Metalation calculators for E. coli strain JM109 (DE3): Aerobic, anaerobic and hydrogen peroxide exposed cells cultured in LB media

Foster, Andrew W; Clough, Sophie E; Aki, Zeynep; Young, Tessa R; Clarke, Alison R; Robinson, Nigel J

Abstract

Three Web-based calculators, and three analogous spreadsheets, have been generated that predict in vivo metal occupancies of proteins based on known metal affinities. The calculations exploit estimates of the availabilities of the labile buffered pools of different metals inside a cell. Here, metal availabilities have been estimated for a strain of Escherichia coli that is commonly used in molecular biology and biochemistry research, e.g. in the production of recombinant proteins. Metal availabilities have been examined for cells grown in Luria-Bertani (LB) medium aerobically, anaerobically, and in response to H2O2 by monitoring the abundance of a selected set of metal-responsive transcripts by quantitative polymerase chain reaction (qPCR). The selected genes are regulated by DNA-binding metal sensors that have been thermodynamically characterized in related bacterial cells enabling gene expression to be read out as a function of intracellular metal availabilities expressed as free energies for forming metal complexes. The calculators compare these values with the free energies for forming complexes with the protein of interest, derived from metal affinities, to estimate how effectively the protein can compete with exchangeable binding sites in the intracellular milieu. The calculators then inter-compete the different metals, limiting total occupancy of the site to a maximum stoichiometry of 1, to output percentage occupancies with each metal. In addition to making these new and conditional calculators available, an original purpose of this article was to provide a tutorial that discusses constraints of this approach and presents ways in which such calculators might be exploited in basic and applied research, and in next-generation manufacturing.

Citation

Foster, A. W., Clough, S. E., Aki, Z., Young, T. R., Clarke, A. R., & Robinson, N. J. (2022). Metalation calculators for E. coli strain JM109 (DE3): Aerobic, anaerobic and hydrogen peroxide exposed cells cultured in LB media. Metallomics, 14(9), https://doi.org/10.1093/mtomcs/mfac058

• Journal Article Type: Article
• Acceptance Date: Jul 24, 2022
• Online Publication Date: Sep 1, 2022
• Publication Date: 2022-09
• Deposit Date: Aug 15, 2022
• Publicly Available Date: Oct 25, 2022
• Journal: Metallomics
• Print ISSN: 1756-5901
• Electronic ISSN: 1756-591X
• Publisher: Oxford University Press
• Peer Reviewed: Peer Reviewed
• Volume: 14
• Issue: 9
• DOI: https://doi.org/10.1093/mtomcs/mfac058
• Public URL: https://durham-repository.worktribe.com/output/1197108