title: Greatly enhanced detection of a volatile ligand at femtomolar levels using bioluminescence resonance energy transfer (BRET).
abstract: Our goal is to develop a general transduction system for G-protein coupled receptors (GPCRs). GPCRs are present in most eukaryote cells and transduce diverse extracellular signals. GPCRs comprise not only the largest class of integral membrane receptors but also the largest class of targets for therapeutic drugs. In all cases studied, binding of ligand to a GPCR leads to a sub-nanometer intramolecular rearrangement. Here, we report the creation of a novel chimaeric BRET-based biosensor by insertion of sequences encoding a bioluminescent donor and a fluorescent acceptor protein into the primary sequence of a GPCR. The BRET(2)-ODR-10 biosensor was expressed in membranes of Saccharomyces cerevisiae. Assays conducted on isolated membranes indicated an EC(50) in the femtomolar range for diacetyl. The response was ligand-specific and was abolished by a single point mutation in the receptor sequence. Novel BRET-GPCR biosensors of this type have potential application in many fields including explosive detection, quality control of food and beverage production, clinical diagnosis and drug discovery.
authors.affiliation: CSIRO Food Futures National Research Flagship & CSIRO Ecosystem Sciences, Australia, PO Box 1700, Canberra, ACT 2601, Australia. helen.dacres@csiro.au
authors.forename: Helen
authors.initials: H
authors.lastname: Dacres
authors.forename: Jian
authors.initials: J
authors.lastname: Wang
authors.forename: Virginia
authors.initials: V
authors.lastname: Leitch
authors.forename: Irene
authors.initials: I
authors.lastname: Horne
authors.forename: Alisha R
authors.initials: AR
authors.lastname: Anderson
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Trowell
mesh_terms: D000818:Animals; D060807:Bioluminescence Resonance Energy Transfer Techniques / Q000379:methods*; D015374:Biosensing Techniques / Q000379:methods*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D049410:Luciferases, Renilla / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D018035:Receptors, Odorant / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
chemical_list: D029742:Caenorhabditis elegans Proteins; D008024:Ligands; C109039:ODR-10 protein, C elegans; D043562:Receptors, G-Protein-Coupled; D018035:Receptors, Odorant; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D049410:Luciferases, Renilla