Resolving coarse–grained dynamic distance constraints of the transport cycle of NSS transporters using EPR spectroscopy

Tsalagradas, Petros (2025) Resolving coarse–grained dynamic distance constraints of the transport cycle of NSS transporters using EPR spectroscopy. Doctoral thesis, University of East Anglia.

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Abstract

The amino acid transporter LeuT from Aquifex aeolicus is an extensively studied bacterial homologue of the neurotransmitter:sodium symporters (NSS), particularly the Solute Carrier 6 (SLC6) family. SLC6 transporters possess a pivotal role in the nervous system by regulating neurotransmission and their dysfunction leads to a wide range of neurological diseases rendering them important pharmacological targets. LeuT was the first SLC6 protein to be crystallised and a unique LeuT–fold was identified which was conserved across species. So far, LeuT remains as the principal reference to generate transport cycle models and make inferences regarding the structural dynamics for the eukaryotic counterparts. Since crystallographic information lacks the ability to provide dynamic insights on membrane transporters, alternative biophysical methodologies are necessary for an in–depth understanding of these systems. In this study, the combination of Site–Directed Spin Labelling (SDSL) technology and a variety of both experimental and computational Electron Paramagnetic Resonance (EPR) spectroscopic methods was utilised on selective LeuT cysteine mutants to monitor the structural dynamics underpinning the transport cycle in the conventional detergent micelles as well as the more native proteoliposomes. More specifically, the optimisation of the entire sample preparation procedure resulted in high–quality, spin labelled protein material and subsequent continuous wave EPR spectroscopic data linked with spectral simulations, in silico predictions, spin label accessibility and coarse–grained distance restraints obtained by pulsed EPR measurements demonstrated global/subtle, ion/ligand–dependent conformational rearrangements on the extracellular and cytoplasmic sides of LeuT. Furthermore, distances between coupled spin labels on single–labelled cysteine mutants revealed the formation of dimeric complexes in detergent micelles. Although this study provided invaluable structural, dynamic insights on LeuT, it also set the foundations for more elaborate experimental designs, such as the implementation of orthogonal spin labelling schemes, before generalised conclusions can be drawn on their physiological relevance.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Chemistry, Pharmacy and Pharmacology
Depositing User:	Chris White
Date Deposited:	10 Feb 2026 15:33
Last Modified:	10 Feb 2026 15:33
URI:	https://ueaeprints.uea.ac.uk/id/eprint/101896
DOI:

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