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Quantitative cross-linking/mass spectrometry reveals subtle protein conformational changes

By Zhuo A Chen, Lutz Fischer, Salman Tahir, Jimi-Carlo Bukowski-Wills, Paul N. Barlow, Juri Rappsilber

Posted 25 May 2016
bioRxiv DOI: 10.1101/055418 (published DOI: 10.12688/wellcomeopenres.9896.1)

We have developed quantitative cross-linking/mass spectrometry (QCLMS) to interrogate conformational rearrangements of proteins in solution. Our workflow was tested using a structurally well-described reference system, the human complement protein C3 and its activated cleavage product C3b. We found that small local conformational changes affect the yields of cross-linking residues that are near in space while larger conformational changes affect the detectability of cross-links. Distinguishing between minor and major changes required robust analysis based on replica analysis and a label-swapping procedure. By providing workflow, code of practice and a framework for semi-automated data processing, we lay the foundation for QCLMS as a tool to monitor the domain choreography that drives binary switching in many protein-protein interaction networks.

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