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.

Rational antibody design for undruggable targets using kinetically controlled biomolecular probes

“…Second, short synthetic peptides can have limited structure similarity with the native target. A potential approach that would use larger antigens but still allow for precision targeting would be to first immunize animals with the full-length protein positioned in a lipid system (e.g, nanodisc) and incorporate smaller peptide antigens during screening (47)(48)(49).…”

Miniaturized weak affinity chromatography for ligand identification of nanodiscs-embedded G-protein coupled receptors

“…The SPA assay was adapted from Hansen et al 34 . Briefly, 100 ng of AA2AR-nanodiscs, 100 ng of biotinylated-AA2AR purified in detergent, 60 ng of empty nanodiscs or 10 µg of membrane preparation samples were incubated in triplicates in 96well plates (ProxiPlate-96, Perkin Elmer) in presence of 10 µL of streptavidin-coated yttrium silicate SPA beads (Perkin Elmer) and 25 nM of [3H]-ZM241385 in 100 µL final volume of binding buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 1 mM EDTA).…”

“…MSP1E3D1) was in vitro biotinylated using a NHS-PEG4-Biotin reagent. While the resulting AA2AR nanodisc brings the receptor a stable membrane-mimicking environment compatible with an extensive use in dynamic conditions 34 , it also allows a strong immobilization on streptavidin-coated supports via the biotin residues grafted on the MSP belt. Thanks to the presence of the PEG spacer arm that further maintains the assembly at distance from the support, the overall particle design thus ensures a full accessibility of the extramembrane domains of the receptor to the solvent and to any potential ligands it may contains.…”

Preparation of Recombinant Membrane Proteins from Pichia pastoris for Molecular Investigations

“…Applying preparative procedures similar to those described in this article, a large number of these IMPs could then be extracted from P. pastoris membranes and isolated to quantity and quality levels compatible with their thorough characterization at the molecular level. A rapid, far from exhaustive, survey conducted on the last decade actually retrieved an abundance of information on a variety of IMPs produced in these conditions, covering screening and functional characterization of compounds (Wöhri et al 2013;Logez et al 2014;Scalise et al 2014;Dekki-Shalaly et al 2015;Zehnpfenning et al 2015;Zollmann et al 2015;Westh Hansen et al 2016;Igonet et al 2018), interactions with and regulation by proteins (Bornert et al 2014;Rosell et al 2014;Doshi et al 2017;Damian et al 2018) and lipids (Schölz et al 2011;Brohawn et al 2014), functional impact of critical mutations (Kapri-Pardes et al 2011;Ampah-Korsah et al 2017;Yang et al 2017;Christenson et al 2018), as well as structural insights and mechanistic details observed at the atomic level (Hino et al 2012;Kodan et al 2014;Fan et al 2011;Vinothkumar et al 2016;Wang et al 2016;Lolicato et al 2017;Deng et al 2018;Eddy et al 2018;Ye et al 2018;Garaeva et al 2019;Wang et al 2020).…”

“…Similarly, specific activity assays are a mean of choice to evaluate the stability of IMP in solution. As an example, a time-resolved scintillation proximity assay was developed in a comparative analysis of the human kappa opioid receptor solubilized in various environment, showing that receptor stability was dramatically improved in nanodiscs compared to detergent (Westh Hansen et al 2016).…”