Real time monitoring of membrane GPCR reconstitution by plasmon waveguide resonance: on the role of lipids.
G-protein coupled receptors (GPCRs) are important therapeutic targets since more than 40% of the drugs on the market exert their action through these proteins. To decipher the molecular mechanisms of activation and signaling, GPCRs often need to be isolated and reconstituted from a detergent-solubilized state into a well-defined and controllable lipid model system. Several methods exist to reconstitute membrane proteins in lipid systems but usually the reconstitution success is tested at the end of the experiment and often by an additional and indirect method. Irrespective of the method used, the reconstitution process is often an intractable and time-consuming trial-and-error procedure. Herein, we present a method that allows directly monitoring the reconstitution of GPCRs in model planar lipid membranes. Plasmon waveguide resonance (PWR) allows following GPCR lipid reconstitution process without any labeling and with high sensitivity. Additionally, the method is ideal to probe the lipid effect on receptor ligand binding as demonstrated by antagonist binding to the chemokine CCR5 receptor.
Max Planck Institute for the Science of Light, Erlangen, Germany.|Friedrich Alexander University (FAU) Erlangen-Nürnberg, Erlangen, Germany.|Chemistry and Biology of Membranes and Nanoobjects, UMR 5248 CNRS, University of Bordeaux, Bat. B14 allée Geoffroy St. Hilaire, 33600 Pessac, France.
Pierre
P
Calmet
Department of Developmental Biology, Friedrich Alexander University of Erlangen-Nürnberg, Erlangen, Germany.
Monica
M
De Maria
Chemistry and Biology of Membranes and Nanoobjects, UMR 5248 CNRS, University of Bordeaux, Bat. B14 allée Geoffroy St. Hilaire, 33600 Pessac, France.
Etienne
E
Harté
Heptares Therapeutics Ltd, BioPark, Broadwater Road, Welwyn Garden City, Hertfordshire AL7 3AX, UK.
Daniel
D
Lamb
Heptares Therapeutics Ltd, BioPark, Broadwater Road, Welwyn Garden City, Hertfordshire AL7 3AX, UK.
Maria
M
Serrano-Vega
Heptares Therapeutics Ltd, BioPark, Broadwater Road, Welwyn Garden City, Hertfordshire AL7 3AX, UK.
Ali
A
Jazayeri
Department of Developmental Biology, Friedrich Alexander University of Erlangen-Nürnberg, Erlangen, Germany.|NanoTemper Technologies GmbH, Munich, Germany.
Nuska
N
Tschammer
Chemistry and Biology of Membranes and Nanoobjects, UMR 5248 CNRS, University of Bordeaux, Bat. B14 allée Geoffroy St. Hilaire, 33600 Pessac, France.
Isabel D
ID
Alves
D006801:Humans; D008563:Membrane Lipids / Q000737:chemistry*; D008567:Membranes, Artificial; D019713:Receptors, CCR5 / Q000737:chemistry*; D020349:Surface Plasmon Resonance / Q000379:methods*
C584933:CCR5 protein, human; D008563:Membrane Lipids; D008567:Membranes, Artificial; D019713:Receptors, CCR5