Bergsdorf, Christian, Fiez-Vandal, Cedric, Sykes, David, Bernet, Pascal, Aussenac, Sonia, Charlton, Steven, Schopfer, Ulrich, Ottl, Johannes and Duckely, Myriam (2016) An alternative thiol-reactive dye to analyze ligand interactions with the chemokine receptor CXCR2 using a new thermal shift assay format. Journal of Biomolecular Screening, 21 (3). pp. 243-251. ISSN 1087-05711552-454X

Abstract

Integral membrane proteins (IMP) play an important role in many cellular events and are involved in numerous pathological processes. Therefore, understanding the structure and function of IMPs is a crucial prerequisite to enable successful targeting of these proteins with low molecular weight (LMW) ligands early on in the discovery process. To optimize IMP purification/crystallization and to identify/characterize LMW ligand/target interactions, robust, reliable, high-throughput and sensitive biophysical methods are needed. Here, we describe a differential scanning fluorimerty (DSF) screening method using the thiol-reactive BODIPY®-FL-cystine dye to monitor thermal unfolding of the G-protein coupled receptor (GPCR), CXCR2. To validate this method the seven-transmembrane protein CXCR2 was analyzed with a set of well-characterized antagonists. This study showed that the new DSF assay assessed reliably the stability of CXCR2 in 384well format. The analysis of 14 ligands with a potency range over 4 log units demonstrated the detection/characterization of LMW ligands binding to the membrane protein target. Furthermore, DSF results cross-validated with the label-free differential static light scattering (DSLS) thermal denaturation method. These results underline the potential of the BODIPY® assay format as a general tool to investigate membrane proteins and their interaction partners.

Item Type:	Article
Keywords:	membrane protein, thermal shift assay, ligand interaction, thiol-reactive dye
Date Deposited:	25 Apr 2016 23:45
Last Modified:	04 Jul 2016 23:45
URI:	https://oak.novartis.com/id/eprint/25765