Detergent screening of a G-protein-coupled receptor using serial and array biosensor technologies.
We describe the benefits and limitations of two biosensor approaches for screening solubilization conditions for G-protein-coupled receptors (GPCRs). Assays designed for a serial processing instrument (Biacore 2000/3000/T100) and an array platform (Biacore Flexchip) were used to examine how effectively 96 different detergents solubilized the chemokine receptor CCR5 while maintaining its binding activity for a conformationally sensitive Fab (2D7). Using the serial processing instrument, we were able to analyze three samples in each 30-min binding cycle, thereby requiring approximately 24h to screen an entire 96-well plate of conditions. In-line capturing allowed us to normalize the 2D7 binding responses for different receptor capture levels. In contrast, with the array system, we could characterize the effects of all 96 detergents simultaneously, completing the assay in less than 1h. But the current array technology requires that we capture the GPCR preparations off-line, making it more challenging to normalize for receptor capture levels. Also, the array platform is less sensitive than the serial platforms, thereby limiting the size of the analyte to larger molecules (>5000Da). Overall, the two approaches proved to be highly complementary; both assays identified identical detergents that produced active solubilized CCR5 as well as those detergents that either were ineffective solubilizers or inactivated the receptor.
Rich
Rebecca L
RL
Center for Biomolecular Interaction Analysis, School of Medicine, University of Utah, Salt Lake City, 84132, USA.
Miles
Adam R
AR
Gale
Bruce K
BK
Myszka
David G
DG
D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D003902:Detergents / Q000592:standards*; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D008722:Methods; D019713:Receptors, CCR5 / Q000737:chemistry; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*; D012107:Research Design; D012995:Solubility
D003902:Detergents; D007140:Immunoglobulin Fab Fragments; D019713:Receptors, CCR5; D043562:Receptors, G-Protein-Coupled