TY - JOUR T1 - Measurement of non-purified GPCR thermostability using the homogenous ThermoBRET assay JF - bioRxiv DO - 10.1101/2020.08.05.237982 SP - 2020.08.05.237982 AU - Bradley L. Hoare AU - Amandeep Kaur AU - Clare R. Harwood AU - Nicola C. Dijon AU - Nicholas D. Holliday AU - David A. Sykes AU - Dmitry B. Veprintsev Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/08/17/2020.08.05.237982.abstract N2 - 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.Competing Interest StatementThe authors have declared no competing interest. ER -