Dataset Information

In Situ Structural Restraints from Cross-Linking 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. Cross-linking mass spectrometry (CLMS) is contributing to this effort, typically through the use of mass spectrometry (MS)-cleavable cross-linkers. Here, we apply the popular noncleavable cross-linker disuccinimidyl suberate (DSS) to human mitochondria and identify 5518 distance restraints between protein residues. Each distance restraint on proteins or their interactions provides structural information within mitochondria. Comparing these restraints to protein data bank (PDB)-deposited structures and comparative models reveals novel protein conformations. Our data suggest, among others, substrates and protein flexibility of mitochondrial heat shock proteins. Through this study, we bring forward two central points for the progression of CLMS towards large-scale in situ structural biology: First, clustered conflicts of cross-link data reveal in situ protein conformation states in contrast to error-rich individual conflicts. Second, noncleavable cross-linkers are compatible with proteome-wide studies.

SUBMITTER: Ryl PSJ

PROVIDER: S-EPMC7010328 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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In Situ Structural Restraints from Cross-Linking Mass Spectrometry in Human Mitochondria.

Ryl Petra S J PSJ Bohlke-Schneider Michael M Lenz Swantje S Fischer Lutz L Budzinski Lisa L Stuiver Marchel M Mendes Marta M L MML Sinn Ludwig L O'Reilly Francis J FJ Rappsilber Juri J

Journal of proteome research 20191219 1

The field of structural biology is increasingly focusing on studying proteins in situ, i.e., in their greater biological context. Cross-linking mass spectrometry (CLMS) is contributing to this effort, typically through the use of mass spectrometry (MS)-cleavable cross-linkers. Here, we apply the popular noncleavable cross-linker disuccinimidyl suberate (DSS) to human mitochondria and identify 5518 distance restraints between protein residues. Each distance restraint on proteins or their interact ...[more]

PMID: 31746214

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Project description:Protein cross-linking and the analysis of cross-linked peptides by mass spectrometry is currently receiving much attention. Not only is this approach applied to isolated complexes to provide information about spatial arrangements of proteins, but it is also increasingly applied to entire cells and their organelles. As in quantitative proteomics, the application of isotopic labeling further makes it possible to monitor quantitative changes in the protein-protein interactions between different states of a system. Here, we cross-linked mitochondria from Saccharomyces cerevisiae grown on either glycerol- or glucose-containing medium to monitor protein-protein interactions under non-fermentative and fermentative conditions. We investigated qualitatively the protein-protein interactions of the 400 most abundant proteins applying stringent data-filtering criteria, i.e. a minimum of two cross-linked peptide spectrum matches and a cut-off in the spectrum scoring of the used search engine. The cross-linker BS3 proved to be equally suited for connecting proteins in all compartments of mitochondria when compared with its water-insoluble but membrane-permeable derivative DSS. We also applied quantitative cross-linking to mitochondria of both the growth conditions using stable-isotope labeled BS3. Significant differences of cross-linked proteins under glycerol and glucose conditions were detected, however, mainly because of the different copy numbers of these proteins in mitochondria under both the conditions. Results obtained from the glycerol condition indicate that the internal NADH:ubiquinone oxidoreductase Ndi1 is part of an electron transport chain supercomplex. We have also detected several hitherto uncharacterized proteins and identified their interaction partners. Among those, Min8 was found to be associated with cytochrome c oxidase. BN-PAGE analyses of min8Δ mitochondria suggest that Min8 promotes the incorporation of Cox12 into cytochrome c oxidase.

Dataset Information

In Situ Structural Restraints from Cross-Linking Mass Spectrometry in Human Mitochondria.

Publications

In Situ Structural Restraints from Cross-Linking Mass Spectrometry in Human Mitochondria.

Similar Datasets

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