文章基本信息

标题：Saturation transfer difference NMR on the integral trimeric membrane transport protein GltPh determines cooperative substrate binding
本地全文：下载
作者：Jenny L. Hall ; Azmat Sohail ; Eurico J. Cabrita 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2020
卷号：10
期号：1
页码：1-9
DOI：10.1038/s41598-020-73443-z
出版社：Springer Nature
摘要：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.
其他摘要：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 ( K D ). Although widely employed to study soluble proteins, its use remains negligible for membrane proteins. Here the use of STD NMR for K D 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 K D ’s and cooperativity are observed between detergent micelles and proteoliposomes, the physiological implications of which are discussed.