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Proofreading of substrate by the Escherichia coli Twin Arginine Translocase

Jones, Alexander Stephen (2017) Proofreading of substrate by the Escherichia coli Twin Arginine Translocase. Doctor of Philosophy (PhD) thesis, University of Kent,. (KAR id:65666)

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Abstract

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).

Item Type: Thesis (Doctor of Philosophy (PhD))
Thesis advisor: Robinson, Colin
Thesis advisor: Brown, David
Uncontrolled keywords: Protein translocation, Twin arginine translocase, Tat, General secretory, Sec, proofreading, quality control
Divisions: Divisions > Division of Natural Sciences > Biosciences
SWORD Depositor: System Moodle
Depositing User: System Moodle
Date Deposited: 08 Jan 2018 13:10 UTC
Last Modified: 08 Dec 2022 19:01 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/65666 (The current URI for this page, for reference purposes)

University of Kent Author Information

Jones, Alexander Stephen.

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