Lanthanide resonance energy transfer-based distance measurements in the mammalian glutamate transporter excitatory amino acid transporter 3 (1064.12)

EAAT3 (Excitatory amino acid transporter 3) mediates the regulation of synaptic transmission by reuptake of glutamate in the synaptic cleft. The motivation of the study is to gain insight into the structure function relation of EAAT3. The project utilizes the high resolution crystal structure of GltPh, the bacterial orthologue of Pyrococcus horykoshi to mammalian glutamate transporters. The structural rearrangement of the protein is caused by the helical movements which will be assessed by distance measurements using the technique of lanthanide resonance energy transfer (LRET). LRET relies on Forster's theory of resonance energy transfer where there is distance dependent transfer of energy between the donor fluorochrome to an acceptor fluorochrome. The protein is expressed in Xenopus laevis oocytes. Lanthanide binding tags (LBT) has been inserted into the protein to chelate the lanthanide terbium which serves as the donor element and aminoacid residues in the protein are mutated to cysteine which then rea...