Capture and reconstitution of G protein-coupled receptors on a biosensor surface
Capture and reconstitution of G protein-coupled receptors on a biosensor surface
复制标题
DOI：
10.1016/s0003-2697(03)00046-0
复制
发表时间：
2003-05-15
影响因子：
2.9
通讯作者：
Myszka, DG
中科院分区：
文献类型：
--
作者：
Stenlund, P;Babcock, GJ;Myszka, DG
表面等离子体共振（SPR）生物传感器提供了一个独特的机会，研究G蛋白偶联受体（GPCR）的结合活性在真实的时间与最少的样品制备。使用两种趋化因子受体（CXCR 4和CCR 5）作为模型系统，我们将粗细胞制剂中的蛋白质捕获到生物传感器表面上，并重建脂质环境以维持受体活性。使用构象依赖性抗体并通过表征天然趋化因子配体（基质细胞衍生因子批次）的结合特性来探测受体的构象状态。结果表明，洗涤剂增溶的受体是积极的配体结合在存在和不存在的重建双层。使用这种受体捕获方法有三个优点：（1）在固定到生物传感器表面之前不需要纯化受体，（2）通过捕获步骤受体被均匀地固定，和（3）受体可以以足够高的密度被捕获以允许研究相对低分子量的配体（2000-4000 Da）。我们还证明了受体对增溶条件敏感，这说明了使用SPR生物传感器快速筛选GPCR增溶条件的潜力。(C)2003 Elsevier Science（美国）。All rights reserved.
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.