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Interaction between SecA and SecYEG in micellar solution and formation of the membrane-inserted state

机译：SecA和SecYEG在胶束溶液中的相互作用和膜插入状态的形成

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Preprotein translocation in Escherichia coli is mediated by the translocase with SecA as peripheral ATPase and SecY, SecE, and SecG as membrane domain. To facilitate large-scale purification of the SecYEG heterotrimer, SecY was fused at its amino terminus with a hexahistidine tag and co-overexpressed with SecE and SecG. The presence of the His tag allowed purification of homogeneously pure SecYEG complex by a single anion-exchange chromatographic step starting from octyl glucoside-solubilized inner membranes. Endogenous levels of SecD and SecF copurified with the SecYEG protein. Purified SecYEG complex retained a nativelike, alpha-helical conformation in octyl glucoside and in micellar solution binds SecA with high affinity. In the presence of the nonhydrolyzable nucleotide analogue adenosine 5'-(beta,gamma-imidotriphosphate), octyl glucoside-solubilized SecYEG is nearly as effective as the reconstituted enzyme in inducing the formation of a proteinase K-protected 30 kDa fragment of I-125-labeled SecA, while SecYEG is proteolyzed to fragments smaller than 6 kDa. These data demonstrate that the 30-kDa SecA fragment is not protected by the lipid phase nor by SecYEG but rather indicate that it represents a SecYEG- and nucleotide-induced stable conformational state of a SecA domain. [References: 63]
机译：大肠杆菌中的前蛋白易位是由以SecA为外围ATPase，以SecY，SecE和SecG为膜结构域的转位酶介导的。为了促进SecYEG异源三聚体的大规模纯化，将SecY在其氨基末端与一个六组氨酸标签融合，并与SecE和SecG共表达。 His标签的存在允许通过单一的阴离子交换色谱步骤从辛基糖苷溶解的内膜开始纯化均一的SecYEG复合物。 SecD和SecF的内源性水平与SecYEG蛋白共纯化。纯化的SecYEG复合物在辛基糖苷中保留了天然的α-螺旋构象，并且在胶束溶液中以高亲和力结合SecA。在存在不可水解的核苷酸类似物腺苷5'-（β，γ-亚氨基三磷酸）的情况下，辛基葡萄糖苷溶解的SecYEG在诱导形成蛋白酶K保护的I-125片段时，与重组酶几乎一样有效。标记的SecA，而SecYEG被蛋白水解成小于6 kDa的片段。这些数据证明30-kDa SecA片段不受脂质相或SecYEG的保护，而是表明它代表SecYEG和核苷酸诱导的SecA结构域的稳定构象状态。 [参考：63]

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