ThermoBRET: A Ligand‐Engagement Nanoscale Thermostability Assay Applied to GPCRs**

Abstract

AbstractMeasurements 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 to 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 β2‐adrenoceptor (β2AR) 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 °C versus 59 °C). ThermoBRET allows the determination of GPCR thermostability, which is useful for protein purification optimisation and drug discovery screening.