Mapping Cell Envelope and Periplasm Protein Interactions of Escherichia coli Respiratory Formate Dehydrogenases by Chemical Cross-Linking and Mass Spectrometry
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- 作者：Michael Zorn ; Christian H. Ihling ; Ralph Golbik ; R. Gary Sawers ; Andrea Sinz
- 刊名：Journal of Proteome Research
- 出版年：2014
- 出版时间：December 5, 2014
- 年：2014
- 卷：13
- 期：12
- 页码：5524-5535
- 全文大小：505K
- ISSN：1535-3907
文摘
During anaerobic growth Escherichia coli synthesizes two large, highly homologous respiratory formate dehydrogenases (Fdh鈥檚), Fdh-N and Fdh-O, which are associated with the inner membrane but have their respective active site located within the periplasm. The Fdh-N enzyme extends 90 脜 into the periplasmic compartment, which in E. coli ranges between 100 and 150 脜 from the inner to the outer membrane leaflet. To date, little is known about the interaction partners of Fdh-N and Fdh-O in the periplasmic space that might be involved in stabilizing these enzymes after maturation and translocation across the cytoplasmic membrane has occurred. To address this question, we performed chemical cross-linking in combination with mass spectrometry. We present for the first time the identification of cell envelope interaction partners of Fdh-N and -O from anaerobically grown E. coli using a heterobifunctional amine/photo-reactive cross-linker followed by mass spectrometric analysis of the cross-linked products. We additionally mapped the interface regions within the Fdh/protein complexes for four selected Fdh-binding partners, the chaperone Skp, the l,d-transpeptidase ErfK, OppA, and TolB. Our work yields first structural and functional insights into the mechanisms that support the postmaturation of the multisubunit enzymes Fdh-N and Fdh-O in the periplasm of E. coli.