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bioRxiv · 10.1101/2020.07.07.191957

ThermoFRET: A novel nanoscale G protein coupled receptor thermostability assay functional in crude solubilised membrane preparations

David N Tippett·Brad Hoare·Tamara Miljus·David A Sykes·Dmitry B. Veprintsev
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Abstract

Sensitive protein stability assays for membrane proteins are crucial for developing purification protocols, for structural and biophysical characterisation and drug discovery. Here, we describe a novel high-throughput 384-well FRET-based thermostability methodology, ThermoFRET, allowing for the ultrasensitive determination of G protein coupled receptor (GPCR) stability. This method measures FRET between a terbium-cryptate labelled GPCR and BODIPY-FL-Cystine, a thiolreactive dye that reacts with cysteine residues exposed upon protein unfolding in response to thermal denaturation. ThermoFRET is functional in crude solubilised membrane preparations, without protein purification and can detect receptor stabilising ligands, making it ideally suited for orphan receptor screening.

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David N Tippett, Brad Hoare, Tamara Miljus, David A Sykes, Dmitry B. Veprintsev. 2020-07-08. ThermoFRET: A novel nanoscale G protein coupled receptor thermostability assay functional in crude solubilised membrane preparations. https://doi.org/10.1101/2020.07.07.191957

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