stability (thermal- and urea-induced denaturation) of the mutant proteins. The replacement of Trp-20 did not impact on the protein's gross structural properties. Functionally, the W20F was non-disruptive, whereas the cavity-creating W20A mutation was. Both mutants destabilised the native state with W20A exerting the greatest effect. Reduced m-values as well as the protein concentration dependence of the urea unfolding transitions for W20F GSTA1-1 suggest the presence of a dimeric intermediate at equilibrium that is not observed with wild-type protein. Unfolding kinetics monitored by stopped-flow tyrosine fluorescence was mono-exponential and corresponded to the global unfolding of the protein during which the dimeric intermediate unfolds to two unfolded monomers. The similar unfolding kinetics data for wild-type and W20F A1-1 indicates that the global unfolding event was not affected by amino acid replacement. We propose that the packing interactions at the conserved Trp-20 plays an important role in stabilising the intrasubunit domain I-domain II interface of class alpha GSTs.
二聚体谷胱甘肽转移酶（GST）的折叠和组装涉及每个亚基中两个结构不同的结构域的缔合。α类GST中一个显著且保守的结构域-结构域相互作用是由结构域I中Trp-20的吲哚侧链堆积到结构域II的一个疏水口袋中形成的。稳定性研究表明，在人GSTA1-1的解折叠动力学过程中，靠近Trp-20的结构域的部分解离是一个初始快速事件（Wallace等人，《生物化学》37 (1998) 5320 - 5328；Wallace等人，《生物化学杂志》336 (1998) 413 - 418）。通过将Trp-20突变为苯丙氨酸（W20F）或丙氨酸（W20A），并测定突变蛋白的功能（催化和非底物配体结合）和稳定性（热诱导和尿素诱导变性），研究了Trp-20对稳定结构域-结构域界面的贡献。Trp-20的替换对蛋白质的总体结构性质没有影响。在功能上，W20F没有破坏性，而产生空洞的W20A突变则有。两个突变体都使天然状态不稳定，其中W20A的影响最大。W20F GSTA1-1的m值降低以及尿素解折叠转变对蛋白质浓度的依赖性表明，在平衡状态下存在一种二聚体中间体，而野生型蛋白质中未观察到这种中间体。通过停流酪氨酸荧光监测的解折叠动力学是单指数的，并且对应于蛋白质的整体解折叠，在此过程中，二聚体中间体解折叠为两个未折叠的单体。野生型和W20F A1-1相似的解折叠动力学数据表明，整体解折叠事件不受氨基酸替换的影响。我们认为，保守的Trp-20处的堆积相互作用在稳定α类GST亚基内结构域I-结构域II界面中起重要作用。
