Understanding ligand recognition in a mammalian membrane transporter (360G-Wellcome-211765_Z_18_Z)

GltPh is an homotrimeric aspartate transporter from the thermophile Pyrococcus horikoshii. This archaeal homolog of mammalian glutamate transporters belongs to the SLC1 transporter family, which also includes the human Excitatory Amino Acid Transporters (EAAT). The EAATs have been implicated in various neurological diseases including epilepsy and Alzheimer's disease. Crystal structures suggest large scale conformational changes and recent Electron Paramagnetic Resonance (EPR) spectroscopic studies have suggested a heterogeneous mixture of distances and have therefore remained inconclusive with respect to conformational changes associated with substrate transport. To dissect the underlying conformations, which contribute to the EPR spectra, we aim to purify and spin-label this transporter. Mutations have been designed to block the transporter in specific conformations. In silico predictions of further mutations for EPR experiments will be performed to identify suitable variants and subsequent spin labelling will be performed. The overall aim is to obtain a complete set of EPR results in various intermediate conformations of the transport cycle, both in detergent and reconstituted into proteolipsomes. In other studies, preliminary data indicate that the conformational stability of GltPh also depends on the status of ligand binding. We aim to extend our EPR studies to also address this ligand-dependant conformational stability.