Denoncin Katleen, Nicolaes Valérie, Cho Seung-Hyun, Leverrier Pauline, Collet Jean-François
Brussels Center for Redox Biology and de Duve Institute, Université Catholique de Louvain, Brussels, Belgium.
Methods Mol Biol. 2013;966:325-36. doi: 10.1007/978-1-62703-245-2_20.
Many proteins secreted to the bacterial cell envelope contain cysteine residues that are involved in disulfide bonds. These disulfides either play a structural role, increasing protein stability, or reversibly form in the catalytic site of periplasmic oxidoreductases. Monitoring the in vivo redox state of cysteine residues, i.e., determining whether those cysteines are oxidized to a disulfide bond or not, is therefore required to fully characterize the function and structural properties of numerous periplasmic proteins. Here, we describe a reliable and rapid method based on trapping reduced cysteine residues with 4'-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS), a maleimide compound. We use the Escherichia coli DsbA protein to illustrate the method, which can be applied to all envelope proteins.
许多分泌到细菌细胞包膜的蛋白质含有参与二硫键形成的半胱氨酸残基。这些二硫键要么发挥结构作用，增强蛋白质稳定性，要么在周质氧化还原酶的催化位点可逆形成。因此，要全面表征众多周质蛋白的功能和结构特性，就需要监测半胱氨酸残基的体内氧化还原状态，即确定这些半胱氨酸是否被氧化成二硫键。在此，我们描述了一种基于用4'-乙酰氨基-4'-马来酰亚胺基芪-2,2'-二磺酸（AMS，一种马来酰亚胺化合物）捕获还原型半胱氨酸残基的可靠且快速的方法。我们用大肠杆菌DsbA蛋白来说明该方法，该方法可应用于所有包膜蛋白。
