Investigating the functional dynamics of membrane transport and associated proteins using advanced magnetic resonance spectroscopy

Protein structures were originally determined by X-ray crystallography, a technique which uses a regularly arranged lattice to measure the electron density of a protein in three-dimensional space. NMR later emerged as an alternative for solving solution-based structures but is generally limited to smallproteins or complexes.Membrane transporters are often multimeric, macromolecular systems that undergo large conformational changes. To understand these ‘machineries’ and their dynamic nature we must use an approach that is capable of capturing their various conformations.Site-directed spin labelling can be used in combination with continuous wave EPR techniques to investigate the structure, topology and chemical environments of the strategically placed probes. Pulsed Electron-Electron Double Resonance spectroscopy can precisely measure distances between two labels, thus allowing the conformations of these transporters to be mapped as they undergo their dynamic rearrangements.Here this approach has been applied to various systems in order to gain better insight into the functional dynamics of membrane-bound or -associated proteins. This includes members of the generally well-studied substrate binding proteins from ATP-binding cassette (ABC)-type transporters, where crystal structures have been published and an accepted model of function exists. This subclass of proteins is responsible for binding a huge range of natural substrates, and the reported inter-lobe flexibilities can vary widely. Further to this, preliminary investigations of a multimeric secondary active transporter (the first of its family to have its structure resolved) are reported here, with the view to work towards answering several questions such as binding order and potential synchronisation of the monomers.The systems studied here are of interest due to their medical relevance; substrate binding domains are vital for sequestering nutrients in many cell types that are involved in disease (e.g. pathogens and cancer), and the malfunction of many mammalian homologues of secondary active transporters have been linked to neurological disorders