Lanthanide resonance energy transfer-based distance

Transcription

Lanthanide resonance energy transfer-based distance
© Prof. Harald Sitte, 2011
moltag.univie.ac.at
1
Lanthanide resonance energy transfer-based distance
measurements in the glutamate transporter
E-mail: [email protected]
Address:
Medical University of Vienna, Center for Physiology and Pharmacology
Institute of Pharmacology
Währingerstrasse 13a
1090 VIENNA
AUSTRIA
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 cosubstrates, 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.
© Prof. Harald Sitte, 2011
moltag.univie.ac.at
2
Lanthanide resonance energy transfer (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.
"MolTag PhD Project SIT1" is the reference code for this vacancy.
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 channelproperties. The EAAC1-expressing oocytes will subsequently be exploited in LRET experiments.
"MolTag PhD Project SIT2" is the reference code for this vacancy.
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, LBTtolerating 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.
© Prof. Harald Sitte, 2011
moltag.univie.ac.at
3
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.
For scientific questions about the projects please contact Prof. H. Sitte.
Answers to administrative questions might be found under Frequently Asked Questions.
If you need additional information contact our office via e-mail ([email protected]).

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