TY - JOUR
T1 - In Situ Structural Restraints from Cross-Linking Mass Spectrometry in Human Mitochondria
AU - Ryl, Petra S.J.
AU - Bohlke-Schneider, Michael
AU - Lenz, Swantje
AU - Fischer, Lutz
AU - Budzinski, Lisa
AU - Stuiver, Marchel
AU - Mendes, Marta M.L.
AU - Sinn, Ludwig
AU - O'Reilly, Francis J.
AU - Rappsilber, Juri
N1 - Funding Information:
The authors thank Andrea Graziadei for the technical support for protein modeling and helpful discussions. The authors thank Dr. Philipp Selenko for the opportunity of using the cell culture and lab equipment for mitochondria isolation at the FMP, Berlin. The authors thank Dr. Janine Kirstein for valuable discussion and comments on their data on heat shock proteins. Furthermore, the authors thank Drs. James E. Bruce and Juan Chavez for critically reading the manuscript. This work was supported by the Einstein Foundation, the DFG (RA 2365/4-1, SFB-740 1-5000019-01-TP), and the Wellcome Trust through a Senior Research Fellowship to J.R. (103139) and a multiuser equipment grant (108504). The Wellcome Centre for Cell Biology is supported by core funding from the Wellcome Trust (203149).
Publisher Copyright:
Copyright © 2019 American Chemical Society.
PY - 2020/1/3
Y1 - 2020/1/3
N2 - 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.
AB - 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.
KW - comparative modeling
KW - cross-linking mass spectrometry
KW - human mitochondria
KW - in situ large-scale structural biology
KW - noncleavable DSS cross-linker
UR - http://www.scopus.com/inward/record.url?scp=85077475393&partnerID=8YFLogxK
U2 - 10.1021/acs.jproteome.9b00541
DO - 10.1021/acs.jproteome.9b00541
M3 - Article
C2 - 31746214
AN - SCOPUS:85077475393
SN - 1535-3893
VL - 19
SP - 327
EP - 336
JO - Journal of Proteome Research
JF - Journal of Proteome Research
IS - 1
ER -