Elucidating the mechanism of glutamate transporters using magnetic resonance spectroscopy

Hepworth, Shona (2023) Elucidating the mechanism of glutamate transporters using magnetic resonance spectroscopy. Doctoral thesis, University of East Anglia.

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The homotrimeric aspartate membrane transporters from Thermococcus kodakarensis (GltTk) and Pyrococcus horikoshii (GltPh) are archaeal homologues of mammalian glutamate transporters belonging to the Solute Carrier 1 (SLC1) family which includes the mammalian excitatory amino acid transporters (EAATs). Dysfunction of these systems can lead to neurological disorders such as schizophrenia, Alzheimer’s disease, and epilepsy. The EAATs regulate neurotransmitter levels in the synaptic cleft to prevent over-stimulation. GltPh is often used as a model system for the EAATs and is well studied, however, GltTk has been less well characterised especially with biophysical spectroscopic techniques.

This thesis works to further elucidate the proposed alternating access mechanism of the EAATs using a range of magnetic resonance techniques. Site-directed spin labelling using the cysteine specific spin probe MTSL has been used to study the functional dynamics of GltTk during the transport cycle. Continuous wave (cw) EPR spectroscopy was used to investigate the changes in local environment of the spin probe, and double electron-electron resonance (DEER) spectroscopy to follow conformational changes during the transport cycle. Different membrane mimetics were studied to investigate the importance of ‘biologically relevant’ environments on both protein conformation and dynamics. These included detergent micelles, proteoliposomes, nanodiscs and styrene maleic acid lipid particles (SMALPs). This work provides evidence of independent function of the three protomers in trimeric GltTk with evidence of potential cooperativity or steric effects when inhibitor bound. Saturation-Transfer Difference (STD) NMR, a ligand-based NMR technique, was used to determine apparent KD values for L-aspartate and L-glutamate with GltTk.

An orthogonal labelling approach was developed to further investigate the functional dynamics of GltPh. Successful Cu2+-NTA labelling was assessed using cw-EPR and DEER spectroscopy with the future aim of using advanced EPR (iDEER) methods to extract novel distance distributions.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Chemistry
Depositing User: Nicola Veasy
Date Deposited: 26 Jun 2024 10:50
Last Modified: 26 Jun 2024 10:50
URI: https://ueaeprints.uea.ac.uk/id/eprint/95688

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