Abstract
Measurements of membrane protein thermostability allows indirect detection of ligand binding. Current thermostability assays require protein purification or rely on pre-existing radiolabelled or fluorescent ligands, limiting their application to established target proteins. Alternative methods detect protein aggregation which requires sufficiently high level of protein expression.
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 β2-adrenoceptor (β2AR) 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 vs 59 ⁰C). ThermoBRET allows determination of GPCR thermostability, which is useful for protein purification optimisation and as part of drug discovery screening strategies.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Significant revision of the text and addition of new data
↵# The full-length CB1 receptor contains an unusually long (around 117 amino acids) and likely unstructured N-terminal domain. It was therefore truncated at the N-terminus in order to bring the Nluc tag in proximity with the transmembrane helices.