Abstract
Breakthroughs in crystallography and electron microscopy have allowed structural information on membrane proteins to become readily available, yet elucidating their detailed mechanisms of action remains a challenge. Secondary transporters are a class of membrane proteins that utilise the energy from coupling to transport substances from one side of the membrane to the other. The coupling of the downhill transport of an ion to the uphill accumulation of a substrate is a fundamental process that controls countless metabolic responses. Understanding how this happens from information obtained only from static structural snapshots is often impossible. As a consequence, linking structure to molecular mechanisms requires probing the conformational dynamics of membrane proteins; this however remains a considerable challenge using the existing biophysical toolkit.This thesis aims to investigate the molecular mechanism of a prototypical sugar transporter XylE using the synergistic power of hydrogen-deuterium exchange mass spectrometry (HDX- MS) and molecular dynamics (MD) simulations.
The first part of this thesis (chapter 2) focused on characterising the transport cycle and conformational dynamics of XylE upon binding to endogenous ligands (substrate-xylose, inhibitor-glucose). HDX-MS data together with MD simulations revealed that protonation at D27 controls the conformational transition and that only the substrate xylose can conformationally transition between states via an allosteric coupling.
The second part of this thesis (chapter 3) established a workflow combining integrative modelling with HDX-MS data and MD simulations to quantify the conformational landscape of XylE. The methodology was applied to characterise the structural dynamics of XylE upon binding to four different ligands, including xylose, glucose and two known glucose transporter (GLUT) inhibitors (phloretin and phloridzin). The results suggested substantially different binding effects for XylE between exogenous ligands (phloretin and phloridzin) and endogenous ligands (xylose and glucose).
Overall, I characterised the structural dynamics and ligand interaction of XylE, highlighting the ability of combining HDX-MS and MD simulations to explore the molecular mechanism of sugar transporters. Moreover, I demonstrated the potential of integrative modelling in interpreting HDX-MS data quantitatively and objectively.
| Date of Award | 1 Feb 2023 |
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| Original language | English |
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| Supervisor | Argyris Politis (Supervisor), Paula Booth (Supervisor) & Eamonn Reading (Supervisor) |