Multidimensional Cross-Linking and Real-Time Informatics for Multiprotein Interaction Studies

Chemical cross-linking combined with mass spectrometry is a technique used to study protein structures and identify protein complexes. Traditionally, chemical cross-linkers contain two reactive groups, allowing them to covalently bond a pair of proximal residues, either within a protein or between t...

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Veröffentlicht in:Journal of proteome research 2024-01, Vol.23 (1), p.107-116
Hauptverfasser: Mohr, Jared P., Caudal, Arianne, Tian, Rong, Bruce, James E.
Format: Artikel
Sprache:eng
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Zusammenfassung:Chemical cross-linking combined with mass spectrometry is a technique used to study protein structures and identify protein complexes. Traditionally, chemical cross-linkers contain two reactive groups, allowing them to covalently bond a pair of proximal residues, either within a protein or between two proteins. The output of a cross-linking experiment is a list of interacting site pairs that provide structural constraints for modeling of new structures and complexes. Due to the binary reactive nature of cross-linking reagents, only pairs of interacting sites can be directly observed, and assembly of higher-order structures typically requires prior knowledge of complex composition or iterative docking to produce a putative model. Here, we describe a new tetrameric cross-linker bearing four amine-reactive groups, allowing it to covalently link up to four proteins simultaneously and a real-time instrument method to facilitate the identification of these tetrameric cross-links. We applied this new cross-linker to isolated mitochondria and identified a number of higher-order cross-links in various OXPHOS complexes and ATP synthase, demonstrating its utility in characterizing complex interfaces. We also show that higher-order cross-links can be used to effectively filter models of large protein assemblies generated by using Alphafold. Higher-dimensional cross-linking provides a new avenue for characterizing multiple protein interfaces, even in complex samples such as intact mitochondria.
ISSN:1535-3893
1535-3907
1535-3907
DOI:10.1021/acs.jproteome.3c00455