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.
Highlights
Domain rearrangements of individual proteins act as molecular switches, govern the assembly of complexes and regulate the activity of networks
Cross-linking combined with mass spectrometry and database searching is a tool that can reveal the topology and other structural details of proteins and their complexes [1,2,3]. It is currently unclear if dynamic information could be obtained from such straight applications of cross-linking/mass spectrometry (CLMS) analysis
We suggest a code of practice for the use of quantitative CLMS (QCLMS) in the study of protein dynamics
Summary
Domain rearrangements of individual proteins act as molecular switches, govern the assembly of complexes and regulate the activity of networks. A typical example of a predominantly conformational change-driven finely tuned protein-protein interaction network is the mammalian complement system of ~40 plasma and cell-surface proteins This is responsible for clearance of immune complexes from body fluids along with other hazards to health including bacteria and viruses. Cross-linking combined with mass spectrometry and database searching is a tool that can reveal the topology and other structural details of proteins and their complexes [1,2,3]. It is currently unclear if dynamic information could be obtained from such straight applications of cross-linking/mass spectrometry (CLMS) analysis. The mass spectrometric signals of cross-linked peptides derived from different conformations can be distinguished by their masses and quantified and related to conformational differences (Fig. 1A)
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