title: Direct measurement of thermal stability of expressed CCR5 and stabilization by small molecule ligands.
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.
authors.affiliation: Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
authors.forename: Adam M
authors.initials: AM
authors.lastname: Knepp
authors.forename: Amy
authors.initials: A
authors.lastname: Grunbeck
authors.forename: Sourabh
authors.initials: S
authors.lastname: Banerjee
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Sakmar
authors.forename: Thomas
authors.initials: T
authors.lastname: Huber
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism; D018360:Crystallography, X-Ray; D003510:Cyclohexanes / Q000378:metabolism; D019793:Fluorescein / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D008024:Ligands; D008051:Lipid Bilayers / Q000378:metabolism; D000077592:Maraviroc; D009536:Niacinamide / Q000031:analogs & derivatives / Q000378:metabolism; D010879:Piperazines / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D055550:Protein Stability; D011743:Pyrimidines / Q000378:metabolism; D019713:Receptors, CCR5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013816:Thermodynamics; D014230:Triazoles / Q000378:metabolism
chemical_list: C523280:AD 101; D000911:Antibodies, Monoclonal; D003510:Cyclohexanes; D008024:Ligands; D008051:Lipid Bilayers; D010879:Piperazines; D011743:Pyrimidines; D019713:Receptors, CCR5; D014230:Triazoles; D009536:Niacinamide; D000077592:Maraviroc; C486781:vicriviroc; D019793:Fluorescein