title: Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay.
abstract: Long-term functional stability of isolated membrane proteins is crucial for many in vitro applications used to elucidate molecular mechanisms, and used for drug screening platforms in modern pharmaceutical industry. Compared to soluble proteins, the understanding at the molecular level of membrane proteins remains a challenge. This is partly due to the difficulty to isolate and simultaneously maintain their structural and functional stability, because of their hydrophobic nature. Here we show, how scintillation proximity assay can be used to analyze time-resolved high-affinity ligand binding to membrane proteins solubilized in various environments. The assay was used to establish conditions that preserved the biological function of isolated human kappa opioid receptor. In detergent solution the receptor lost high-affinity ligand binding to a radiolabelled ligand within minutes at room temperature. After reconstitution in Nanodiscs made of phospholipid bilayer the half-life of high-affinity ligand binding to the majority of receptors increased 70-fold compared to detergent solubilized receptors--a level of stability that is appropriate for further downstream applications. Time-resolved scintillation proximity assay has the potential to screen numerous conditions in parallel to obtain high levels of stable and active membrane proteins, which are intrinsically unstable in detergent solution, and with minimum material consumption.
authors.affiliation: Department of Chemistry & Nano-Science Center, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Randi Westh
authors.initials: RW
authors.lastname: Hansen
authors.affiliation: Department of Chemistry & Nano-Science Center, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Xiaole
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Chemistry & Nano-Science Center, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Golab
authors.affiliation: CNRS UMR7242/Laboratoire d'excellence MEDALIS, Institut de Recherche de l'ESBS, Biotechnologie et Signalisation Cellulaire, Université de Strasbourg, Illkirch, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Bornert
authors.affiliation: Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Christine
authors.initials: C
authors.lastname: Oswald
authors.affiliation: CNRS UMR7242/Laboratoire d'excellence MEDALIS, Institut de Recherche de l'ESBS, Biotechnologie et Signalisation Cellulaire, Université de Strasbourg, Illkirch, France.
authors.forename: Renaud
authors.initials: R
authors.lastname: Wagner
authors.affiliation: Department of Chemistry & Nano-Science Center, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Karen Laurence
authors.initials: KL
authors.lastname: Martinez
mesh_terms: D003902:Detergents / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D049329:Nanostructures / Q000737:chemistry*; D010843:Pichia / Q000235:genetics; D011485:Protein Binding; D055550:Protein Stability; D017473:Receptors, Opioid, kappa / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
chemical_list: D003902:Detergents; D008024:Ligands; D008051:Lipid Bilayers; D017473:Receptors, Opioid, kappa; D019204:GTP-Binding Proteins