Understanding movement and mechanism in essential mammalian membrane transporters

The research presented within this thesis has utilised advanced magnetic resonance techniques to (a) probe fundamental unknowns of transport mechanisms in membrane transport proteins, and (b) test alternative approaches and magnetic resonance techniques for the study of these complex molecular machineries. The cation diffusion facilitator (CDF) proteins contribute to maintenance of divalent cation homeostasis, and are conserved throughout all domains of life. Malfunction of CDF proteins in humans is linked to a number of serious diseases, including Type-II diabetes and neurodegenerative diseases. MamM is a (magnetosome associated) CDF protein of magnetotactic bacteria and has more recently been used as a system on which to model the structure and function of the human proteins. Research within this thesis has extended studies of MamM’s C-terminal domain via electron paramagnetic resonance (EPR) spectroscopy. This has afforded insights into metal binding and metal type-dependent conformational change, metal binding stoichiometry and relevant affinities, and has aided in the demonstration (for the first time) that C-terminal domains of CDF proteins act as a first site for recognition and metal selectivity in CDF proteins. A separate section of this thesis presents the study of membrane transporter GltPh, a both structurally and functionally well-characterised member of the solute carrier 1 (SLC1) family. Whilst GltPh typically represents a model system for understanding of mammalian excitatory amino acid transporters (EAATs), here it has been used as a system upon which to test alternative strategies to probe conformational change, and magnetic resonance approaches typically not used for the study of membrane proteins. This work describes the potential use of conformation ’locking’ variants for deconvolution of complex EPR data, affording clearer insights into the solution state conformations adopted by this trimer under various conditions. A ligand-based NMR technique (known as STD NMR) has also been applied to demonstrate its use for KD determination of two competing ligands, in different protein environments. This work has also provided evidence for potential cooperativity in GltPh trimers, a behaviour which is unique to protein reconstituted into proteoliposomes