Understanding mis-metalation of native versus heterologously expressed protein

了解天然与异源表达蛋白质的错误金属化

• 批准号: BB/W015749/1
• 负责人: Nigel Robinson
• 金额: $ 58.16万
• 依托单位: Durham University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW015749%2F1
• 关键词: Understanding; mis; metalation; native; versus

All known life depends on metals, microbial and plant. The special chemical properties of different metals expand the repertoire of what proteins can do unaided. This requires the chemically correct metal(s) to associate with each metalloprotein. It is tempting to assume that such metalation is a sole function of the proteins themselves, but purified proteins typically bind wrong metals many orders of magnitude more tightly than the correct ones. We now appreciate that correct metalation is a combined function of proteins and their biological surroundings. Proteins compete with other molecules for each metal. Over the millennia this competition has evolved such that the correct metals generally win and mis-metalation is largely avoided: But does this mean that proteins are liable to mis-metalation when expressed in different organisms or if the cellular milieu is somehow altered? DNA-binding metal sensors are tuned to compete for available metal with the intracellular milieu (Nature Chemical Biology 2017 13:409-417). Over the course of nearly a decade, supported by the BBSRC, a series of thermodynamic values were collected for a set of DNA-binding metal sensors (from Salmonella), making it possible to calculate the magnitude of intracellular competition for each metal (Nature Chemical Biology 2019 15:241-0249). By reference to these values, it has become possible to make predictions about the metalation of metalloproteins. We have recently developed a metalation calculator that provides such calculations in Salmonella and closely related E. coli (Nature Communications 2021 12: 1195). It is now necessary to test such calculations in other organisms.Some metals are embedded into cofactors. Proteins that supply metals to cofactors, such as cobalt to vitamin B12, must also somehow acquire the correct metal. Notably, plants do not make B12 generating interest in the supply of this vitamin for the increasing numbers of individuals turning to low meat diets. E. coli also does not make B12 but the CoI has engineered (by introduction of more than 30 genes) E. coli to generate a B12 hyper-producing strain. We have shown that in the synthetic E. coli system the cobalt delivery protein for B12 is susceptible to mis-metalation with zinc but mismetalation is overcome when cells are supplemented with surplus cobalt. Is such mis-metalation a function of a mis-match between the introduced proteins and the cellular milieu? We will express, purify and prepare the deduced or known Rhizobium proteins involved in sensing zinc and cobalt, and in cobalt delivery for B12, in a manner suitable for determining metal affinities and DNA-affinities for the sensors. Sensor abundance will also be determined. A hybrid Rhizobium metalation calculator will thus be produced that can determine metalation and mis-metalation of the B12 pathway with cobalt and zinc (or iron) in this native host system. B12 production will be measured and metalation calculated both in free living Rhizobium and in root nodules. One central purpose of this work is to further develop (and exemplify the utility of) the metalation calculator with widespread applications/implications in biology. This work also has implications for the supply of B12 to those on plant-based diets (refer to closing statement on advancing to higher TRLs). Rhizobium makes B12 in nodules of leguminous plant roots and thus cobalt promotes legume growth. An ability to design seed dressings (potentially containing cobalt or with a zinc chelator), to enhance nitrogen fixation also has implications for global sustainability since the manufacture of nitrogen fertiliser represents up to 2% of energy demands. The PI and CoI directed phase I, and now direct phase II, of a BBSRC Network in Industrial Biotechnology and Bioenergy with the express purpose of accelerating the exploitation of such advances in metals in biology to support biotechnology.

