Stowers Institute for Medical Research, Kansas City, MO 64110.
Stowers Institute for Medical Research, Kansas City, MO 64110;
Proc Natl Acad Sci U S A. 2020 Dec 15;117(50):31861-31870. doi: 10.1073/pnas.2007931117. Epub 2020 Nov 30.
Streamlined characterization of protein complexes remains a challenge for the study of protein interaction networks. Here we describe serial capture affinity purification (SCAP), in which two separate proteins are tagged with either the HaloTag or the SNAP-tag, permitting a multistep affinity enrichment of specific protein complexes. The multifunctional capabilities of this protein-tagging system also permit in vivo validation of interactions using acceptor photobleaching Förster resonance energy transfer and fluorescence cross-correlation spectroscopy quantitative imaging. By coupling SCAP to cross-linking mass spectrometry, an integrative structural model of the complex of interest can be generated. We demonstrate this approach using the Spindlin1 and SPINDOC protein complex, culminating in a structural model with two SPINDOC molecules docked on one SPIN1 molecule. In this model, SPINDOC interacts with the SPIN1 interface previously shown to bind a lysine and arginine methylated sequence of histone H3. Our approach combines serial affinity purification, live cell imaging, and cross-linking mass spectrometry to build integrative structural models of protein complexes.
蛋白质复合物的简化描述仍然是蛋白质相互作用网络研究的一个挑战。在这里，我们描述了串联捕获亲和纯化（SCAP），其中两个不同的蛋白质分别被 HaloTag 或 SNAP-tag 标记，允许特定蛋白质复合物的多步亲和富集。这种蛋白质标记系统的多功能特性还允许使用受体光漂白Förster 共振能量转移和荧光互相关光谱定量成像对相互作用进行体内验证。通过将 SCAP 与交联质谱法相结合，可以生成感兴趣的复合物的综合结构模型。我们使用 Spindlin1 和 SPINDOC 蛋白质复合物来演示这种方法，最终得到一个结构模型，其中有两个 SPINDOC 分子结合在一个 SPIN1 分子上。在该模型中，SPINDOC 与先前显示与组蛋白 H3 的赖氨酸和精氨酸甲基化序列结合的 SPIN1 界面相互作用。我们的方法结合了串联亲和纯化、活细胞成像和交联质谱法，构建了蛋白质复合物的综合结构模型。
