Comparative membrane metalloproteomics of respiring anaerobes

厌氧呼吸微生物的能量守恒在很大程度上依赖于含金属的多聚膜结合蛋白复合体，它允许电子供体的氧化、电子的结构传导和末端电子受体的还原。虽然许多呼吸复合体已经在选定的生物中进行了详细的研究，但金属蛋白、含金属辅因子和金属离子在呼吸厌氧菌中的总体分布尚不清楚。尽管如此，在厌氧菌基因组中编码的、被预测位于细胞膜中的大量金属蛋白复合体没有附属功能。含钼的FeS簇蛋白和11亚基复合体I的同源物都是在原核基因组中丰富编码的，它们都是其中之一。我们建议用电感耦合等离子体质谱(ICPMS)和液质联用(LC-MS/MS)分别定量金属和蛋白质亚基，研究膜结合金属蛋白复合体在不同原核生物中的存在和分布。这项研究将包括三个工作包：一是全细胞膜制备中金属和蛋白质的比较分析，即鸟枪膜金属蛋白质组学；二是天然凝胶电泳法分离金属蛋白络合物后金属和蛋白质的比较分析；三是与金属蛋白相关的含金属有机辅因子的质谱分析。我们将专注于含有金属Fe、Ni、Co和Mo的蛋白质络合物以及含有金属的辅因子F430、钴胺和钼蝶呤，这些都参与了厌氧节能过程的关键反应。该项目与SPP提出的一系列其他项目在主题和方法上都是相联系的。通过调查细菌，其中特定的金属蛋白由SPP的其他小组进行研究，我们可以将这些小组的结果整合到我们的工作中，以组装金属蛋白质组学的总体图景，同时为这些小组提供补充信息。其他合作主题包括钼辅因子和辅因子F430分析，以及在大肠杆菌中检测异源过表达和组装的金属蛋白。最后，展望了金属质谱学、蛋白质质谱学和二次离子质谱仪(NanSIMS，ToF-SIMS)的方法学合作。

Energy conservation in anaerobically respiring microorganisms highly depends on metal-containing multimeric membrane-bound protein complexes allowing the oxidation of electron donors, the structured conduction of electrons and the reduction of terminal electron acceptors. Although many respiratory complexes have been studied in detail in selected organisms, the overall distribution of metalloproteins, metal-containing cofactors and metal ions in respiring anaerobes is unknown. Still, a significant number of metalloprotein complexes encoded in the genomes of anaerobes and predicted to be located in the membrane have no affiliated function. The Mo-containing FeS cluster proteins and the 11-subunit complex I homologues, both abundantly encoded in prokaryotic genomes, are among them. We propose to study the occurrence and distribution of membrane-bound metalloprotein complexes in diverse prokaryotes by ICP-MS (inductively coupled plasma mass spectrometry) and LC-MS/MS (liquid chromatography mass spectrometry) to quantify metals and protein subunits, respectively. The study will consist of three work packages: First, the comparative analysis of metals and proteins in membrane preparations of whole cells, i.e. shotgun membrane metalloproteomics; second, the comparative analysis of metals and proteins in metalloprotein complexes after separation of the complexes by native gel electrophoresis; and third, the mass spectrometric analysis of metal-containing organic cofactors associated with metalloproteins. We will focus on protein complexes containing the metals Fe, Ni, Co and Mo and the metal-containing cofactors F430, cobalamin and Mo-pterins, all involved in key reactions of anaerobic energy-conserving processes.The project is connected to a series of other projects proposed within the SPP, both with regard to their topic and methodologically. By investigating bacteria, in which specific metalloproteins are investigated by other groups of the SPP, we can integrate results of these groups into our work to assemble an overall picture of metalloproteomics and at the same time provide supplementary information to these groups. Other cooperative topics are Mo-cofactor and cofactor F430 analysis as well as the detection of heterologously overexpressed and assembled metalloproteins in E. coli. Finally, methodological cooperation in metal mass spectrometry, protein mass spectrometry and secondary ion mass spectrometry (nanoSIMS, ToF-SIMS) is projected.