Measurement of non-purified GPCR thermostability using the homogenous ThermoBRET assay

Author:

Hoare Bradley L.ORCID,Kaur Amandeep,Harwood Clare R.,Dijon Nicola C.ORCID,Holliday Nicholas D.ORCID,Sykes David A.ORCID,Veprintsev Dmitry B.ORCID

Abstract

ABSTRACTSensitive 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.

Publisher

Cold Spring Harbor Laboratory

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