Surface plasmon resonance study of G protein/receptor coupling in a lipid bilayer-free system
Surface plasmon resonance study of G protein/receptor coupling in a lipid bilayer-free system
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DOI：
10.1021/ac051629t
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发表时间：
2006-02-15
影响因子：
7.4
通讯作者：
Koch, KW
中科院分区：
文献类型：
--
作者：
Komolov, KE;Senin, II;Koch, KW
表面等离子共振（SPR）光谱是一种实时研究蛋白质-蛋白质相互作用的技术；然而，就受体在生物传感器表面的功能性和均匀固定而言，应用SPR光谱研究膜受体是很困难的。在当前的研究中，我们开发了一种简单、直接、基于生物传感器的方法来监测 G 蛋白转导蛋白 (G) 和视紫红质 (Rho)（一种典型的 G 蛋白偶联受体 (GPCR)）之间的分子相互作用。洗涤剂溶解的暗适应 Rho 通过凝集素相互作用被捕获到生物传感器表面，从而实现受体的定点固定，使其细胞质表面可与偶联 G 蛋白接触。该系统在受体密度、闪光或恒定光照射后 G(t) 的结合、Gt 与 Rho 的快速 GTP 依赖性解离、Rho 的再生以及 Gt 结合对光强度和 Gt 浓度的依赖性方面与自然系统相似。 G(t)/Rho 相互作用的表观 K-D 为 13.6 nM。我们的结果验证了 SPR 光谱作为研究 GPCR 系统中 G 蛋白激活的工具的用途，并且可以扩展到 GPCR 的其他相互作用伙伴的应用。
Surface plasmon resonance (SPR) spectroscopy is a technique to study protein-protein interactions in real time; however, application of SPR spectroscopy for investigations of membrane receptors is difficult with respect to functional and uniform immobilization of receptors on a biosensor surface. In the current study, we developed a simple, direct, biosensor-based approach to monitor the molecular interactions between G protein transducin (G) and rhodopsin (Rho), a prototypical G protein-coupled receptor (GPCR). Detergent-solubilized dark-adapted Rho was captured onto a biosensor surface via lectin interaction, enabling site-directed immobilization of the receptor that made its cytoplasmic surface accessible to a coupling G protein. The system resembled the natural system with respect to receptor density, binding of G(t) following flash or constant light application, fast GTP-dependent dissociation of Gt from Rho, regeneration of Rho, and dependence of Gt binding on light intensity and on concentration of Gt. The apparent K-D of the G(t)/Rho interaction was 13.6 nM. Our results validate the use of SPR spectroscopy as a tool to study G protein activation in GPCR systems and could be extended for application to other interaction partners of GPCRs.