Structural studies of potential new-generation antibiotic targets and the use of one of them, TonB as a model protein in protein engineering

Only a few years after the introduction of the first antibiotic, penicillin, in 1940, an antibiotic resistant strain of Staphylococcus aureus was identified. Over time bacteria have developed various mechanisms to inactivate antibiotics. Such antibiotic resistance in bacteria is a natural and inevitable process and represents a global threat. At the present time resistance to every main class of antibiotic has been observed. Therefore, the continuous development of new-generation antibiotics is crucial to combat the rise of antibiotic resistant strains. Identification of potential antibiotic targets and investigation of their structure and function represent a rational approach to developing a better understanding of the essential processes in which they are involved, and may lead to finding a mechanism to inhibit these processes. The first part of this thesis covers structural characterization and functional studies of the potential antibiotic targets TonB protein and the cell shape determining protein MreC. TonB is needed for TonB-dependent uptake of scarce nutrients, such as iron and vitaminB12. The cell shape determining protein MreC is involved in cell wall synthesis, which is the target of penicillin and its derivatives. In this study, the three dimensional structures of the C-terminal domain of Helicobacter pylori TonB of different lengths and the C-terminal domain of Bacillus subtilis MreC were determined using nuclear magnetic resonance (NMR) spectroscopy. Additionally, interaction studies of MreC with penicillin-binding proteins were done using NMR spectroscopy and a bacterial two-hybrid system. NMR spectroscopy is a versatile tool to investigate protein structure, dynamics and interactions. One of the advantages of NMR is that proteins can be studied in solution under nearly physiological conditions. However, with increasing molecular size (> 25 – 30 kDa), structural investigation by NMR becomes more complex and often requires specific labelling techniques and alternative methodologies for NMR measurements, such as transverse-relaxation optimized spectroscopy (TROSY). Segmental isotopic labelling, where only a part of the protein is stable isotopic labelled, is an attractive method to overcome the challenges of studying large proteins by NMR. Segmental isotopic labelling allows, e.g. investigation of individual protein domains in a full-length context. For ligation of the protein domains different methods can be used. Furthermore, NMR is a powerful tool to study the integrity of a protein. Certain requirements, however, have to be fulfilled: the protein has to be soluble at high concentrations and stable over the whole measurement time. Therefore it is important to optimize protein production in order to obtain soluble, properly folded proteins at high concentrations. In the second part of this study, TonB has been used as a model protein to show traceless intein-mediated segmental isotopic labelling by salt induced protein trans splicing using a halophilic intein. This approach facilitates structural investigations of TonB by NMR. TonB consists of a well-structured C-terminal domain and a flexible proline-rich region, which would severely interfere with spectral quality in a uniformly labelled sample. Furthermore TonB was used as a model protein to show the benefit in protein expression of using tandem SUMO fusion vectors as tools for the expression of more soluble proteins, which tend to be expressed in an insoluble form. Both of these applications are beneficial for structural investigation of proteins by NMR and can be applied to other proteins.