title: Use of NMR saturation transfer difference spectroscopy to study ligand binding to membrane proteins.
abstract: Detection of weak ligand binding to membrane-spanning proteins, such as receptor proteins at low physiological concentrations, poses serious experimental challenges. Saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy offers an excellent way to surmount these problems. As the name suggests, magnetization transferred from the receptor to its bound ligand is measured by directly observing NMR signals from the ligand itself. Low-power irradiation is applied to a (1)H NMR spectral region containing protein signals but no ligand signals. This irradiation spreads quickly throughout the membrane protein by the process of spin diffusion and saturates all protein (1)H NMR signals. (1)H NMR signals from a ligand bound transiently to the membrane protein become saturated and, upon dissociation, serve to decrease the intensity of the (1)H NMR signals measured from the pool of free ligand. The experiment is repeated with the irradiation pulse placed outside the spectral region of protein and ligand, a condition that does not lead to saturation transfer to the ligand. The two resulting spectra are subtracted to yield the difference spectrum. As an illustration of the methodology, we review here STD-NMR experiments designed to investigate binding of ligands to the human sweet taste receptor, a member of the large family of G-protein-coupled receptors. Sweetener molecules bind to the sweet receptor with low affinity but high specificity and lead to a variety of physiological responses.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA.
authors.forename: Rani Parvathy
authors.initials: RP
authors.lastname: Venkitakrishnan
authors.forename: Outhiriaradjou
authors.initials: O
authors.lastname: Benard
authors.forename: Marianna
authors.initials: M
authors.lastname: Max
authors.forename: John L
authors.initials: JL
authors.lastname: Markley
authors.forename: Fariba M
authors.initials: FM
authors.lastname: Assadi-Porter
mesh_terms: D000818:Animals; D005947:Glucose / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism; D013223:Statistics as Topic
chemical_list: D008024:Ligands; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D005947:Glucose