Maskos Klaus, Huber-Wunderlich Martina, Glockshuber Rudi
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, CH-8093 Zürich, Switzerland.
J Mol Biol. 2003 Jan 17;325(3):495-513. doi: 10.1016/s0022-2836(02)01248-2.
Oxidative protein folding in the periplasm of Escherichia coli is catalyzed by the thiol-disulfide oxidoreductases DsbA and DsbC. We investigated the catalytic efficiency of these enzymes during folding of proteins with a very complex disulfide pattern in vivo and in vitro, using the Ragi bifunctional inhibitor (RBI) as model substrate. RBI is a 13.1 kDa protein with five overlapping disulfide bonds. We show that reduced RBI can be refolded quantitatively in glutathione redox buffers in vitro and spontaneously adopts the single correct conformation out of 750 possible species with five disulfide bonds. Under oxidizing redox conditions, however, RBI folding is hampered by accumulation of a large number of intermediates with non-native disulfide bonds, while a surprisingly low number of intermediates accumulates under optimal or reducing redox conditions. DsbC catalyzes folding of RBI under all redox conditions in vitro, but is particularly efficient in rearranging buried, non-native disulfide bonds formed under oxidizing conditions. In contrast, the influence of DsbA on the refolding reaction is essentially restricted to reducing redox conditions where disulfide formation is rate limiting. The effects of DsbA and DsbC on folding of RBI in E.coli are very similar to those observed in vitro. Whereas overexpression of DsbA has no effect on the amount of correctly folded RBI, co-expression of DsbC enhanced the efficiency of RBI folding in the periplasm of E.coli about 14-fold. Addition of reduced glutathione to the growth medium together with DsbC overexpression further increased the folding yield of RBI in vivo to 26-fold. This shows that DsbC is the bacterial enzyme of choice for improving the periplasmic folding yields of proteins with very complex disulfide bond patterns.
大肠杆菌周质中的氧化蛋白质折叠由硫醇 - 二硫键氧化还原酶DsbA和DsbC催化。我们使用拉吉双功能抑制剂（RBI）作为模型底物，在体内和体外研究了这些酶在具有非常复杂二硫键模式的蛋白质折叠过程中的催化效率。RBI是一种13.1 kDa的蛋白质，具有五个重叠的二硫键。我们表明，还原型RBI在体外谷胱甘肽氧化还原缓冲液中可以定量重折叠，并从750种可能的具有五个二硫键的物种中自发地采用单一正确构象。然而，在氧化还原条件下，RBI折叠受到大量具有非天然二硫键的中间体积累的阻碍，而在最佳或还原氧化还原条件下积累的中间体数量惊人地少。DsbC在体外所有氧化还原条件下都催化RBI的折叠，但在重排氧化条件下形成的埋藏的非天然二硫键方面特别有效。相比之下，DsbA对重折叠反应的影响基本上仅限于还原氧化还原条件，其中二硫键形成是限速步骤。DsbA和DsbC对大肠杆菌中RBI折叠的影响与体外观察到的非常相似。虽然DsbA的过表达对正确折叠的RBI数量没有影响，但DsbC的共表达提高了大肠杆菌周质中RBI折叠的效率约14倍。在生长培养基中添加还原型谷胱甘肽并同时过表达DsbC进一步将体内RBI的折叠产率提高到26倍。这表明DsbC是提高具有非常复杂二硫键模式的蛋白质周质折叠产率的首选细菌酶。
