title: Direct NMR detection of the binding of functional ligands to the human sweet receptor, a heterodimeric family 3 GPCR.
abstract: We present a robust method for monitoring the binding of ligands to the heterodimeric (T1R2+T1R3) human sweet receptor (a family 3 GPCR receptor). The approach utilizes saturation transfer difference (STD) NMR spectroscopy with receptor proteins expressed on the surface of human epithelial kidney cells. The preparation investigated by NMR can contain either live cells or membranes isolated from these cells containing the receptor. We have used this approach to confirm the noncompetitive binding of alitame and cyclamate to the receptor and to determine that greatly reduced receptor binding affinity compared to wild-type brazzein explains the lack of sweetness of brazzein mutant A16C17. This approach opens new avenues for research on the mechanism of action of the sweet receptor and for the design of new noncalorigenic sweeteners.
authors.affiliation: National Magnetic Resonance Facility at Madison, Biochemistry Department, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706, USA. fariba@nmrfam.wisc.edu
authors.forename: Fariba M
authors.initials: FM
authors.lastname: Assadi-Porter
authors.forename: Marco
authors.initials: M
authors.lastname: Tonelli
authors.forename: Emeline
authors.initials: E
authors.lastname: Maillet
authors.forename: Klaas
authors.initials: K
authors.lastname: Hallenga
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
mesh_terms: D006801:Humans; D007700:Kinetics; D008024:Ligands; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism; D013549:Sweetening Agents / Q000737:chemistry* / Q000378:metabolism; D000098706:Taste Receptors, Type 2
chemical_list: D008024:Ligands; D010940:Plant Proteins; D043562:Receptors, G-Protein-Coupled; D013549:Sweetening Agents; C090521:brazzein protein, Pentadiplandra brazzeana; C431926:taste receptors, type 1; D000098706:Taste Receptors, Type 2