The GntR super-family of transcriptional regulators are ubiquitously distributed throughout the prokaryotic world. There are currently 231,015 sequences in the Pfam database pertaining to GntR proteins (PF000392) from over 17,000 bacterial species. The proteobacteria, firmicutes and actinobacetria account for 95% of all GntR sequences in the database. Yet despite their wide distribution, very few have been studied in depth. The general structure of GntR proteins is a highly homologous helix-turn-helix (HTH) domain linked to a C-terminal effector binding/oligomerisation (Eb/O domain) by a flexible linker. Binding of effector molecules to the Eb/O domain causes conformational changes within the protein allowing binding or release of specific DNA operator sequences which controls gene transcription or repression. The work described here aims to address some of the unknowns relating to GntR-like regulators in terms of structural and dynamical information about their general mechanism of function of this highly diverse family of proteins. Thirty target GntR proteins were selected from sequence analysis of PF000392. These thirty targets were extensively analysed in silico revealing four proteins with novel C-terminal topologies (Gp26, FucR, Reut_B4779 and Colbol_00895) for which there is no structural information. Four proteins (HutC, DevA, DevE and Gp26) were also studied experimentally by using protein biochemistry and x-ray crystallography. The structure of HutC has been solved. However, it has not been fully refined due to the publication of a homologous structure during the course of this work. DevE crystals were obtained and examined by x-ray, further refinement of crystallography conditions and selenomethionine protein preparation resulted in poor phasing and low resolution defracting crystals. Work in this thesis also aimed to refine methodology of 2D-IR spectroscopy to examine protein molecular dynamics at femtosecond resolution with a view to applying this to GntR-like proteins. The model system for this work was InhA and isoniazid.
|Date of Award||16 Feb 2016|
- University Of Strathclyde
|Sponsors||University of Strathclyde & BBSRC (Biotech & Biological Sciences Research Council)|
|Supervisor||Paul Hoskisson (Supervisor) & Neil Hunt (Supervisor)|