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.
Highlights
L-glutamate is the primary excitatory neurotransmitter in the mammalian central nervous system
GltPh, a bacterial homolog of mammalian glutamate transporters from Pyrococcus horikoshii was the first member of the Solute Carrier 1 (SLC1) family of membrane transporters to have been crystallized while very recently a first crystal structure of human EAAT1 was determined facilitating extrapolation of results obtained for GltPh to its human counterparts[5,6,7,8]
Saturation-transfer difference (STD) NMR signals from γ- and β-protons of L-glutamate are observable in the presence but not in the absence of N a+
Summary
L-glutamate is the primary excitatory neurotransmitter in the mammalian central nervous system. ΑSTD and KD for L-glutamate are determined by fitting the observed experimental curve using Eq (2), and the resulting KD values for L-glutamate are 414 and 1034 μM for detergent-solubilised and proteoliposome-reconstituted GltPh respectively (Fig. 3) using the γ-protons (very similar results of 458 μM and 984 μM respectively were obtained if the β-protons were used).
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