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Title: A structural investigation of bacterial twin-arginine translocation (tat) complexes by single-particle electron microscopy
Author: Beck, Daniel K.
ISNI:       0000 0004 2748 4850
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2012
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The Twin arginine translocase (Tat) pathway was first characterised in chloroplast thylakoid membranes in the late 1990s. It has since been identified in the plasma membranes of both Gram-positive and Gram-negative bacteria. Substrates of this transport system contain a critical twin-arginine motif within their cleavable Nterminal signal sequence and the majority are large co-factor containing proteins. There is now considerable evidence that Tat systems can transport such globular proteins in a fully folded state. The minimal components required for transport in E.coli are TatA, TatB and TatC; these three integral membrane proteins are thought to form an active translocon. In Bacillus subtilis only TatA and TatC subunits are present, with TatA acting in a bifunctional manner to replace TatB. Little structural information is known about these multimeric integral membrane protein complexes due to the inherent difficulty in purifying them and their compositional variability. Complexes formed by B. subtilis TatAd and TatAyCy and E. coli TatE were investigated by single-particle EM analysis. An image processing protocol was developed to analyse and separate out individual Tat complexes based on their size. Using this method 3D electron density maps were generated of TatAd and TatE, which appear as small, ring-shaped complexes. Unlike E. coli TatA complexes, that have been shown to vary widely in size, those observed here appear small and homogeneous. These data conflict with the widely accepted ‘size-fitting pore’ model of Tat mediated translocation and rather support the alternative transient coalescent model. Additionally the first structural characterisation of a TatA-type mutant protein was performed revealing a dramatic polymerisation phenotype and indicating a primary role for the N-terminus in forming protein-protein interactions.
Supervisor: Not available Sponsor: Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC) ; Wellcome Trust (London, England) (055663/Z/98/Z)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QP Physiology