title: Quantitative cross-linking/mass spectrometry reveals subtle protein conformational changes.
abstract: Quantitative cross-linking/mass spectrometry (QCLMS) probes protein structural dynamics in solution by quantitatively comparing the yields of cross-links between different conformational statuses. We have used QCLMS to understand the final maturation step of the proteasome lid and also to elucidate the structure of complement C3(H2O). Here we benchmark our workflow 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.
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, EH9 3BF, UK.
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, EH9 3BF, UK.
authors.forename: Lutz
authors.initials: L
authors.lastname: Fischer
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, EH9 3BF, UK.
authors.forename: Salman
authors.initials: S
authors.lastname: Tahir
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, EH9 3BF, UK.
authors.forename: Jimi-Carlo
authors.initials: JC
authors.lastname: Bukowski-Wills
authors.affiliation: Schools of Chemistry and Biological Sciences, University of Edinburgh, Edinburgh, EH9 3JJ, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Barlow
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, EH9 3BF, UK.|Institute of Biotechnology, Technische Universität Berlin, Berlin, 13355, Germany.
authors.forename: Juri
authors.identifier: 0000-0001-5999-1310
authors.initials: J
authors.lastname: Rappsilber
keywords: automated data process; conformational change; cross-linking/mass spectrometry; isotope labeled cross-linkers; quantitative protein structure