Abstract
The transport of solutes across biological membranes is a fundamental process in nature. This thesis concerns a novel membrane transport protein known as LicB that is essential for the persistence and virulence of pathogenic bacteria in the human respiratory tract. LicB catalyses the uptake of choline from the host, which is an essential first step in a short metabolic pathway that attaches choline to the lipopolysaccharide. LicB is a putative high-affinity choline transporter with no significant sequence homology or functional similarity to other choline permeases and is a potential drug target. However, the structure and function of LicB remain unknown.Here, LicB was successfully overexpressed in Escherichia coli, solubilised in dodecyl-maltoside detergent and purified by affinity chromatography. Size exclusion chromatography, as well as blue native PAGE, showed that LicB could be purified as a monomer, although some nonspecific oligomerisation occurred. Purified LicB was experimentally determined to contain ten transmembrane alpha-helices, in agreement with bioinformatic predictions. A novel fluorescence assay for ligand binding in vitro was introduced that allows the first studies of the ligand binding affinity and specificity of LicB. LicB was selective in binding to choline and acetylcholine over related small molecules, confirming previous reports on substrate specificity. Finally, choline uptake assays were established after LicB was successfully reconstituted in synthetic proteoliposomes. This work thus provides the first experimental characterisation of the Choline Uptake Transporter (LicB-T) protein family.
A parallel project was focused on further optimisation of the expression and purification of a class of peripheral membrane-bound proteins, the acyl-CoA:alcohol O-acyltransferases, here exemplified by the Saccharomyces cerevisiae Atf1 and specific enzymes from papaya, melon and strawberry fruit. Efforts to express, purify and characterise these proteins are described in the final chapter of the thesis.
| Date of Award | 28 Sept 2021 |
|---|---|
| Original language | English |
| Awarding Institution |
|
| Supervisor | Paul Curnow (Supervisor) & Paul Race (Supervisor) |