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

Abstract

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.

Probing drug-target engagement of soluble Gαi1protein using the SolThermoBRET thermal shift assay

“…The tsNLuc [20] was inserted into the human 10His-Gαi1 construct in pJ411 bacterial expression vector described earlier [9] between the his-tag and N terminus of the Gαi1. Sequence was confirmed by Sanger sequencing.…”

“…To address the critical need for an ultrasensitive thermostability assay applicable to non-purified soluble proteins, we adapted the ThermoBRET approach that we previously developed to measure the stability of membrane proteins, [19] and applied it to soluble proteins. SolThermoBRET monitors changes in BRET between a thermally stabilised nano-luciferase (tsNLuc) [20] tagged protein and SYPRO Red, a fluorescent dye that binds hydrophobic surfaces exposed as a soluble protein undergoes thermal denaturation. In the case of SolThermoBRET, the fluorescent properties of the dye SYPRO Red are better suited for excitation by the tsNLuc protein due to its lower reported background signal.…”