A functional His-tagged c subunit of the Escherichia coli F-type ATPase/Synthase.
A functional His-tagged c subunit of the Escherichia coli F-type ATPase/Synthase.
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大肠杆菌 F 型 ATP 酶/合酶的功能性 His 标记 c 亚基。
DOI：
10.1006/abbi.2000.2251
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Brusilow,WS
中科院分区：
文献类型：
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作者：
Tomashek,JJ;Poposki,JA;Brusilow,WS
大肠杆菌f0的c亚基在其c端被标记为六组氨酸基序。标记的亚基能够形成功能性的f0复合物，在没有f1复合物的情况下转运质子。在F1存在的情况下，这两个区关联并显示野生型酶的所有生化活性：dccd抑制的ATP酶活性、ATP合成酶活性和ATP依赖的质子泵送。该酶可以在固定金属亲和层析树脂（IMAC）上作为完整的配合物在天然条件下溶解和纯化。纯化的复合物可以重新合并到脂质体中，并表现出atp依赖的质子泵活性。相比之下，位于n端的六组氨酸标签都是无活性的。这些实验证明了标记c亚基对进一步研究f0的可行性，并表明c亚基的n端在蛋白质的组装或功能中起重要作用。
The c subunit of the Escherichia coli F0has been tagged with a hexahistidine motif at its C-terminus. The tagged subunit is capable of forming functional F0complexes that translocate protons in the absence of the F1complex. In the presence of F1, the two sectors associate and display all biochemical activities of the wildtype enzyme: DCCD-inhibitable ATPase activity, ATP synthase activity, and ATP-dependent proton pumping. The enzyme can be solubilized and purified as an intact complex under native conditions on immobilized-metal affinity chromatography (IMAC) resin. The purified complex can be reincorporated into liposomes and demonstrates ATP-dependent proton pumping activity. Hexahistine tags placed at the N-terminus, in contrast, were all inactive. These experiments demonstrate the feasibility of tagging the c subunit for further studies of the F0and suggest an important role for the N-terminus of the c subunit in either assembly or function of the protein.