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Title: An investigation into the structure and localisation of the bacterial twin arginine translocase (Tat) complexes by electron microscopy
Author: Smith, Sarah M.
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2015
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The twin arginine translocase (Tat) pathway is expressed in the inner membrane of both Gram-negative and Gram-positive bacteria. The model organism for each of these bacteria is Escherichia coli and Bacillus subtilis, respectively. In B. subtilis the Tat machinery is comprised of only TatA and TatC subunits. Namely, TatAdCd and TatAyCy translocases, which act in parallel, yet possess different substrate specificities. Little structural information is known about these multimeric integral membrane protein complexes owing to the difficulty in their purifications, small size and compositional heterogeneity. A primary structural investigation was performed on TatAyCy to gauge the true molecular weight and heterogeneity of this complex, providing fundamental insight into the effect that detergent solubilisation can have on a detergent-solubilised protein. Therefore, I circumvented the use of detergent and analysed Tat machinery in its native cellular environment. To achieve this, I developed a novel combinatorial approach of immunogold labelling with the volumetric microscopy technique- array tomography. The E. coli Tat system is comprised of three proteins: TatA, TatB and TatC. TatA is believed to constitute the protein-conducting element of the translocase machinery. However recent structural data has cast doubt on the biological significance of the properties of this protein, and consequently its role in the translocation process. Therefore, by using array tomography, I endeavoured to directly visualise TatA in its native cellular environment to gain insight into the localisation and distribution of this protein in E. coli. TatA was shown to cluster along the inner membrane in a TatBC-dependent manner. This is the first time that any Tat component has been visualised in 3D space, in situ. Additionally, the detailed insight into the exploitation of Tat machinery for the export of high value, biotechnologically-relevant protein was investigated; with a potential for this translocase to be used in cell surface display technologies revealed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology