Hatton, Caitlin (2021) Structural and functional characterisation of a cytosine transporter. PhD thesis, University of Warwick.

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Abstract

CodB, the putative cytosine transporter from Escherichia coli, is a member of the LeuT-fold and evolutionarily clusters with the hydantoin transporter Mhp1 from Microbacterium liquefaciens. Mhp1 was the first active secondary-transporter structurally characterised in all conformations of the alternating-access model. The aim of this thesis was to structurally and functionally characterise CodB to expand understanding of transport mechanism and the role of sodium in the nucleobase cation symporter 1 (NCS1) family. Chapter 3 describes the expression, solubilisation, and purification of CodB from different bacterial species to identify suitable constructs for downstream experiments. CodB from Proteus vulgaris was acknowledged as the best candidate for structural and functional studies due its ability to be solubilised and purified in appropriate quantities. Chapter 4 details the structural determination and analysis of CodB to a final resolution of 2.4 A, this structure is an outward-open conformation bound to both cytosine and sodium. This structure identifies the amino acids required for cytosine and sodium coordination as well as highlighting similarities and differences of CodB with other LeuT-fold proteins. Chapter 5 focuses on ligand binding and transport kinetics of CodB. Cytosine has been confirmed as a ligand for CodB and CodB mediated transport was confirmed to be sodium dependent, whilst other potential ligands and binding partners were screened;. Amino acids in the substrate and cation sites were mutated to understand their role in binding and transport.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QP Physiology Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Cytosine, Escherichia coli, Ligand binding (Biochemistry), ATP-binding cassette transporters, Membrane proteins
Official Date:	March 2021
Dates:	Date Event March 2021 UNSPECIFIED
Institution:	University of Warwick
Theses Department:	School of Life Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Cameron, Alexander
Format of File:	pdf
Extent:	193 leaves : illustrations
Language:	eng

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