title: Direct detection of ligand binding to Sepharose-immobilised protein using saturation transfer double difference (STDD) NMR spectroscopy.
abstract: We report an easy and direct application of 'Saturation Transfer Double Difference' (STDD) NMR spectroscopy to identify ligands that bind to a Sepharose-immobilised target protein. The model protein, cytidine 5'-monophosphate sialic acid (CMP-Sia) synthetase, was expressed as a Strep-Tag II fusion protein and immobilised on Strep-Tactin Sepharose. STD NMR experiments of the protein-enriched Sepharose matrix in the presence of a binding ligand (cytidine 5'-triphosphate, CTP) and a non-binding ligand (alpha/beta-glucose) clearly show that CTP binds to the immobilised enzyme, whereas glucose has no affinity. This approach has three major advantages: (a) only low quantities of protein are required, (b) no specialised NMR technology or the application of additional data analysis by non-routine methods is required, and (c) easy multiple use of the immobilised protein is available.
authors.affiliation: Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Qld 9726, Australia.
authors.forename: Thomas
authors.initials: T
authors.lastname: Haselhorst
authors.forename: Anja K
authors.initials: AK
authors.lastname: Münster-Kühnel
authors.forename: Melanie
authors.initials: M
authors.lastname: Oschlies
authors.forename: Joe
authors.initials: J
authors.lastname: Tiralongo
authors.forename: Rita
authors.initials: R
authors.lastname: Gerardy-Schahn
authors.forename: Mark
authors.initials: M
authors.lastname: von Itzstein
mesh_terms: D001681:Biological Assay / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D000216:N-Acylneuraminate Cytidylyltransferase / Q000737:chemistry*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D012685:Sepharose / Q000737:chemistry*
chemical_list: D004800:Enzymes, Immobilized; D008024:Ligands; D012685:Sepharose; D000216:N-Acylneuraminate Cytidylyltransferase