Strukturelle und funktionelle Untersuchungen zur Kupfer-Homöostase in Corynebacterium glutamicum

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• Xenia Schuplezow

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• Manuels
• Manuels universitaires

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Copper is a crucial trace element for many organisms but becomes toxic at high concentrations. This thesis addresses three aspects of copper homeostasis in Corynebacterium glutamicum, a Gram-positive soil bacterium used as a model in microbial biotechnology. The first focus is on determining the structure of the sensor kinase CopS, a membrane-bound histidine kinase integral to the CopSR two-component signal transduction system. CopS is essential for sensing elevated copper levels and activating genes related to the copper stress response. It is believed that CopS detects extracellular copper through a short extracytoplasmic domain situated between its two transmembrane helices. Two strategies were employed for elucidating CopS's structure. Initially, the complete CopS protein was overproduced in Escherichia coli, purified, and crystallized; however, the crystals exhibited inner asymmetry, preventing X-ray structure determination. Subsequently, solid-state NMR was explored for structure analysis. Due to the size of the complete CopS, 14 C-terminally shortened derivatives were created and evaluated for overproduction and purification. One variant, NGHMCopS206, was identified as suitable for further analysis. The methods for its overproduction, purification, and reconstitution into proteoliposomes were optimized to maximize protein yield. However, solid-state NMR measurements indicated that the sample's mobility was too high, all

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• Année de publication: 2015