Volume 13 Supplement 1
Decrypting structural and functional changes in LeuTAaat atomic level employing LRET
© Sohail et al; licensee BioMed Central Ltd. 2012
Published: 17 September 2012
Neurotransmitter:sodium symporters (NSS) are integral membrane proteins that mediate the reuptake of monoamine neurotransmitters previously released into the synaptic cleft. They are of pharmacological significance because they are the target of many clinically important drugs. LeuT Aa , a leucine/alanine transporter is a bacterial homolog to NSS. Crystal structures of LeuT Aa with open to outward, occluded and inward-facing states have already been resolved at high resolution. Hence, LeuT Aa serves as a good paradigm for exploring the structure-function relationship of NSS proteins.
Methods and results
To investigate the structure-function relation in LeuT at atomic level we employ lanthanide-based resonance energy transfer (LRET). LRET-based measurements require the introduction of an LBT (lanthanide binding tag) to accommodate terbium as the donor element and fluorophores chemically linked to a cysteine residue as the acceptor element. LBT tags and cysteine are introduced at selected positions in LeuT Aa . Introduction of an LBT tag may lead to a functionally disturbed host protein. So to screen functional LBT-LeuT mutants we established the scintillation proximity assay in the lab. To date, after screening functional LBT mutants of LeuT Aa , we have measured the intramolecular distances at atomic level of LeuT in micelles. In order to validate these distances we want to see the distance changes after reconstitution into liposomes, a more native environment that allows to establish a sodium gradient; this is important since the NSS operates along a chemical gradient.
Our LRET measurements are expected to help us validate or propose models of substrate transport. Our future plan focusses on the reconstitution of LeuT Aa in liposomes to allow for distance measurements in a more native environment.
Supported by the Austrian Science Fund FWF (grant SFB3506 to H.H.S.) and partially funded by HEC (A.S.).
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.