Strukturelle und funktionelle Untersuchungen zur Kupfer-Homöostase in Corynebacterium glutamicum

Copper is an important trace element for many organisms, but toxic at elevated concentrations. This thesis covered three issues of copper homeostasis in Corynebacterium glutamicum, a Gram-positive soil bacterium serving as a model organism in microbial biotechnology. The first subject aimed at the structure determination of the sensor kinase CopS. CopS is a membrane-bound histidine kinase with two transmembrane helices and part of the two-component signal transduction system CopSR, which is required for sensing elevated copper concentrations and activating transcription of a set of genes involved in the copper stress response. It is assumed that CopS senses the extracellular copper concentration via a very short extracytoplasmic domain located between the two transmembrane helices. Two different approaches were pursued for CopS structure elucidation. On the one hand, the complete CopS protein was overproduced in Escherichia coli, purified and crystallized. However, the crystals showed inner asymmetry and therefore could not be used for X-ray structure determination. On the other hand, attempts were made to apply solid-state NMR for structure determination. As the complete CopS was too large for this approach, a series of 14 C-terminally shortened CopS derivatives were constructed and tested with respect to overproduction and purification. One of the variants turned out to be suitable for further analysis, termed NGHMCopS206. The methods for overproduction, purification, and reconstitution into proteoliposomes of this derivative were carefully optimized to achieve the maximal protein yield. The solid-state NMR measurement of the resulting proteoliposomes showed that the mobility of the sample was too high and only the amino acids of the two transmembrane helices were detectable