posted on 2023-12-26, 15:07authored byJared
P. Mohr, Arianne Caudal, Rong Tian, James E. Bruce
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.