Direct Measurement of Thermal Stability of Expressed CCR5 and Stabilization by Small Molecule Ligands
- 14 December 2010
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 50 (4), 502-511
- https://doi.org/10.1021/bi101059w
Abstract
The inherent instability of heptahelical G protein-coupled receptors (GPCRs) during purification and reconstitution is a primary impediment to biophysical studies and to obtaining high-resolution crystal structures. New approaches to stabilizing receptors during purification and screening reconstitution procedures are needed. Here we report the development of a novel homogeneous time-resolved fluorescence assay (HTRF) to quantify properly folded CC-chemokine receptor 5 (CCR5). The assay permits high-throughput thermal stability measurements of femtomole quantities of CCR5 in detergent and in engineered nanoscale apolipoprotein-bound bilayer (NABB) particles. We show that recombinantly expressed CCR5 can be incorporated into NABB particles in high yield, resulting in greater thermal stability compared with that of CCR5 in a detergent solution. We also demonstrate that binding of CCR5 to the HIV-1 cellular entry inhibitors maraviroc, AD101, CMPD 167, and vicriviroc dramatically increases receptor stability. The HTRF assay technology reported here is applicable to other membrane proteins and could greatly facilitate structural studies of GPCRs.Keywords
This publication has 42 references indexed in Scilit:
- NMR Analyses of the Interaction between CCR5 and Its Ligand Using Functional Reconstitution of CCR5 in Lipid BilayersJournal of the American Chemical Society, 2010
- Detergent screening of a G-protein-coupled receptor using serial and array biosensor technologiesAnalytical Biochemistry, 2009
- Chemokine receptor antagonists: overcoming developmental hurdlesNature Reviews Drug Discovery, 2008
- Microscale Fluorescent Thermal Stability Assay for Membrane ProteinsStructure, 2008
- Targeting Chemokine Receptors in HIV: A Status ReportAnnual Review of Pharmacology and Toxicology, 2008
- Conformational thermostabilization of the β1-adrenergic receptor in a detergent-resistant formProceedings of the National Academy of Sciences, 2008
- Monomeric G protein-coupled receptor rhodopsin in solution activates its G protein transducin at the diffusion limitProceedings of the National Academy of Sciences, 2007
- A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G proteinProceedings of the National Academy of Sciences, 2007
- Production and characterization of monoclonal antibodies sensitive to conformation in the 5HT2c serotonin receptorProceedings of the National Academy of Sciences of the United States of America, 2007
- Physiological Roles of G Protein–Coupled Receptor Kinases and ArrestinsAnnual Review of Physiology, 2007