Methyl TROSY of alanine residues in large protein complexes: development and application

DNA is the building block of the life and contains all the information that the cells require to manufacture proteins. An intermediate language exists in the sequence of RNA that translates a gene's message into a protein's amino acid sequence. The process of producing RNA is called transcription and is the most important regulatory step in gene expression in organisms ranging from simple bacteria to humans. The protein RNA polymerase (RNAP) is the enzyme which catalyses transcription and is the target, directly or indirectly, of most regulation of gene expression. The cells that make up our bodies are highly regulated and dynamic systems. In and around cells, functions are determined by the interplay of different types of biomolecules, such as proteins. These interactions are possible through the recognition of specific, complementary surfaces at the atomic level. For a better understanding of these cellular mechanisms, researchers need to develop new technologies for the study of biomolecules. In particular, modern molecular techniques like nuclear magnetic resonance (NMR) offer a powerful method to determine the different shapes (or conformations) and motion (or dynamics) of molecules and the interactions between them. In this proposal we aim to develop a novel application of NMR that can be applied and analysed on very large biomolecules, such as RNAP. The structural detail provided by these techniques is also sufficient for the elucidation of enzymatic mechanisms (e.g. the series of steps required for the synthesis and degradation of new molecules) and to analyse the interaction of biomolecules. We will exploit this new methodology to understand more about how viruses can interfere with the activity for the RNAP in order to alter gene expression to serve its own needs

NMR allows detailed atomic pictures of proteins, ligand interactions and other complex cellular components to be elucidated in a way that is highly complementary to X-ray crystallography. In addition, NMR can provide information regarding the conformational dynamics, aggregation states, binding equilibria and folding. A novel strategy will be developed and tested that will enable NMR spectra of alanine residues to be obtained for very large proteins (>>100 kDa), yielding structural, dynamic and interactional information. Furthermore, methods will be devised for providing robust site-specific assignments in very large systems. The proposed technology will have broad applicability in the study of protein-protein interactions and to illustrate this we have chosen two exemplar systems to develop the technique. The first is a fusion protein of glutathione-S-transferase (GST) with the tenth fibronectin type III domain (10FnIII) and the second are small protein-inhibitors of RNA polymerase (RNAP). The 72 kDa GST-10FnIII will enable our approach to be refined and tested, as comprehensive structural, dynamic and interactional information is available. We will use the road-tested methodology to provide new insight into function, interactions and mode of inhibition of bacterial RNAP by protein modulators (SRBPs) from T4 and T7 bacteriophages,