Zapun A, Bardwell J C, Creighton T E
European Molecular Biology Labortory, Heidelberg, Germany.
Biochemistry. 1993 May 18;32(19):5083-92. doi: 10.1021/bi00070a016.
The protein DsbA facilitates disulfide bond formation in the periplasm of Escherichia coli. It has only two cysteine residues that are separated in the sequence by two other residues and are shown to form a disulfide bond reversibly. Chemical modification studies demonstrate that only one of the cysteine residues has an accessible thiol group in the reduced protein. Equilibrium and kinetic characterization of thiol-disulfide exchange between DsbA and glutathione showed that the DsbA disulfide bond was 10(3)-fold more reactive than a normal protein disulfide. Similarly, the mixed disulfide between the accessible cysteine residue and glutathione was 10(4)-fold more reactive than normal. The overall equilibrium constant for DsbA disulfide bond formation from GSSG was only 8 x 10(-5) M. These properties indicate that disulfide-bonded DsbA is a potent oxidant and ideally suited for generating protein disulfide bonds. Disulfide bonds generally increase the stabilities of folded proteins, when the folded conformation reciprocally stabilizes the disulfide bonds. In contrast, the disulfide bond of DsbA was so unstable in the folded state that its stability increased by 4.5 +/- 0.1 kcal.mol-1 when the protein unfolded. This implies that the disulfide bond destabilizes the folded conformation of DsbA. This was confirmed by demonstrating that the reduced protein was 3.6 +/- 1.4 kcal.mol-1 more stable than that with the disulfide bond.
蛋白质DsbA促进大肠杆菌周质中二硫键的形成。它仅有两个半胱氨酸残基，在序列中被另外两个残基隔开，且显示能可逆地形成二硫键。化学修饰研究表明，在还原态蛋白质中只有一个半胱氨酸残基具有可及的巯基。DsbA与谷胱甘肽之间硫醇 - 二硫键交换的平衡和动力学特征表明，DsbA的二硫键比正常蛋白质二硫键的反应活性高10³倍。同样，可及半胱氨酸残基与谷胱甘肽之间的混合二硫键比正常情况的反应活性高10⁴倍。由谷胱甘肽二硫化物（GSSG）形成DsbA二硫键的总体平衡常数仅为8×10⁻⁵ M。这些特性表明，二硫键结合的DsbA是一种强效氧化剂，非常适合用于生成蛋白质二硫键。一般来说，当折叠构象反过来稳定二硫键时，二硫键会增加折叠蛋白的稳定性。相比之下，DsbA的二硫键在折叠状态下非常不稳定，以至于当蛋白质展开时其稳定性增加了4.5±0.1 kcal·mol⁻¹。这意味着二硫键使DsbA的折叠构象不稳定。这一点通过证明还原态蛋白质比具有二硫键的蛋白质稳定3.6±1.4 kcal·mol⁻¹得到了证实。
