Gildenhuys Samantha, Wallace Louise A, Dirr Heini W
Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa.
Biochemistry. 2008 Oct 7;47(40):10801-8. doi: 10.1021/bi801272t. Epub 2008 Sep 13.
Glutaredoxin 2 (Grx2) from Escherichia coli is monomeric and an atypical glutaredoxin that takes part in the monothiol deglutathionylation of proteins. Unlike its orthologs, Grx2 is a larger molecule with a canonical glutathione transferase (GST) fold that consists of two structurally distinct domains, an N-terminal glutaredoxin domain and a C-terminal alpha-helical domain. While GSTs are dimeric proteins, the conformational stability and unfolding kinetics of Grx2 were investigated to establish the contribution made by the domain interface to the stability of the tertiary structure of GST-like proteins without any influence from quaternary interactions. Equilibrium unfolding transitions for Grx2, using urea as a denaturant, are monophasic and exhibit coincidence of the fluorescence and CD data indicative of a concerted loss or formation of tertiary and secondary structure. The data fit well to a two-state N <--> U model with no evidence that an intermediate is being formed. The experimental m value [2.7 kcal mol (-1) (M urea) (-1)] is in excellent agreement with a predicted value of 2.5 kcal mol (-1) (M urea) (-1) based on the amount of surface area expected to become exposed during unfolding. These findings provide evidence that the two structurally distinct domains of Grx2 behave as a single cooperative folding unit. The unfolding kinetics are complex which, as a result of native-state heterogeneity, are characterized by two observable unfolding reactions that occur in parallel. A major population representing one distinct nativelike form unfolds on a fast track to denatured Grx2 with cis-Pro49. This is followed by a spectroscopically silent cis-trans proline isomerization reaction as determined by interrupted unfolding experiments. A minor population representing the other distinct nativelike form unfolds slowly with its rate being limited by an undetermined structural isomerization reaction. Further, there is no evidence indicating that unfolding proceeds via a high-energy intermediate that might suggest independent unfolding of the two nonidentical domains in Grx2. The kinetics data are, therefore, consistent with the existence of cooperativity between the domains, in agreement with the equilibrium data.
来自大肠杆菌的谷氧还蛋白2（Grx2）是单体的，并且是一种非典型的谷氧还蛋白，它参与蛋白质的单硫醇去谷胱甘肽化反应。与它的直系同源物不同，Grx2是一个更大的分子，具有典型的谷胱甘肽转移酶（GST）折叠结构，由两个结构不同的结构域组成，一个N端谷氧还蛋白结构域和一个C端α螺旋结构域。虽然GST是二聚体蛋白，但对Grx2的构象稳定性和去折叠动力学进行了研究，以确定结构域界面在没有四级相互作用影响的情况下对类GST蛋白三级结构稳定性的贡献。以尿素作为变性剂时，Grx2的平衡去折叠转变是单相的，并且荧光和圆二色性数据显示出一致性，表明三级和二级结构同时丧失或形成。数据很好地拟合了两态N⇔U模型，没有证据表明正在形成中间体。实验得到的m值[2.7千卡摩尔（-1）（摩尔尿素）（-1）]与基于去折叠过程中预期暴露的表面积预测值2.5千卡摩尔（-1）（摩尔尿素）（-1）非常一致。这些发现提供了证据，表明Grx2的两个结构不同的结构域表现为一个单一的协同折叠单元。去折叠动力学很复杂，由于天然态的异质性，其特征是两个可观察到的去折叠反应同时发生。代表一种不同的类天然形式的主要群体在快速轨道上去折叠成具有顺式-Pro49的变性Grx2。随后是由中断去折叠实验确定的光谱学上沉默的顺反脯氨酸异构化反应。代表另一种不同的类天然形式的次要群体缓慢去折叠，其速率受未确定的结构异构化反应限制。此外，没有证据表明去折叠通过可能暗示Grx2中两个不同结构域独立去折叠的高能中间体进行。因此，动力学数据与结构域之间存在协同性一致，与平衡数据相符。
