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Title: Functional analysis and structural investigations of MTB DosS sensory domain
Author: Sardiwal, Sunita
ISNI:       0000 0004 2669 0011
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2008
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Mycobacterium tuberculosis (MTB) is a very successful pathogen, causing the deaths of approximately two million people a year world-wide. Survival of the pathogen in vivo is dependant on its ability to respond and adapt to changes within its environment. One method of adaptation is through two-component signal transduction systems, the phosphotransfer pathways that couple stimuli to responses. Generic two-component systems involve two conserved elements, a membrane bound histidine kinase, which is the sensory protein, and a response-regulator protein that controls the response usually by altering the expression of genes required for adaptive responses. The aim of this thesis is to investigate the structure and function of the sensory protein DosS (DevS). DosS is induced by exposure to hypoxia, NO and ethanol and is the only one of the 11 paired MTB two component systems for which inducers have been identified but the precise chemical nature of the signal is unknown. The N-terminal input region of the DosS sensor contains two putative GAF domains. Various fragments of the N-terminal region were cloned, expressed and purified to homogeneity. Ultraviolet-Visible spectral analysis reveals that full length DosS binds a classical high spin b haem cofactor. Mutagenesis identified histidine 149 of DosS, which is within the N-terminal GAF domain, as critical to haem-binding. This is the first known GAF domain to bind haem and the presence of a haem co-factor is consistent with the postulated involvement of DosS in oxygen and redox sensing. Based on this data a model for histidine kinase activation is suggested. The second GAF domain of DosS was analysed using NMR spectroscopy. Triple resonance NMR experiments enabled the identification and sequential assignment for 99 out of the 141 backbone amide proton and nitrogen resonances. In addition, both of the GAF domains were also subjected to protein crystallisation while the full length DosS was investigated using electron microscopy.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available