Unfolding the Structure of LeuT Employing Luminescence Resonance Energy Transfer

Abstract

Background: Neurotransmitter sodium symporters (NSS) are located in the brain and retrieve neurotransmitters from the synaptic cleft to end synaptic transmission. Solute carier class six proteins (SCLC6) are of great pharmacological importance in terms of their localization and function. The crystal structures obtained from a bacterial homolog, the leucine transporter LeuTAa, in open to outward, occluded and open to inward conformations are present in frozen state with high resolution. Due to its close kinship with SLC6 proteins, LeuTAa serves as a paradigm for these transporters.Methods: In order to address the dynamicity of the substrate transport cycle in LeuTAa, we use the Lanthanide based resonance energy transfer (LRET) technique. This method is a spin-off of the fluorescence resonance energy transfer method according to Forster employing the introduction of the genetically encoded lanthanide binding tags (LBT) as donor elements. Exogenous cysteine residues labelled with cysteine specific fluorophores are used as acceptor elements. This technique is an alternative to address the movement of helices, with great resolution and has been employed successfully to examine potassium channels.Results: We screened for the functional LBT_mutants using the scintillation proximity assay. The LeuT_A335-LBT-G336 mutant displayed function in terms of its binding activity. Within this background, we generated cysteine mutants. To date, we have successfully measured the intramolecular distances in different LBT_LeuT_Cys mutants. Furthermore, we observed intramolecular distance changes from these purified proteins in detergent micelles.Conclusion: Our LRET measurements will help us to understand the transport cycle and help to complete the missing steps in substrate transport cycle of LeuTAa. Currently, we focus on the reconstitution of purified LeuTAa into liposomes and have our LRET measurements in a reconstituted system that allows to use more physiological ionic gradients.