Thesis
Structural, functional and mechanistic analysis of the Escherichia coli ammonium transporter AmtB
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2018
- Thesis identifier
- T15111
- Person Identifier (Local)
- 201583137
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- The exchange of ammonium across cellular membranes is an essential process in all kingdoms of life. Ammonium is a major source of nitrogen for bacteria, fungi, and plants, whereas in animal cells it is a cytotoxic waste metabolite, which must be excreted. The transport of ammonium is accomplished by the ubiquitous Ammonium transporter/Methylammonium permease/Rhesus (Amt/Mep/Rh) superfamily of membrane proteins. In addition to their fundamental role in facilitating the transport of metabolites, members of the Amt/Mep/Rh superfamily are important drivers of the virulence of infectious fungi. In humans, malfunctions of Rh proteins are linked to inherited haemolytic anaemia, stomatocytosis, and early onset depressive disorder, amongst many other human pathologies. Therefore, an improved understanding of the function of ammonium transporters has important medical implications alongside the elucidation of a central biological process. In spite of its general importance, a consensus on the pathway and mechanism of ammonium transport by this superfamily has not yet been achieved. The overall aim of this project is to use Escherichia coli AmtB, the paradigmatic, most intensely studied member of the Amt/Mep/Rh protein family and an integrative approach combining molecular genetics, biochemistry, biophysics and molecular dynamic simulations to gain valuable structural and functional information on this important protein family. A new in-solution structural approach combining Small Angle Neutron/X-ray Scattering with Molecular Dynamics simulation has been successfully developed providing a new tool to study membrane protein dynamics in solution. Moreover, a new mechanism for the deprotonation/translocation associated with ammonium transport through AmtB was suggested and demonstrated by using Solid Supported Membrane Electrophysiology (SSME) and Molecular Dynamics simulations on genetic variants. Furthermore, the influence of 1-palmitoyl-2-oleoyl phosphatidylglycerol (PG), a specific lipid known to structurally interact with AmtB, was assessed and it was suggested that this lipid is essential for translocation of ammonium across AmtB. Last but not least, the electrophysiological technique developed on AmtB has been extended to study the bacterial Rh and fungal Mep proteins to explore whether the mechanism I propose for AmtB is a common feature for the Amt/Mep/Rh protein family. Given the importance of this protein family in various fundamental biological processes and human diseases, this work may, in the long term, lead to the development of new therapeutic interventions.
- Advisor / supervisor
- Hoskisson, Paul
- Javelle, Arnaud
- Resource Type
- Note
- This thesis was previously held under moratorium from 28th June 2019 until 12th February 2024
- DOI
- Date Created
- 2018
- Former identifier
- 9912691288102996
Relations
Items
| Thumbnail | Title | Date Uploaded | Visibility | Actions |
|---|---|---|---|---|
|
|
PDF of thesis T15111 | 2021-07-02 | Public | Download |