Saturation transfer difference NMR on the integral trimeric membrane transport protein GltPh determines cooperative substrate binding

Hall, Jenny L., Sohail, Azmat, Cabrita, Eurico J., Macdonald, Colin, Stockner, Thomas, Sitte, Harald H., Angulo, Jesus and Macmillan, Fraser (2020) Saturation transfer difference NMR on the integral trimeric membrane transport protein GltPh determines cooperative substrate binding. Scientific Reports, 10 (1). ISSN 2045-2322

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Abstract

Saturation-transfer difference (STD) NMR spectroscopy is a fast and versatile method which can be applied for drug-screening purposes, allowing the determination of essential ligand binding affinities (KD). Although widely employed to study soluble proteins, its use remains negligible for membrane proteins. Here the use of STD NMR for KD determination is demonstrated for two competing substrates with very different binding affinities (low nanomolar to millimolar) for an integral membrane transport protein in both detergent-solubilised micelles and reconstituted proteoliposomes. GltPh, a homotrimeric aspartate transporter from Pyrococcus horikoshii, is an archaeal homolog of mammalian membrane transport proteins—known as excitatory amino acid transporters (EAATs). They are found within the central nervous system and are responsible for fast uptake of the neurotransmitter glutamate, essential for neuronal function. Differences in both KD’s and cooperativity are observed between detergent micelles and proteoliposomes, the physiological implications of which are discussed.

Item Type: Article
Faculty \ School: Faculty of Science > School of Chemistry
Faculty of Science
Faculty of Science > School of Pharmacy
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Depositing User: LivePure Connector
Date Deposited: 13 Oct 2020 00:05
Last Modified: 19 Oct 2020 23:57
URI: https://ueaeprints.uea.ac.uk/id/eprint/77224
DOI: 10.1038/s41598-020-73443-z

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