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Title: Proofreading of substrate by the Escherichia coli Twin Arginine Translocase
Author: Jones, Alexander Stephen
ISNI:       0000 0004 6497 3602
Awarding Body: University of Kent
Current Institution: University of Kent
Date of Award: 2017
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The Twin Arginine Translocase (Tat) is one of two protein translocation mechanisms in E. coli to move proteins across the inner bacterial membrane, from the cytosol to the periplasm. A unique feature of the Tat pathway is its ability to translocate fully folded proteins, indeed, in E. coli the Tat pathway preferentially transports correctly folded proteins. This 'proofreading' mechanism, as it has been dubbed, is of interest to the biopharmaceutical industry, however little is known of the mechanism by which Tat proofreads a substrates conformational state. Initial studies (chapter 3) addressed if the Tat proofreading mechanism sensed the surface charge or hydrophobicity of a substrate. To this end, surface residues of an scFv were mutated to create areas of charge and hydrophobicity without altering tertiary structure. Expression of these variants in E. coli revealed that Tat proofreading is tolerant of surface charge and hydrophobicity, but dependent on conformational flexibility. Further studies utilising a maquette in various folding states, confirmed Tat proofreading is sensitive to the structural rigidity of substrates (chapter 4). Investigations then went on to assess the quality of protein entering the periplasm via the Tat pathway by comparing it to the same protein transported by the General Secretory (Sec) pathway (chapter 5). This revealed, at least for a relatively simple biotheraputic, Tat-translocated protein is of the same quality to Sec-translocated protein. Finally, the question of what is responsible for the proofreading ability of Tat began to be addressed through C-terminal truncation studies of the Tat components that attempted to restore export of export-incompatible substrates (chapter 6).
Supervisor: Robinson, Colin ; Brown, David Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available