ThermoBRET: A Ligand-Engagement Nanoscale Thermostability Assay Applied to GPCRs

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 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 beta 2-adrenoceptor (beta 2AR) 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 degrees C versus 59 degrees C). ThermoBRET allows the determination of GPCR thermostability, which is useful for protein purification optimisation and drug discovery screening. ThermoBRET assay detects unfolding of detergent solubilised non-purified GPCRs with genetically encoded N-terminus tsNluc. As the protein unfolds in the gradient PCR thermocycler due to thermal denaturation, sulfo-Cy3 maleimide (SCM) reacts with newly exposed cysteine residues putting the sulfo-Cy3 acceptor fluorophore in proximity with the tsNluc donor. Fitting the melting curve to a Boltzmann sigmoidal equation yields a melting point (Tm), that is increased in the presence of ligands that bind the receptor.**+image

Automated summary

Measurements of membrane protein thermostability can reflect ligand binding, but current methods have limitations. This study presents a new method, ThermoBRET, for quantifying the relative thermostability of GPCRs, which does not require purified proteins or labelled ligands.