Zhang Chunli, Allegretti Matteo, Vonck Janet, Langer Julian D, Marcia Marco, Peng Guohong, Michel Hartmut
Max Planck Institute of Biophysics, Department of Molecular Membrane Biology, Max-von-Laue-Str. 3, D-60438 Frankfurt am Main, Germany.
Biochim Biophys Acta. 2014 Jan;1840(1):34-40. doi: 10.1016/j.bbagen.2013.08.023. Epub 2013 Sep 1.
F1FO ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane. A number of ATP synthases have been characterized to date. The one from the hyperthermophilic bacterium Aquifex aeolicus presents unique features, i.e. a putative heterodimeric stalk. To complement previous work on the native form of this enzyme, we produced it heterologously in Escherichia coli.
We designed an artificial operon combining the nine genes of A. aeolicus ATP synthase, which are split into four clusters in the A. aeolicus genome. We expressed the genes and purified the enzyme complex by affinity and size-exclusion chromatography. We characterized the complex by native gel electrophoresis, Western blot, and mass spectrometry. We studied its activity by enzymatic assays and we visualized its structure by single-particle electron microscopy.
We show that the heterologously produced complex has the same enzymatic activity and the same structure as the native ATP synthase complex extracted from A. aeolicus cells. We used our expression system to confirm that A. aeolicus ATP synthase possesses a heterodimeric peripheral stalk unique among non-photosynthetic bacterial F1FO ATP synthases.
Our system now allows performing previously impossible structural and functional studies on A. aeolicus F1FO ATP synthase.
More broadly, our work provides a valuable platform to characterize many other membrane protein complexes with complicated stoichiometry, i.e. other respiratory complexes, the nuclear pore complex, or transporter systems.
F1FO型ATP合酶催化由跨膜离子驱动力驱动的ADP和无机磷酸合成ATP。迄今为止，已有多种ATP合酶得到了表征。嗜热细菌水生栖热菌的ATP合酶具有独特的特征，即推测为异源二聚体的柄。为了补充之前对该酶天然形式的研究，我们在大肠杆菌中进行了异源表达。
我们设计了一个人工操纵子，将水生栖热菌ATP合酶的九个基因组合在一起，这些基因在水生栖热菌基因组中被分成四个簇。我们表达了这些基因，并通过亲和色谱和尺寸排阻色谱法纯化了酶复合物。我们通过天然凝胶电泳、蛋白质印迹和质谱对复合物进行了表征。我们通过酶促测定研究了其活性，并通过单颗粒电子显微镜观察了其结构。
我们表明，异源产生的复合物具有与从水生栖热菌细胞中提取的天然ATP合酶复合物相同的酶活性和相同的结构。我们利用我们的表达系统证实，水生栖热菌ATP合酶具有一个在非光合细菌F1FO型ATP合酶中独特的异源二聚体外周柄。
我们的系统现在允许对水生栖热菌F1FO型ATP合酶进行以前不可能进行的结构和功能研究。
更广泛地说，我们的工作提供了一个有价值的平台，用于表征许多其他具有复杂化学计量的膜蛋白复合物，即其他呼吸复合物、核孔复合物或转运系统。
