Capture and reconstitution of G protein-coupled receptors on a biosensor surface.
Surface plasmon resonance (SPR) biosensors offer a unique opportunity to study the binding activity of G protein-coupled receptors (GPCRs) in real time with minimal sample preparation. Using two chemokine receptors (CXCR4 and CCR5) as model systems, we captured the proteins from crude cell preparations onto the biosensor surface and reconstituted a lipid environment to maintain receptor activity. The conformational states of the receptors were probed using conformationally dependent antibodies, and by characterizing the binding properties of a native chemokine ligand (stromal cell-derived factor 1alpha). The results suggest that the detergent-solubilized receptors are active for ligand binding in the presence and absence of a reconstituted bilayer. There are three advantages to using this receptor-capturing approach: (1) there is no need to purify the receptor prior to immobilization on the biosensor surface, (2) the receptors are homogeneously immobilized through the capturing step, and (3) the receptors can be captured at high enough densities to allow the study of relatively low-molecular-mass ligands (2000-4000Da). We also demonstrated that the receptors are sensitive to the solubilizing conditions, which illustrates the potential for using SPR biosensors to rapidly screen solublization conditions for GPCRs.
Stenlund
Peter
P
Center for Biomolecular Interaction Analysis, University of Utah, Salt Lake City 84132, USA.
Babcock
Gregory J
GJ
Sodroski
Joseph
J
Myszka
David G
DG
D000911:Antibodies, Monoclonal / Q000737:chemistry; D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D054377:Chemokine CXCL12; D019743:Chemokines, CXC / Q000032:analysis / Q000378:metabolism; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D019713:Receptors, CCR5 / Q000737:chemistry / Q000378:metabolism; D019718:Receptors, CXCR4 / Q000737:chemistry / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D013997:Time Factors
D000911:Antibodies, Monoclonal; C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; D019743:Chemokines, CXC; D008051:Lipid Bilayers; D019713:Receptors, CCR5; D019718:Receptors, CXCR4; D043562:Receptors, G-Protein-Coupled