所有已知的寿命都取决于金属，微生物和植物。不同金属的特殊化学特性扩大了蛋白质可以单独进行的曲目。这需要化学正确的金属才能与每种金属蛋白相关。假设这种金属化是蛋白质本身的唯一功能，但是纯化的蛋白质通常比正确的蛋白质更紧密地结合了许多数量级。现在，我们感谢正确的金属化是蛋白质及其生物学环境的综合功能。蛋白质与每种金属的其他分子竞争。在几千年中，这种竞争的发展已经发展，以至于正确的金属通常会赢得胜利，并且在很大程度上避免了误差：但是，这是否意味着在不同生物体中表达或在某种程度上改变了细胞环境时，蛋白质在表达时可能会误解？ DNA结合金属传感器被调整为与细胞内环境的可用金属竞争（自然化学生物学2017 13：409-417）。在近十年的时间里，在BBSRC的支持下，为一组DNA结合金属传感器（来自沙门氏菌）收集了一系列热力学值，从而可以计算每种金属的细胞内竞争幅度（自然化学生物学2019 15：241-0249）。参考这些值，可以对金属蛋白的金属进行预测。我们最近开发了一种金属化计算器，该计算器可在沙门氏菌和密切相关的大肠杆菌中提供此类计算（自然通信2021 12：1195）。现在有必要在其他生物体中测试此类计算。一些金属嵌入辅助因子中。向辅因子供应金属的蛋白质，例如维生素B12的钴，也必须以某种方式获取正确的金属。值得注意的是，植物不会使B12对这种维生素的供应产生兴趣，以至于越来越多地转向肉类饮食的个体数量越来越多。大肠杆菌也不制造B12，但是COI已设计（通过引入30多个基因）大肠杆菌生成B12超生产菌株。我们已经表明，在合成大肠杆菌系统中，B12的钴递送蛋白容易与锌合作误差，但是当细胞中补充了多余的钴时，克服了不易裂。如此错误的位置是否是引入蛋白质与细胞环境之间错误匹配的函数？我们将以适合确定传感器的金属亲和力和传感器的DNA率的方式表达，净化和制备与传感锌和钴相关的推导或已知的根瘤菌蛋白，以及B12的钴递送。传感器丰度也将被确定。因此，将产生一种杂交根茎金属化计算器，该计算器可以确定该天然宿主系统中用钴和锌（或铁）的B12途径的金属化和错误位置。将测量B12的产生，并在自由生活根茎和根结节中计算金属化。这项工作的一个核心目的是进一步发展（并体现）金属化计算器的实用性，并在生物学中具有广泛的应用/含义。这项工作也对B12的供应对基于植物的饮食的供应有影响（请参阅有关前进到更高TRL的结束声明）。根茎在豆科植物根部的结节中生产B12，因此钴促进了豆科植物的生长。设计种子敷料的能力（可能含有钴或锌螯合剂），以增强氮固定，也对全球可持续性产生了影响，因为氮肥的生产占能源需求的2％。 PI和COI在工业生物技术和生物能源方面的BBSRC网络的第一阶段（现在是II阶段）的明确目的是加速生物技术中金属的这种进步以支持生物技术。

项目成果

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

大肠杆菌菌株 JM109 (DE3) 的金属化计算器：在 LB 培养基中培养的需氧、厌氧和过氧化氢暴露细胞

• DOI: 10.1101/2022.05.06.490408
• 发表时间: 2022
• 作者: Foster A

Protein metalation in a nutshell.

• DOI: 10.1002/1873-3468.14500
• 发表时间: 2023-01
• 期刊: FEBS LETTERS
• 影响因子: 3.5
• 作者: Osman, Deenah;Robinson, Nigel J.
• 通讯作者: Robinson, Nigel J.

Hyperaerated metalation calculator for E. coli strain JM109 (DE3) grown in LB media

用于在 LB 培养基中生长的大肠杆菌菌株 JM109 (DE3) 的过度通气金属化计算器

• DOI: 10.1101/2022.09.02.506343
• 发表时间: 2022
• 作者: Clough S

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

• DOI: 10.1093/mtomcs/mfac058
• 发表时间: 2022-09-01
• 期刊: METALLOMICS
• 影响因子: 3.4
• 作者: Foster, Andrew W.;Clough, Sophie E.;Aki, Zeynep;Young, Tessa R.;Clarke, Alison R.;Robinson, Nigel J.
• 通讯作者: Robinson, Nigel J.