The Escherichia coli trp repressor is a member of the "helix-turn-helix" family of DNA-binding proteins, which is allosterically activated by the binding of L-tryptophan. In this thesis, I have used NMR spectroscopy to investigate the mechanism of allosteric activation of the trp repressor. I report here the results of the binding to trp repressor of tryptophan analogues such as 5-methyltryptophan, L- and D-tryptophan, which are corepressors, and inducers such as indole-3-propionic acid and 5-methylindole. A comparison of the mode of binding of indole-3-propionic acid with other analogues of tryptophan (inducers and corepressors) has also been carried out. The chemical shifts of the indole NH and 4-protons differs between corepressors and the inducer, indole-3-propionic acid. The pattern of intermolecular NOEs between protons of the repressor and those of the ligand also differ between the two classes of ligand. These results suggests that the orientation of the indole ring in the binding site differs by approximately 180 between the two kinds of ligand. The chemical shifts and intermolecular NOE patterns for the inducer, 5-methylindole, suggests that this ligand can bind in both L-tryptophan and indole-3-propionic orientations. In order to facilitate the determination of ligand induced shifts, protein-ligand NOEs and spectral analysis, I have also used isotopic labelling strategies such as random fractional and selective protonation to minimise spectral overlap. These labelling strategies will eventually be useful for NMR studies of the repressor-oligonucleotide complex, which has a Mr of 38 kDa and should also improve spectral resolution. However, for the purpose of efficient isotopic labelling, strong expression systems in E. coli are required. Therefore, I have constructed an improved T7 expression plasmid for producing isotopically labelled trp repressor.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:674291 |
| Date | January 1993 |
| Creators | Frederick, Ronnie Oliverie |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/35224 |
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