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J Proteome Res. 2019 Nov 20. doi: 10.1021/acs.jproteome.9b00541. [Epub ahead of print]

In Situ Structural Restraints from Crosslinking Mass Spectrometry in Human Mitochondria.

Abstract

The field of structural biology is increasingly focusing on studying proteins in situ, i.e. in their greater biological context. Crosslinking mass spectrometry is contributing to this effort, typically through the use of MS-cleavable crosslinkers. Here, we apply the popular non-cleavable crosslinker disuccinimidyl suberate to human mitochondria and identify 5,518 distance restraints between protein residues. Each distance restraint on proteins or their interactions provides structural information within mitochondria. Comparing these restraints PDB deposited structures and comparative models detects novel protein conformations. Our data suggest amongst others substrates and protein flexibility of mitochondrial heat shock proteins. Through this study we bring forward two central points for the move of crosslinking mass spectrometry towards large-scale in situ structural biology: clustered better than error-rich individual conflicts of crosslink data with other structural data reveals in situ protein conformation states and non-cleavable crosslinkers are compatible with proteome-wide studies. This opens the field for in situ photo-crosslinking or other functionalised crosslinkers which were excluded so far from large-scale studies.

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