Membrane-bound toxin-antitoxin systems

Toxin-Antitoxin (TA) systems have been found on various plasmids but also bacterial chromosomes. On plasmids they guarantee preferential growth of plasmid-carrying cells in a bacterial population by killing newborn bacteria that have not inherited a plasmid at cell division. Chromosomal TA systems have been postulated to help bacteria deal with nutritional stress by adjusting the rates of protein and DNA synthesis via down-regulation of translation and replication. In the presented project we plan to carry out a comprehensive structural investigation of a series of small membrane-bound TA components by high-resolution NMR spectroscopy. Due to inability to crystallize these polypeptides there is currently absolutely no structural information available on any membrane-bound TA system. To understand the biological function not only the structure but also the interaction with membranes or membrane- mimetic systems (micelles) is important. We will use relaxation enhancements exerted by a freely soluble paramagnetic agent to probe the distances of nuclei in the peptide from the micelle surface to orient helical polypeptides through our recently introduced paramagnetic waves. As part of this project we will also develop the use of paramagnetic relaxation enhancements (PREs) to fully position a structurally characterized polypeptide in a micelle and use PRE as actually structural restraints. By using these PRE values the number of necessary NOEs can be reduced significantly to obtain the polypeptide structure. Preliminary experiments on the use of PRE restraints for solution structure determinations have been carried out on an antimicrobial peptide and proved their enormous potential. To work with optimal size micelles for NMR experiments on a specific polypeptide we have synthesized a homologous series of deuterated phosphocholines. Besides the establishment of novel techniques the presented project will provide a deeper understanding of how membrane-bound TA systems work and about differences between plasmidic and chromosomal toxin-antitoxin modules. Apart from these fundamental biological questions, killing systems play an increasing role in biotechnology due to their use for stabilizing autonomously replicating vectors employed in recombinant bacteria. In addition, structural studies on TA systems hold promise for the design of new antibiotics and might open a new route towards fighting antibiotic resistance, which uses TA systems to ensure their persistence during host replication.

Toxin-Antitoxin (TA) systems have been found on various plasmids but also bacterial chromosomes. On plasmids they guarantee preferential growth of plasmid-carrying cells in a bacterial population by killing newborn bacteria that have not inherited a plasmid at cell division. Chromosomal TA systems have been postulated to help bacteria deal with nutritional stress by adjusting the rates of protein and DNA synthesis via down-regulation of translation and replication. In the presented project we plan to carry out a comprehensive structural investigation of a series of small membrane-bound TA components by high-resolution NMR spectroscopy. Due to inability to crystallize these polypeptides there is currently absolutely no structural information available on any membrane-bound TA system. To understand the biological function not only the structure but also the interaction with membranes or membrane- mimetic systems (micelles) is important. We will use relaxation enhancements exerted by a freely soluble paramagnetic agent to probe the distances of nuclei in the peptide from the micelle surface to orient helical polypeptides through our recently introduced paramagnetic waves. As part of this project we will also develop the use of paramagnetic relaxation enhancements (PREs) to fully position a structurally characterized polypeptide in a micelle and use PRE as actually structural restraints. By using these PRE values the number of necessary NOEs can be reduced significantly to obtain the polypeptide structure. Preliminary experiments on the use of PRE restraints for solution structure determinations have been carried out on an antimicrobial peptide and proved their enormous potential. To work with optimal size micelles for NMR experiments on a specific polypeptide we have synthesized a homologous series of deuterated phosphocholines. Besides the establishment of novel techniques the presented project will provide a deeper understanding of how membrane-bound TA systems work and about differences between plasmidic and chromosomal toxin-antitoxin modules. Apart from these fundamental biological questions, killing systems play an increasing role in biotechnology due to their use for stabilizing autonomously replicating vectors employed in recombinant bacteria. In addition, structural studies on TA systems hold promise for the design of new antibiotics and might open a new route towards fighting antibiotic resistance, which uses TA systems to ensure their persistence during host replication.