期刊
JOURNAL OF PROTEOME RESEARCH
卷 16, 期 7, 页码 2457-2471出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.7b00068
关键词
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
资金
- Australian Research Council (ARC) [DP110100190]
- The Netherlands Organization for Scientific Research (NWO) [STW-Vici 12128]
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 delta and beta' subunit of RNA polymerase close to the downstream DNA channel, providing a clue into how delta 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.
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