Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.566316
Title: Structural studies on proteins from pathogenic bacteria
Author: Salmon, Richard Michael
ISNI:       0000 0004 2732 0362
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2012
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Abstract:
Burkholderia pseudomallei is a Class III bacterial pathogen of humans and the causative agent of melioidosis, a disease endemic to South East Asia and Australia. This work describes X-ray crystallographic and biochemical studies of the BPSL1204 protein from B. pseudomallei. The amino acid sequence of BPSL1204 possesses an N-terminal glyceryl-cysteine lipid-anchoring motif for covalent association with a membrane. After genetic truncation of the anchoring sequence, the BPSL1204 truncate was cloned, over-expressed and purified from E. coli before being subjected to crystallisation trials. The 1.05 A resolution crystal structure of BPSL1204 was solved by Selenomethionine MAD and revealed a two-domain fold similar to that of β Lactamase Inhibitory Protein I (BLIP-I). Like BLIP-I, the BPSL1204 structure consists of two similar domains. However TEM1 β-lactamase assays showed BPSL1204 to lack the general inhibitory activity of BLIP-I. Pull-down assays to uncover biological binding partners of the BPSL1204 homologue BCAL2351 from the paraphyletic Class II B. cenocepacia were undertaken. Although a methionyl-tRNA synthetase was isolated, no complex was seen to form between pure samples of the proteins. Antibodies raised against pure BPSL1204 were used to locate the BCAL2351 homologue in fractionated B. cenocepacia. BCAL2351 was found exclusively in the purified outer membrane by immunoblot analysis. Some evidence of binding of antibodies to whole B. cenocepacia cells was observed by fluorescence microscopy, indicative of external BCAL2351 and BPSL1204. However, other slides containing samples from the same culture showed no such binding, suggestive of internal or absent protein. Surface presence may be cell-cycle dependant. However, no immune response was found when BPSL1204 was challenged with anti-B. mallei human sera. Thus by a cross-disciplinary approach to investigating the structure and function of a protein of unknown function from the pathogenic B. pseudomallei, it was concluded that BPSL1204 is a protein with a BLIP-like fold potentially located on either the periplasmic or possibly outer face of the outer membrane of the bacterium.
Supervisor: Artymiuk, Peter J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.566316  DOI: Not available
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