Capture and reconstitution of G protein-coupled receptors on a biosensor surface

被引:85
作者
Stenlund, P
Babcock, GJ
Sodroski, J
Myszka, DG [1 ]
机构
[1] Univ Utah, Ctr Biomol Interact Anal, Salt Lake City, UT 84132 USA
[2] Harvard Univ, Sch Med, Div Aids, Dept Pathol, Boston, MA 02115 USA
[3] Harvard Univ, Sch Med, Dana Farber Canc Inst, Dept Canc & Immunol & AIDS, Boston, MA 02115 USA
关键词
D O I
10.1016/S0003-2697(03)00046-0
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 lot). 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 (2000-4000 Da). 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. (C) 2003 Elsevier Science (USA). All rights reserved.
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页码:243 / 250
页数:8
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