- Title
- In-culture cross-linking of bacterial cells reveals large-scale dynamic protein-protein interactions at the peptide level
- Creator
- de Jong, Luitzen; de Koning, Edward A.; Hamoen, Leendert W.; de Koster, Chris G.; Roseboom, Winfried; Buncherd, Hansuk; Wanner, Martin J.; Dapic, Irena; Jansen, Petra J.; van Maarseveen, Jan H.; Corthals, Garry L.; Lewis, Peter J.
- Relation
- ARC.DP110100190 http://purl.org/au-research/grants/arc/DP110100190
- Relation
- Journal of Proteome Research Vol. 16, Issue 7, p. 2457-2471
- Publisher Link
- http://dx.doi.org/10.1021/acs.jproteome.7b00068
- Publisher
- American Chemical Society
- Resource Type
- journal article
- Date
- 2017
- Description
- Identification of dynamic protein–protein interactions at the peptide level on a proteomic scale is a challenging approach that is still in its infancy. We have developed a system to cross-link cells directly in culture with the special lysine cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). We used the Gram-positive model bacterium Bacillus subtilis as an exemplar system. Within 5 min extensive intracellular cross-linking was detected, while intracellular cross-linking in a Gram-negative species, Escherichia coli, was still undetectable after 30 min, in agreement with the low permeability in this organism for lipophilic compounds like BAMG. We were able to identify 82 unique interprotein cross-linked peptides with <1% false discovery rate by mass spectrometry and genome-wide database searching. Nearly 60% of the interprotein cross-links occur in assemblies involved in transcription and translation. Several of these interactions are new, and we identified a binding site between the δ and β′ subunit of RNA polymerase close to the downstream DNA channel, providing a clue into how δ might regulate promoter selectivity and promote RNA polymerase recycling. Our methodology opens new avenues to investigate the functional dynamic organization of complex protein assemblies involved in bacterial growth.
- Subject
- bacillus subtilis; in vivo cross-linking; bis(succinimidyl)-3-azidomethyl-glutarate (BAMG); diagonal strong cation exchange chromatography; RNA polymerase; delta subunit; mass spectrometry; NusA; glutamate dehydrogenase; ribosome biogenesis
- Identifier
- http://hdl.handle.net/1959.13/1350075
- Identifier
- uon:30485
- Identifier
- ISSN:1535-3893
- Rights
- This is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License, which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes.
- Language
- eng
- Full Text
- Reviewed
- Hits: 13906
- Visitors: 14737
- Downloads: 522
| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT02 | Publisher version (open access) | 3 MB | Adobe Acrobat PDF | View Details Download |