PXD051661 is an
original dataset announced via ProteomeXchange.
Dataset Summary
Title | Structural analysis of the HypCD apo- and holoproteins by crosslinking mass spectrometry |
Description | [NiFe]-hydrogenases catalyse the reversible splitting of hydrogen. The catalytic metal centre (NiFe(CN)2CO) is unique in biology, and assembled by an intricate protein machinery, in a process that is still being explored. We hypothesised a structural, ATP-dependent, mechanistic explanation for the assembly of the Fe(CN)2CO fragment via the HypCD complex. We carried out a crosslinking mass spectrometry (crosslinking MS) analysis to study the structure of the HypCD complex, both with (holoprotein) and without (apoprotein) the metal cofactor, and both with and without the addition of ATP. We used the UV-photoactivatable crosslinking reagent sulfo-SDA, which has been shown to have excellent performance when studying dynamic and flexible protein complexes. For photoactivation we used a high-powered LED which enabled the use of exceptionally short reaction times (20 seconds) and gave strikingly clear results. From the resulting crosslinked residue pair patterns identified, we were able to unambiguously distinguish between holoprotein with and without ATP. Crosslinks found in holoprotein, in the absence of ATP, suggested a “closed” protein conformation. When the HypCD holoprotein was crosslinked in the presence of ATP, two distinct crosslink bands were almost entirely absent, indicating that the protein conformation had shifted to an “open” conformation. Interestingly, no shift in protein conformation was evident in the crosslinked apoprotein, which implied that the cofactor was central to protein conformational dynamics. Crosslinking MS data helped to explain, and was in agreement with, protein structures predicted by AlphaFold2 (which were subsequently refined by density functional theory (DFT) modeling for placing the cofactor). Considering all the experimental data from this study led to the conclusion that the binding of ATP alone, not its hydrolysis, is required for the transfer of the Fe(CN)2CO fragment to the apo-hydrogenase large subunit. |
HostingRepository | PRIDE |
AnnounceDate | 2024-10-28 |
AnnouncementXML | Submission_2024-10-28_02:10:41.739.xml |
DigitalObjectIdentifier | https://dx.doi.org/10.6019/PXD051661 |
ReviewLevel | Peer-reviewed dataset |
DatasetOrigin | Original dataset |
RepositorySupport | Supported dataset by repository |
PrimarySubmitter | Adam Belsom |
SpeciesList | scientific name: Escherichia coli; NCBI TaxID: 562; |
ModificationList | iodoacetamide derivatized residue |
Instrument | Q Exactive HF |
Dataset History
Revision | Datetime | Status | ChangeLog Entry |
0 | 2024-04-23 06:11:29 | ID requested | |
⏵ 1 | 2024-10-28 02:10:42 | announced | |
Publication List
Keyword List
submitter keyword: [NiFe]-hydrogenase assembly, photocrosslinking MS,HypCD |
Contact List
Juri Rappsilber |
contact affiliation | Technische Universität Berlin, Chair of Bioanalytics, 10623 Berlin, Germany Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, UK Si-M/"Der Simulierte Mensch", a Science Framework of Technische Universität Berlin and Charité - Universitätsmedizin Berlin, Berlin, Germany |
contact email | juri.rappsilber@tu-berlin.de |
lab head | |
Adam Belsom |
contact affiliation | Technische Universität Berlin |
contact email | adam.belsom@tu-berlin.de |
dataset submitter | |
Full Dataset Link List
Dataset FTP location
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PRIDE project URI |
Repository Record List
[ + ]
[ - ]
- PRIDE
- PXD051661
- Label: PRIDE project
- Name: Structural analysis of the HypCD apo- and holoproteins by crosslinking mass spectrometry