Metalation calculators for E. coli strain JM109 (DE3): aerobic, anaerobic, and hydrogen peroxide exposed cells cultured in LB media.
Metalation calculators for E. coli strain JM109 (DE3): aerobic, anaerobic, and hydrogen peroxide exposed cells cultured in LB media.
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DOI：
10.1093/mtomcs/mfac058
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发表时间：
2022-09-01
期刊：
影响因子：
3.4
通讯作者：
Robinson, Nigel J.
中科院分区：
文献类型：
--
作者：
Foster, Andrew W.;Clough, Sophie E.;Aki, Zeynep;Young, Tessa R.;Clarke, Alison R.;Robinson, Nigel J.
关键词：
已经生成了三个基于网络的计算器和三个类似的电子表格，根据已知的金属亲和力预测蛋白质在体内的金属占有率。这些计算利用了对电池内不稳定的不同金属缓冲池的可用性的估计。在这里，估计了一种在分子生物学和生物化学研究中常用的大肠杆菌菌株的金属可利用性，例如在生产重组蛋白中。通过定量聚合酶链式反应(QPCR)监测一组选定的金属反应转录本的丰度，检测了在Luria-Bertani(Luria-Bertani(LB))培养基中有氧、厌氧和对H_2O_2反应的细胞的金属利用率。所选择的基因由DNA结合的金属传感器调控，这些传感器已经在相关细菌细胞中进行了热力学表征，使得基因表达可以作为细胞内金属可获得性的函数读出，表达为形成金属络合物的自由能。计算器将这些值与与目标蛋白质形成络合物的自由能进行比较，这些自由能来自金属亲和力，以估计蛋白质与细胞内环境中可交换的结合部位竞争的效率。然后，计算器对不同的金属进行相互竞争，将场地的总占有率限制在最大化学计量比1，从而输出每种金属的占有率。除了提供这些新的和条件计算器之外，本文最初的目的是提供一个教程，讨论这种方法的限制，并介绍在基础和应用研究以及下一代制造中使用这种计算器的方法。细胞内蛋白质金属化的在线计算器。
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. On-line calculators of protein metalation in cells.
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影响因子：
6.4
作者：
Capdevila DA;Edmonds KA;Giedroc DP
通讯作者：
Giedroc DP
影响因子：
14.8
作者：
Foster AW;Pernil R;Patterson CJ;Scott AJP;Pålsson LO;Pal R;Cummins I;Chivers PT;Pohl E;Robinson NJ
通讯作者：
Robinson NJ
影响因子：
3.6
作者：
Anjem A;Varghese S;Imlay JA
通讯作者：
Imlay JA
影响因子：
64.8
作者：
Moore SJ;Sowa ST;Schuchardt C;Deery E;Lawrence AD;Ramos JV;Billig S;Birkemeyer C;Chivers PT;Howard MJ;Rigby SE;Layer G;Warren MJ
通讯作者：
Warren MJ
影响因子：
7.8
作者：
Foster AW;Young TR;Chivers PT;Robinson NJ
通讯作者：
Robinson NJ