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Title: Structural studies on protein targets from the pathogenic bacterium Burkholderia pseudomallei
Author: Day, Matthew
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2012
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The main body of this thesis deals with a small-scale structural genomics project for proteins from the bacterium, Burkholderia pseudomallei. Potential pathogenicity determinants were selected based on their possession of at least one of two criteria. The first was their expression in the pathogenic Burkholderia pseudomallei but not the closely related, nonpathogenic Burkholderia thailandensis. The second required their expression be controlled as part of a stress response, which controls other known virulence factors. Of the nine selected targets, five were successfully overexpressed, purified and crystallised and the structure of one was determined. The selected targets were all of unknown function and initial sequence analysis highlighted a number of interesting characteristics for certain targets. BPSL1958 contains a highly repetitive sequence conserved at both the protein and DNA level, suggesting the gene was created in a recent duplication event and there has not been enough time for the sequence of the individual repeats to drift. Four of the selected targets, BPSS0211 - BPSS0214, formed an operon, of these BPSS0212 and BPSS0213 were homologous, with BPSS0211 also representing the C-terminal domain of the two proteins. The structure of BPSS0211 was determined to 2.17 Å using multiplewavelength anomalous dispersion experimental phasing. BPSS0211 is a small protein annotated as containing a conserved domain of unknown function, DUF1843. The tertiary structure of BPSS0211 entails two alpha helices stacked together which assemble to form a tetramer comprising a dimer of dimers. The pattern of conservations between residues in DUF1843 within BPSS0211, BPSS0212 and BPSS0213 is such that interactions between the three proteins would be possible. The proposed function of DUF1843 is as an oligomerisation domain, allowing the formation of homo and hetero, dimer and tetramer, complexes of three proteins encoded within the BPSS0211-BPSS0214 operon. Other work included involves the determination of the structure of thioredoxin from Burkholderia pseudomallei as part of an on-going project into this system. The structure was solved to 1.07 Å using molecular replacement and agreed well with previously solved thioredoxin structures from other organisms.
Supervisor: Rice, David Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available