PUBMED

Driving integrative structural modeling with serial capture affinity purification.

Liu, Xingyu, Zhang, Ying, Wen, Zhihui, Hao, Yan, Banks, Charles A S, Lange, Jeffrey J, Slaughter, Brian D, Unruh, Jay R, Florens, Laurence, Abmayr, Susan M, Workman, Jerry L, Washburn, Michael P

Data Revisão: 01/02/2021
Data Publicação: 30/11/2020 - [DOI: 10.1073/pnas.2007931117]
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JournalProceedings of the National Academy of Sciences of the United States of America

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.