Cargando…

Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay

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 p...

Descripción completa

Detalles Bibliográficos
Autores principales: Hansen, Randi Westh, Wang, Xiaole, Golab, Agnieszka, Bornert, Olivier, Oswald, Christine, Wagner, Renaud, Martinez, Karen Laurence
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4817975/
https://www.ncbi.nlm.nih.gov/pubmed/27035823
http://dx.doi.org/10.1371/journal.pone.0150658
_version_ 1782424947708133376
author Hansen, Randi Westh
Wang, Xiaole
Golab, Agnieszka
Bornert, Olivier
Oswald, Christine
Wagner, Renaud
Martinez, Karen Laurence
author_facet Hansen, Randi Westh
Wang, Xiaole
Golab, Agnieszka
Bornert, Olivier
Oswald, Christine
Wagner, Renaud
Martinez, Karen Laurence
author_sort Hansen, Randi Westh
collection PubMed
description 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.
format Online
Article
Text
id pubmed-4817975
institution National Center for Biotechnology Information
language English
publishDate 2016
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-48179752016-04-19 Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay Hansen, Randi Westh Wang, Xiaole Golab, Agnieszka Bornert, Olivier Oswald, Christine Wagner, Renaud Martinez, Karen Laurence PLoS One Research Article 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. Public Library of Science 2016-04-01 /pmc/articles/PMC4817975/ /pubmed/27035823 http://dx.doi.org/10.1371/journal.pone.0150658 Text en © 2016 Hansen et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Hansen, Randi Westh
Wang, Xiaole
Golab, Agnieszka
Bornert, Olivier
Oswald, Christine
Wagner, Renaud
Martinez, Karen Laurence
Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay
title Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay
title_full Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay
title_fullStr Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay
title_full_unstemmed Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay
title_short Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay
title_sort functional stability of the human kappa opioid receptor reconstituted in nanodiscs revealed by a time-resolved scintillation proximity assay
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4817975/
https://www.ncbi.nlm.nih.gov/pubmed/27035823
http://dx.doi.org/10.1371/journal.pone.0150658
work_keys_str_mv AT hansenrandiwesth functionalstabilityofthehumankappaopioidreceptorreconstitutedinnanodiscsrevealedbyatimeresolvedscintillationproximityassay
AT wangxiaole functionalstabilityofthehumankappaopioidreceptorreconstitutedinnanodiscsrevealedbyatimeresolvedscintillationproximityassay
AT golabagnieszka functionalstabilityofthehumankappaopioidreceptorreconstitutedinnanodiscsrevealedbyatimeresolvedscintillationproximityassay
AT bornertolivier functionalstabilityofthehumankappaopioidreceptorreconstitutedinnanodiscsrevealedbyatimeresolvedscintillationproximityassay
AT oswaldchristine functionalstabilityofthehumankappaopioidreceptorreconstitutedinnanodiscsrevealedbyatimeresolvedscintillationproximityassay
AT wagnerrenaud functionalstabilityofthehumankappaopioidreceptorreconstitutedinnanodiscsrevealedbyatimeresolvedscintillationproximityassay
AT martinezkarenlaurence functionalstabilityofthehumankappaopioidreceptorreconstitutedinnanodiscsrevealedbyatimeresolvedscintillationproximityassay