Equilibrium-fluctuation analysis for interaction studies between natural ligands and single G protein-coupled receptors in native lipid vesicles.
G protein-coupled receptors (GPCRs) constitute the most versatile family of cell-membrane receptors and have been increasingly identified as important mediators of many physiological functions. They also belong to one of the most central drug target classes, but current screening technologies are limited by the requirements of overexpression and stabilization of GPCRs. This calls for sensitivity-increased detection strategies preferably meeting single-molecule detection limits. This challenge is here addressed by employing total internal reflection fluorescence microscopy to characterize the interaction kinetics between CXCR3, a GPCR involved in inflammatory responses, and two of its chemokine ligands, CXCL10 and CXCL11. Fluorescence labeling of the lipid membrane, rather than the membrane protein itself, of GPCR-containing native vesicles, and immobilization of the corresponding ligand on the surface, enabled determination of the interaction kinetics using single-molecule equilibrium-fluctuation analysis. With a limit of detection of GPCR-containing vesicles in the low picomolar concentration regime, the results demonstrate the possibility to use inhibition in solution screening of high affinity ligands/drug candidates, which due to target-binding depletion of the inhibiting compounds is demanding using assays with more moderate detection limits.
Department of Applied Physics, Chalmers University of Technology , SE 41296 Gothenburg, Sweden.
Olov
O
Wahlsten
Discovery Sciences, AstraZeneca R&D Mölndal , S-43183 Mölndal, Sweden.
Anders
A
Gunnarsson
Department of Applied Physics, Chalmers University of Technology , SE 41296 Gothenburg, Sweden.
Lisa
L
Simonsson Nyström
Department of Applied Physics, Chalmers University of Technology , SE 41296 Gothenburg, Sweden.
Hudson
H
Pace
Discovery Sciences, AstraZeneca R&D Mölndal , S-43183 Mölndal, Sweden.
Stefan
S
Geschwindner
Department of Applied Physics, Chalmers University of Technology , SE 41296 Gothenburg, Sweden.
Fredrik
F
Höök
D005456:Fluorescent Dyes / Q000737:chemistry; D008024:Ligands; D008055:Lipids / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*; D013499:Surface Properties
D005456:Fluorescent Dyes; D008024:Ligands; D008055:Lipids; D043562:Receptors, G-Protein-Coupled