Rapid Transfer of Transmembrane Proteins for Single Molecule Dimerization Assays in Polymer-Supported Membranes
Rapid Transfer of Transmembrane Proteins for Single Molecule Dimerization Assays in Polymer-Supported Membranes
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DOI：
10.1021/cb5005806
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发表时间：
2014-11-01
影响因子：
4
通讯作者：
Piehler, Jacob
中科院分区：
文献类型：
--
作者：
Roder, Friedrich;Wilmes, Stephan;Piehler, Jacob
跨膜受体的二聚化是细胞通讯的关键调节因素，在明确的体外条件下进行研究仍然具有挑战性。我们开发了一种新策略来探索需要微量样品的受控脂质环境中的膜蛋白相互作用。通过将跨膜蛋白从哺乳动物细胞快速转移到聚合物支持的膜中，膜蛋白可以被有效地荧光标记并在非常低的背景下重构。因此，可以通过单分子成像在生理相关浓度下定量探测I型干扰素（IFN）受体亚基IFNAR1和IFNAR2的差异配体诱导的二聚化。这些测量结果清楚地支持了 IFN 对 IFNAR1 的亲和力对控制受体二聚化水平的调节作用。
Dimerization of transmembrane receptors is a key regulatory factor in cellular communication, which has remained challenging to study under well-defined conditions in vitro. We developed a novel strategy to explore membrane protein interactions in a controlled lipid environment requiring minute sample quantities. By rapid transfer of transmembrane proteins from mammalian cells into polymer-supported membranes, membrane proteins could be efficiently fluorescence labeled and reconstituted with very low background. Thus, differential ligand-induced dimerization of the type I interferon (IFN) receptor subunits IFNAR1 and IFNAR2 could be probed quantitatively at physiologically relevant concentrations by single molecule imaging. These measurements clearly support a regulatory role of the affinity of IFNs toward IFNAR1 for controlling the level of receptor dimerization.