Kojic, I.; (2008) An NMR analysis of the structure and ligand binding characteristics of proteins from Mycobacterium tuberculosis implicated in dormancy. Doctoral thesis , University of London.

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Abstract

Two proteins putatively involved in the persistence of Mycobacterium tuberculosis infection have been investigated by heteronuclear nuclear magnetic resonance (NMR) spectroscopy and other methods. The major aspect of work describes the characterization by NMR of the Mtb thiol peroxidase TPx. TPx (165 residues) is a 36 kD symmetrical homodimer that catalyzes the reduction of a range of reactive oxygen species in vitro. Multidimensional heteronuclear NMR spectroscopy was applied to recombinant wild-type (Wt) and Cys60->Ser (C60S) TPx, both of which are clearly folded but display slightly different spectral characteristics. Triple resonance NMR experiments recorded using 2H, 13C, 15N -labelled C60S yielded assignments for the majority of the backbone resonances. 28 non-proline residues could not be assigned mostly due to the absence of cross peaks. TPxWt spectra showed little difference in peak number indicating that the C60S mutation itself does is not responsible for the missing signals. Consideration of related TPx crystal structures indicates that the missing cross peaks correspond to a region of the protein that must display conformational plasticity necessary for the mechanism of action suggesting that the spectrum is affected by conformational exchange processes. Efforts to modulate this exchange behaviour through the application of putative TPx ligands and chemical modification of the active site cysteine residues are described. In a second aspect, a preliminary characterisation of the non-canonical RNA polymerase sigma factor sigJ from Mtb was performed. Bioinformatic analysis revealed that sigJ likely contains conserved o2 and a4 DNA-binding domains fused to a unique C-terminal domain. NMR spectra of recombinant sigJ recovered from inclusion bodies and in the soluble fraction differed, though both forms yielded dispersed resonances consistent with the protein containing a globular component. However further progress was hampered by signal overlap and heterogeneous line widths. sigJ has a general capability to retard the movement of DNA in an electrophoretic mobility shift assay, though specific target sequence was identified.

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