Comparative membrane metalloproteomics of respiring anaerobes

呼吸厌氧菌的比较膜金属蛋白质组学

• 批准号: 311144115
• 负责人: Professor Dr. Lorenz Adrian
• 金额: --
• 依托单位: Helmholtz-Zentrum für Umweltforschung (UFZ)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/311144115?language=en
• 关键词: Comparative; membrane; metalloproteomics; respiring; anaerobes

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.

厌氧呼吸微生物的能量守恒高度依赖于含金属的多聚体膜结合蛋白质复合物，其允许电子供体的氧化、电子的结构化传导和末端电子受体的还原。虽然许多呼吸复合物已在选定的生物体中进行了详细研究，但金属蛋白、含金属辅因子和金属离子在呼吸性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并预测位于膜中的大量金属蛋白复合物没有附属功能。含钼的FeS簇蛋白和11-亚基复合物I同源物，都在原核基因组中大量编码，其中。我们建议研究的发生和分布的膜结合的金属蛋白复合物在不同的原核生物电感耦合等离子体质谱法（电感耦合等离子体质谱法）和LC-MS/MS（液相色谱质谱法），以量化金属和蛋白质亚基，分别。该研究将包括三个工作包：第一，全细胞膜制备物中金属和蛋白质的比较分析，即鸟枪膜金属蛋白质组学;第二，通过天然凝胶电泳分离复合物后，金属蛋白复合物中金属和蛋白质的比较分析;第三，与金属蛋白相关的含金属有机辅因子的质谱分析。我们将重点关注含有金属Fe、Ni、Co和Mo以及含金属辅因子F430、钴胺素和Mo-pterins的蛋白质复合物，所有这些都涉及厌氧节能过程的关键反应。该项目与SPP内提出的一系列其他项目有关，无论是在主题上还是在方法上。通过研究细菌，其中特定的金属蛋白被SPP的其他组研究，我们可以将这些组的结果整合到我们的工作中，以组装金属蛋白质组学的全貌，同时为这些组提供补充信息。其他合作主题是Mo-辅因子和辅因子F430分析，以及在大肠杆菌中异源过表达和组装的金属蛋白的检测。杆菌最后，预计在金属质谱、蛋白质质谱和二次离子质谱（nanoSIMS、ToF-SIMS）方面开展方法学合作。