Gavutis Martynas, Lata Suman, Piehler Jacob
Institute of Biochemistry, Biocenter N210, Johann Wolfgang Goethe-University, Max-von-Laue Strasse 9, 60438 Frankfurt am Main, Germany.
Nat Protoc. 2006;1(4):2091-103. doi: 10.1038/nprot.2006.270.
This protocol describes an in vitro approach for measuring the kinetics and affinities of interactions between membrane-anchored proteins. This method is particularly established for dissecting the interaction dynamics of cytokines with their receptor subunits. For this purpose, the receptor subunits are tethered in an orientated manner onto solid-supported lipid bilayers by using multivalent chelator lipids. Interaction between the ligand with the receptor subunits was probed by a combination of surface-sensitive spectroscopic detection techniques. Label-free detection by reflectance interferometry is used for following assembly of the membrane and tethering of the receptor subunits in quantitative terms. Total internal reflection spectroscopy is used for monitoring ligand binding to the membrane-anchored receptor, for monitoring ligand-receptor interactions by FRET and for monitoring ligand-exchange kinetics. These assays can be used for determining the affinities and stabilities of ligand-receptor complexes in plane of the membrane. The techniques described in this protocol can be established in 2-3 months.
本方案描述了一种用于测量膜锚定蛋白之间相互作用的动力学和亲和力的体外方法。该方法特别适用于剖析细胞因子与其受体亚基之间的相互作用动力学。为此，通过使用多价螯合脂质，将受体亚基以定向方式连接到固体支持的脂质双层上。通过表面敏感光谱检测技术的组合来探测配体与受体亚基之间的相互作用。采用反射干涉术进行无标记检测，以定量跟踪膜的组装和受体亚基的连接。全内反射光谱用于监测配体与膜锚定受体的结合、通过荧光共振能量转移监测配体 - 受体相互作用以及监测配体交换动力学。这些测定可用于确定膜平面内配体 - 受体复合物的亲和力和稳定性。本方案中描述的技术可在2 - 3个月内建立起来。
