The core of this project is to contribute to the development of a comprehensive understanding of substrate binding and recognition through the experimental determination of ligand binding sites and poses. Membrane proteins are still a major challenge when considering NMR solution studies, mainly due to their large size and the need to use detergents encumbering the use of protein- observed solution NMR experiments.
In this project a variety of ligand and protein solution NMR experiments will be combined to investigate the transport cycle of LeuT and DAT, which the NeuroTrans consortium can purify in large quantities. Project 2 will be mainly focused on ligand-observed NMR experiments combined with protein-observed experiments using 13C LeuT methyl labelling (A and IVL) in order to establish structure/function relationships and derive information concerning the dynamics of the transport cycle.
The main objectives of project 2 are:
- to establish an NMR methodology to study binding/release of substrate and co-transported ions. This will be achieved in close collaboration with project 3, in order to develop protocols for sample preparation suitable for NMR studies, the use of proteoliposomes and encapsulated SMALPS will be explored.
- to determine binding poses of bound ligands based on ligand observation NMR experiments. STD-NMR and water LOGSY NMR experiments will be used in combination with different ligands (including inhibitors) in direct or competition experiments.
- to determine transporter dynamics during ligand binding, occlusion and release. This will be achieved in collaboration with project 9 and 12 in order to integrate magnetic resonance driven information with modelling data to study the conformational changes of the transport cycle.