- Project 1: Molecular insights into the role of lipids and regulatory proteins on sodium
- Project 2: NMR based structural and dynamic investigation of the transport cycle of NSS transporters
- Project 3: Purification, functional characterization and reconstitution of DAT and LeuT
- Project 4: Development of a comprehensive model of GABA transporter function
- Project 5: Electrophysiological characterization of neurotransmitter transporters and formulation of an overall kinetic model
- Project 6: Electrophysiological approaches to study ion coupling and transport by NSS
- Project 7: Structure and kinetic mechanism of neurotransmitter transporters
- Project 8: Develop novel approaches to measure transporter-ligand interactions
- Project 9: Integrative modelling of ligand binding, transporter stability and the transport cycle
- Project 10: Time resolved structural studies of membrane
- Project 11: Single-molecule studies of secondary-active transporters and their environmental dependence
- Project 12: Resolving coarse-grained dynamic distance constraints of the transport cycle of NSS transporters using EPR spectroscopy
- Project 13: Characterization of the molecular mode of action of novel psychoactive substances
- Project 14: Development of a microfluidic cell perfusion station for controlled compound delivery and real time fluorescence monitoring
- Project 15: Assessing the molecular determinants of novel disease causing mutations in DAT
Project 2: NMR based structural and dynamic investigation of the transport cycle of NSS transporters
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.
