Measuring conformational changes in glutamate transporters by a lanthanide resonance energy transfer-based approach
Please notice these positions are no longer vacant.

Background
The tertiary structure of glutamate transporters can only be inferred from indirect evidence: to date, little is known about the molecular basis underlying the coupled translocation of substrate- and co-substrates, it is of more than fundamental interest to understand, how this process can be interpreted in the structural context of the glutamate transporter protein and of mutated versions thereof. Furthermore, it is still unknown, which conformations of the transporter proteins are involved in the various conductive state. However, the high-resolution structure of a glutamate-transporter GltPh, a bacterial orthologue to mammalian glutamate transporters, has recently become available (Yernool et al., 2004). This structure provides a starting point for comparative approaches: it allows assessing by educated guesses which residues are involved in ligand binding and how conformational changes account for the transport cycle.
Objective: the working hypothesis of the project is based on the high-resolution structure of GltPh that provides a structural framework for the determination of helical movements in EAAC1. These helical movements will be assessed in wild type transporter as well as mutants (probing the structural rearrangements) by distance measurements based on lanthanide resonance energy transfer (LRET). These data will allow us to assess a distinct picture of the structure/function relationship in glutamate transporter upon binding of different ligands including inhibitors and substrates.

LRET-based distance measurements in a prokaryotic homolog of a mammalian glutamate transporter
The student will learn to determine the extent/type of movements of helices in GltPh; Lanthanide Binding Tags (LBTs) will be inserted in designated extracellular areas of GltPh (guided by homology modelling and molecular dynamics simulations; together with G. Ecker). Uptake measurements proving the preserved function of the LBT-containing transporters will be conducted in proteoliposomes that contain purified and reconstituted GltPh. Furthermore, electrophysiological experiments will allow to test for the channel-properties in the reconstituted transporters. The reconstituted proteoliposomal preparation will subsequently be exploited in LRET experiments.


LRET-based distance measurements in a eurkaryotic glutamate transporter
The student will learn to determine the extent/type of movements of helices in EAAC1; LBTs will be inserted in designated extracellular areas of EAAC1. Uptake measurements proving the preserved function of the LBT-containing transporters will be conducted in vivo in Xenopus laevis oocytes expressing EAAC1. Furthermore, electrophysiological experiments will allow to test for the channel-properties. The EAAC1-expressing oocytes will subsequently be exploited in LRET experiments.

Methods
To achieve the objectives outlined in the showcases, the PhD-students will insert lanthanide binding tags (LBT) in designated extracellular areas of GltPh and EAAC1 (these areas will be based on a model/homology model established together with Gerhard F. Ecker); cysteines will be inserted to serve as docking points for acceptor fluorophores. Uptake measurements proving the preserved function of the LBT-containing transporters will be conducted and the positively evaluated, LBT-tolerating mutants will subsequently be exploited in LRET experiments to reveal the distances between LBTs chelating terbium (the donor) and Bodipy (the acceptor fluorophore). Alternatively, specific fluorescently labelled transporter ligands generated by Marko Mihovilovic will be used to serve as acceptor fluorophore for several donor LBTs.

Expected Results
The high-resolution crystal structure of GltPh provides a distinct framework to evolve structure/function relationships. LRET measurements, a technique recently established in our laboratory, allow to measure distances in proteins by exploiting the resonance energy transfer between terbium and an acceptor fluorophore. The measurements are of highest specificity because resonance energy transfer declines to the power of 6. Inward and outward facing conformations of the transporters will be tested in the presence and absence of transporter substrates and inhibitors, under different ionic conditions (intra- and extracellular) as well as under the influence of different mutations that alter the conformational equilibrium of the transporters.