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Projekt
3D-Strukturbestimmung und funktionelle Charakterisierung von prokaryontischen Schwermetall-tranlozierenden CTX-ATPasen
Antragsteller
Privatdozent Dr. Mathias Lübben
Fachliche Zuordnung
Strukturbiologie
Förderung
Förderung von 2003 bis 2006
Projektkennung
Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 5402641
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.
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