Abstract:AbstractSensitive protein stability assays for membrane proteins are crucial for developing purification protocols, for structural and biophysical characterisation and drug discovery. Here, we describe a novel high-throughput 384-well FRET-based thermostability methodology, ThermoFRET, allowing for the ultrasensitive determination of G protein coupled receptor (GPCR) stability. This method measures FRET between a terbium-cryptate labelled GPCR and BODIPY-FL-Cystine, a thiolreac… Show more
“…Labeling of the SNAP tag on the N terminus of the receptor with Lumi4-Tb allowed thermostability to be investigated without purifying the receptor. β 2 AR unfolding was initially measured by quantifying TR-FRET between Lumi4-Tb and BODIPY FL L-Cystine that covalently reacted with cysteines that become exposed as the receptor unfolded ( Tippett et al., 2020 ). Figure 4 Thermostability of membrane, DDM and DIBMALP preparations of β2AR (A–C) ThermoFRET thermostability curves in (A) β 2 AR membranes, (B) DDM-solubilized β 2 AR, (C) DIBMALP-β 2 AR in the presence and absence of cyanopindolol (100 μM) and F-propranolol (200 nM).…”
“…Labeling of the SNAP tag on the N terminus of the receptor with Lumi4-Tb allowed thermostability to be investigated without purifying the receptor. β 2 AR unfolding was initially measured by quantifying TR-FRET between Lumi4-Tb and BODIPY FL L-Cystine that covalently reacted with cysteines that become exposed as the receptor unfolded ( Tippett et al., 2020 ). Figure 4 Thermostability of membrane, DDM and DIBMALP preparations of β2AR (A–C) ThermoFRET thermostability curves in (A) β 2 AR membranes, (B) DDM-solubilized β 2 AR, (C) DIBMALP-β 2 AR in the presence and absence of cyanopindolol (100 μM) and F-propranolol (200 nM).…”
“…In comparison to our previous ThermoFRET application using a terbium cryptate labelled receptor as a FRET donor, [29] the ThermoBRET approach offers potential advantages. ThermoFRET requires cell surface labelling of the receptor-fused SNAP tag with the terbium cryptate donor molecule, adding to assay cost, but perhaps more importantly creating an extra labelling step that can be problematic if the tag is not readily exposed at the plasma membrane.…”
Measurements of membrane protein thermostability reflect ligand binding. Current thermostability assays often require protein purification or rely on pre‐existing radiolabelled or fluorescent ligands, limiting their application to established targets. Alternative methods, such as fluorescence‐detection size exclusion chromatography thermal shift, detect protein aggregation but are not amenable for high‐throughput screening.
Here, we present a ThermoBRET method to quantify the relative thermostability of G protein coupled receptors (GPCRs), using cannabinoid receptors (CB1 and CB2) and the b2‐adrenoceptor (b2AR) as model systems. ThermoBRET reports receptor unfolding, does not need labelled ligands and can be used with non‐purified proteins. It uses Bioluminescence Resonance Energy Transfer (BRET) between Nanoluciferase (Nluc) and a thiol‐reactive fluorescent dye that binds cysteines exposed by unfolding. We demonstrate that the melting point (Tm) of Nluc‐fused GPCRs can be determined in non‐purified detergent solubilised membrane preparations or solubilised whole cells, revealing differences in thermostability for different solubilising conditions and in the presence of stabilising ligands. We extended the range of the assay by developing the thermostable tsNLuc by incorporating mutations from the fragments of split‐Nluc (Tm of 87 ⁰C versus 59 ⁰C). ThermoBRET allows the determination of GPCR thermostability, which is useful for protein purification optimisation and for drug discovery screening.
“…In comparison to our previous ThermoFRET application using a Tb3+ labelled receptor as a FRET donor (Tippett, Hoare et al 2020), the ThermoBRET approach offers potential advantages. ThermoFRET requires cell surface labelling of the receptorfused SNAP tag with the Tb3+ donor molecule, adding to assay cost but perhaps more importantly creating an extra labelling step that can be problematic if the tag is not readily exposed.…”
Section: Discussionmentioning
confidence: 99%
“…Recently, we reported a ThermoFRET assay that uses the principles of time-resolved Förster resonance energy transfer (TR-FRET) between a Tb3+ labelled receptor and a thiol-reactive fluorescent dye that covalently binds to the cysteines exposed upon temperature induced unfolding of a GPCR (Tippett, Hoare et al 2020). The extreme distance dependence of FRET (usually occurring only where donor:acceptor distances are <10 nm) allows the detection of bimolecular proximity in a homogenous manner, omitting purification steps to remove components which confound traditional measurements.…”
Sensitive assays to measure the thermostability of membrane proteins are important tools in protein purification optimisation and drug discovery. Here, we present a ThermoBRET method to quantify the relative thermostability of G protein coupled receptors (GPCRs), using cannabinoid receptor 2 (CB2) as an example. This method applies the principles of Bioluminescence Resonance Energy Transfer (BRET) between Nanoluciferase (Nluc) and a thiol-reactive fluorescent dye that covalently binds cysteines in the GPCR transmembrane domain, exposed by unfolding. We demonstrate that the melting point (Tm) of Nluc-fused GPCRs can be determined in non-purified detergent solubilised membrane preparations, revealing differences in thermostability for different detergent solubilising conditions and in the presence of stabilising ligands. In addition, we extended the range of the assay by developing the thermostable tsNLuc by incorporating mutations from the fragments of split-Nluc (Tm of 87 °C vs 59 °C). ThermoBRET allows the high-throughput determination of GPCR thermostability which will be useful for protein purification optimisation strategies and as part of a drug discovery screening platform.
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