ThermoBRET: a ligand-engagement nanoscale thermostability assay applied to GPCRs

Abstract

Measurements of membrane protein thermostability allows indirect detection of ligand 3 binding. Current thermostability assays require protein purification or rely on pre4 existing radiolabelled or fluorescent ligands, limiting their application to established 5 target proteins. Alternative methods detect protein aggregation which requires 6 sufficiently high level of protein expression. 7 Here, we present a ThermoBRET method to quantify the relative thermostability of G 8 protein coupled receptors (GPCRs), using cannabinoid receptors (CB1 and CB2) and 9 the b2-adrenoceptor (b2AR) as model systems. ThermoBRET reports receptor 10 unfolding, does not need labelled ligands and can be used with non-purified proteins. 11 It uses Bioluminescence Resonance Energy Transfer (BRET) between 12 Nanoluciferase (Nluc) and a thiol-reactive fluorescent dye that binds cysteines 13 exposed by unfolding. We demonstrate that the melting point (Tm) of Nluc-fused 14 GPCRs can be determined in non-purified detergent solubilised membrane 15 preparations or solubilised whole cells, revealing differences in thermostability for 16 different solubilising conditions and in the presence of stabilising ligands. We extended 17 the range of the assay by developing the thermostable tsNLuc by incorporating 18 mutations from the fragments of split-Nluc (Tm of 87 ⁰C vs 59 ⁰C). ThermoBRET allows 19 determination of GPCR thermostability, which is useful for protein purification 20 optimisation and as part of drug discovery screening strategies.