Driving Integrative Structural Modeling with Serial Capture Affinity Purification [article]

Xingyu Liu, Ying Zhang, Zhiui Wen, Yan Hao, Charles Banks, Jeffrey J Lange, Brian Slaughter, Jay Unruh, Laurence Florens, Susan Abmayr, Jerry Workman, Michael Washburn
2020 bioRxiv   pre-print
Streamlined characterization of protein complexes remains a challenge for the study of protein interaction networks. Here, we describe Serial Capture Affinity Purification (SCAP) where two separate proteins are tagged with either the HaloTag or the SNAP-tag, permitting a multi-step affinity enrichment of specific protein complexes. The multifunctional capabilities of these protein tagging systems also permit in vivo validation of interactions using FRET and FCCS quantitative imaging. When
more » ... ng SCAP to cross-linking mass spectrometry, an integrated structural model of the complex of interest can be generated. We demonstrate this approach using the Spindlin1 and SPINDOC chromatin associated protein complex, culminating in a structural model with two SPINDOC 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 Taken together, we present an integrated affinity purification, live cell imaging, and cross linking mass spectrometry approach for the building of integrative structural models of protein complexes.
doi:10.1101/2020.04.08.032151 fatcat:wc5kxu7obvhe3nr7j37itq5mz4