3D structure determination and functional characterization of prokaryotic heavy metal translocating CPX-ATPases

原核重金属转位 CPX-ATP 酶的 3D 结构测定和功能表征

• 批准号: 5402641
• 负责人: Privatdozent Dr. Mathias Lübben
• 金额: --
• 依托单位: Lehrstuhl für Biophysik
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2003
• 资助国家: 德国
• 起止时间: 2002-12-31 至 2005-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5402641?language=en
• 关键词: 3D; structure; determination; functional; characterization

The P-type CPX-ATPases are responsible for the transport of heavy metal ions in archaea, bacteria and eukaryotes. These ATPases are also involved in bacterial pathogenesis and certain diseases. We have chosen one of the two CPX-ATPases of the thermophile Sulfolobus solfataricus, which we named "CopB", for the investigation of its 3D structure and the molecular mechanism of this integral membrane protein. We expressed and purified the protein as well as three different functional domains of it, which provide the cation binding site (CopB-C), and form a phosphatase (CopB-A) and an ATP binding/phosphorylation site (CopB-B). 3D crystals capable of diffracting X-rays of the 30 kDa CopB-B-protein have been produced; crystallization conditions also of the 20 kDa CopB-A-protein were identified. We propose the determination of the 3D structure of this prototype of CPX-ATPase by combination of the structural information of these individual domains. We have expressed other CPX-ATPases as a whole in Escherichia coli (e. g. CopA of E. coli), amongst other things fused to a GFP-variant, which is regarded as "folding-sensitive". With this method, further homologues of the CPX-ATPase family from different species will be expressed, purified and subjected to 3D crystallization as well. In parallel, we wish to use homologous (over)expressing producers, which are useful alternative sources of proteins and will be necessary for comparative purposes in the course of further investigation of catalytic properties in vitro and in vivo. In addition, the response of Sulfolobus cells exposed to "metal stress" will be studied by proteomic techniques, because we want to understand the biological role of CopB, CopA and possibly of other yet unidentified metal-related proteins.

P型CPX-ATP酶在古细菌、细菌和真核生物中负责重金属离子的转运。这些ATP酶也参与细菌的发病机制和某些疾病。我们选择了嗜热硫化叶菌的两种CPX-ATP酶中的一种，我们将其命名为“CopB”，以研究其三维结构和这种整合膜蛋白的分子机制。我们表达并纯化了该蛋白及其三个不同的功能结构域，它们提供阳离子结合位点（CopB-C），并形成磷酸酶（CopB-A）和ATP结合/磷酸化位点（CopB-B）。已经产生了能够衍射30 kDa CopB-B-蛋白的X射线的3D晶体;还鉴定了20 kDa CopB-A-蛋白的结晶条件。我们建议通过组合这些单个结构域的结构信息来确定CPX-ATP酶原型的3D结构。我们已经在大肠杆菌中表达了其他CPX-ATP酶作为一个整体（e. G. E. coli），其中与GFP变体融合，其被认为是“折叠敏感的”。通过这种方法，来自不同物种的CPX-ATPase家族的其他同系物将被表达、纯化并进行3D结晶。同时，我们希望使用同源（过）表达生产者，这是有用的蛋白质的替代来源，并将是必要的比较目的，在体外和体内的催化性能的进一步研究过程中。此外，将通过蛋白质组学技术研究硫化叶菌细胞对“金属应激”的反应，因为我们想了解CopB、CopA和可能的其他尚未鉴定的金属相关蛋白的生物学作用。