Reconstitution of a disulfide isomerization system
Reconstitution of a disulfide isomerization system
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DOI：
10.1074/jbc.m203028200
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发表时间：
2002-07-26
影响因子：
4.8
通讯作者：
Bardwell, JCA
中科院分区：
文献类型：
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作者：
Collet, JF;Riemer, J;Bardwell, JCA
二硫键的异构化对于具有多个二硫键的蛋白质的正确折叠至关重要。在原核生物中，负责二硫键异构化的催化途径涉及硫氧还蛋白、硫氧还蛋白还原酶和DsbC、DsbG和DsbD蛋白。为了作为异构酶具有活性，DsbC和DsbG必须保持还原。这一任务由细胞质膜蛋白DsbD执行。DsbD又被细胞质硫氧还蛋白还原，由三个结构域组成。β结构域是膜包埋的，而α和γ结构域定位于周质。有人提出电子在DsbD内通过三个结构域之间的一系列二硫键交换反应转移。为了使用生物化学方法测试该模型，我们将对应于α、β、γ和β γ结构域的不同多肽纯化至同质。使用这些结构域，我们可以重建DsbD活性，并首次在体外重建从NADPH和硫氧还蛋白到DsbC和DsbG的电子传递途径。我们发现电子从硫氧还蛋白转移到β结构域，然后依次转移到γ结构域、α结构域，最后转移到DsbC或DsbG。我们还测定了γ结构域的氧化还原电位为-241 mV，并且发现α结构域的氧化还原电位为-229 mV。这表明DsbD内的电子流方向是受驱动的。
Isomerization of disulfide bonds is vital for the proper folding of proteins that possess multiple disulfides. In prokaryotes, the catalytic pathway responsible for disulfide isomerization involves thioredoxin, thioredoxin reductase, and the DsbC, DsbG, and DsbD proteins. To be active as isomerases, DsbC and DsbG must be kept reduced. This task is performed by the cytoplasmic membrane protein DsbD. DsbD in turn is reduced by the cytoplasmic thioredoxin and is composed of three domains. The beta domain is membrane-embedded, whereas the a and gamma domains are localized to the periplasm. It had been proposed that electrons are transferred within DsbD by a succession of disulfide exchange reactions between the three domains. To test this model using biochemical methods, we purified to homogeneity different polypeptides corresponding to the alpha, beta, gamma, and betagamma domains. Using these domains, we could reconstitute a DsbD activity and, for the first time, reconstitute in vitro the electron transport pathway from NADPH and thioredoxin to DsbC and DsbG. We showed that electrons are transferred from thioredoxin to the beta domain then successively to the gamma domain, the alpha domain, and finally on to DsbC or DsbG. We also determined the redox potential of the gamma domain to be -241 mV, and that of the a domain was found to be -229 mV. This shows that the direction of electron flow within DsbD is thermodynamically driven.