Micropatterning for quantitative analysis of protein-protein interactions in living cells
Micropatterning for quantitative analysis of protein-protein interactions in living cells
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DOI：
10.1038/nmeth.1268
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发表时间：
2008-12-01
期刊：
影响因子：
48
通讯作者：
Schuetz, Gerhard J.
中科院分区：
文献类型：
--
作者：
Schwarzenbacher, Michaela;Kaltenbrunner, Martin;Schuetz, Gerhard J.
我们提出了一种方法来识别和表征活哺乳动物细胞中荧光团标记的蛋白质（“猎物”）和膜蛋白（“诱饵”）之间的相互作用。将细胞铺在用针对诱饵细胞外结构域的抗体功能化的微图案表面上。通过荧光猎物的重新分布来测定诱饵-猎物相互作用。我们使用该方法来表征人 CD4（T 细胞激活中的主要共受体）和人 Lck（早期 T 细胞信号传导所必需的蛋白酪氨酸激酶）之间的相互作用。我们通过量化 Lck 对 CD4 微图案的重新分布来测量平衡关联，并通过光漂白实验和单分子成像研究相互作用动力学。除了已知的锌扣结构外，Lck 膜锚对 Lck-CD4 相互作用尤其具有重大影响，介导直接结合并进一步稳定其他 Lck 结构域的相互作用。总的来说，膜锚定将相互作用寿命延长了两个数量级。
We present a method to identify and characterize interactions between a fluorophore-labeled protein ('prey') and a membrane protein ('bait') in live mammalian cells. Cells are plated on micropatterned surfaces functionalized with antibodies to the bait extracellular domain. Bait-prey interactions are assayed through the redistribution of the fluorescent prey. We used the method to characterize the interaction between human CD4, the major co-receptor in T-cell activation, and human Lck, the protein tyrosine kinase essential for early T-cell signaling. We measured equilibrium associations by quantifying Lck redistribution to CD4 micropatterns and studied interaction dynamics by photobleaching experiments and single-molecule imaging. In addition to the known zinc clasp structure, the Lck membrane anchor in particular had a major impact on the Lck-CD4 interaction, mediating direct binding and further stabilizing the interaction of other Lck domains. In total, membrane anchorage increased the interaction lifetime by two orders of magnitude.