A calculator for metalation inside a cell

细胞内金属化计算器

• 批准号: BB/V006002/1
• 负责人: Nigel Robinson
• 金额: $ 63.79万
• 依托单位: Durham University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FV006002%2F1
• 关键词: calculator; metalation; inside; cell

Biotechnology is heavily dependent upon metal-containing enzymes since almost a half of all enzymes are estimated to require one or more bound metal. Sometimes the metals are embedded within cofactors, such as cobalt in vitamin B12, iron in heme or nickel in cofactor F430. Loading metalloenzymes, or loading the proteins that supply metals to cofactors, with the correct metal is a remarkable achievement of all living cells. Our long-term goal has been to understand intracellular metalation and to make this process predictable and exploitable. An easy-to-use, computer-based, calculator of metalation in vivo will be produced and tested.The availabilities of metals inside cells is a dominant factor in determining which metals bind to which proteins (Nature 2008 455: 1138-1142). The intracellular milieu buffers some metals to lower availabilities than other metals and DNA-binding metal sensors are tuned to these availabilities (Nature Chemical Biology 2017 13: 409-417). Over the course of seven years, supported by the BBSRC (plus Procter and Gamble), a series of thermodynamic values were collected for a set of DNA-binding metal sensors, making it possible to calculate the intracellular availabilities of metals in a bacterium (Nature Chemical Biology 2019 15: 241-249). By reference to these values for intracellular metal-availability, it has become possible to make coarse-grain predictions about the metalation of metalloproteins, specifically identifying the right metal for a protein and not merely the tightest-binding metal. Now we will refine (and test) these predictions to understand how metalation and mis-metalation changes as a function of culture conditions. An ability to make such predictions is relevant to the optimisation of metalation in synthetic biology, is of value to industrial biotechnology, but also relevant to basic understanding of cells in systems biology and more generally.Calculation of intracellular metal availabilities will exploit three conceptual advances: First that the cell biology of metals is rapid and associative via a labile buffer; second that the thermodynamic cycles of metal-sensors are mathematically tractable if metals are buffered (hence excluded from mass balance calculations); third that reference to these values for available metal enables calculation of the in vivo metalation state of a protein (or other molecule) that can account for inter-metal competition. The PI and CoI Warren directed a phase I, and now phase II, BBSRC Network in Industrial Biotechnology and Bioenergy with the purpose of accelerating the exploitation of advances in metals in biology research to support bio-manufacturing and other facets of biotechnology. A purpose of the work in this proposal is to make the optimisation of metalation accessible to a platform of industries, as well as to academics, who need not be experts in the sub-discipline. Applications could include the optimisation of metalation in bioprocesses or the optimisation of mis-metalation by antimicrobial ionophores, as examples. We will use three different systems to test three distinct types of predictions of the calculator. We have worked with all of the systems previously and obtained prior data supporting their choice for this work. One of the systems is E. coli that has been engineered by Warren to heterologously produce non-native tetrapyrroles: These tetrapyrroles include cofactor F430 and vitamin B12. The latter has immediate practical relevance. We have evidence (unpublished) that in the synthetic E. coli system the cobalt delivery protein for B12 becomes mis-metalated with zinc but that this can be overcome when cells are supplemented with surplus cobalt. A secondary purpose of this work is to manipulate this strain to enhance the supply of vitamin B12 for the increasing number of individuals turning to low meat diets (plants neither make nor require vitamin B12), in turn exemplifying the merit of the metalation calculator.

生物技术严重依赖含金属酶，因为据估计几乎一半的酶都需要一种或多种结合金属。有时这些金属嵌入辅因子中，如维生素B12中的钴、血红素中的铁或辅因子F430中的镍。装载金属酶，或装载为辅因子提供金属的蛋白质，是所有活细胞的一项了不起的成就。我们的长期目标是了解细胞内金属化，并使这一过程可预测和可利用。一个易于使用的，基于计算机的，体内金属化计算器将被制作和测试。细胞内金属的可用性是决定金属与蛋白质结合的主要因素（Nature 2008 455: 1138-1142）。细胞内环境将一些金属缓冲到比其他金属更低的可用性，dna结合金属传感器调整到这些可用性（Nature Chemical Biology 2017 13: 409-417）。在七年的时间里，在BBSRC（以及宝洁公司）的支持下，为一组dna结合金属传感器收集了一系列热力学值，从而可以计算细菌中金属的细胞内可用性（Nature Chemical Biology 2019 15: 241-249）。通过参考细胞内金属可用性的这些值，可以对金属蛋白的金属化进行粗粒度预测，特别是确定蛋白质的正确金属，而不仅仅是结合最紧密的金属。现在，我们将完善（并测试）这些预测，以了解金属化和错金属化如何随着培养条件的变化而变化。做出这种预测的能力与合成生物学中金属化的优化有关，对工业生物技术有价值，但也与系统生物学中对细胞的基本理解有关，甚至更广泛。细胞内金属可用性的计算将利用三个概念上的进步：首先，金属的细胞生物学是快速的，并通过一个不稳定的缓冲剂联想；其次，如果金属被缓冲（因此被排除在质量平衡计算之外），金属传感器的热力学循环在数学上是可处理的；第三，参考这些可用金属的值，可以计算蛋白质（或其他分子）的体内金属化状态，从而解释金属间竞争。PI和CoI Warren指导了第一阶段，现在是第二阶段，BBSRC工业生物技术和生物能源网络，目的是加速利用生物研究中金属的进步，以支持生物制造和生物技术的其他方面。本提案中工作的目的是使金属化的优化能够为行业平台以及学者提供便利，他们不需要成为该子学科的专家。例如，应用可以包括生物过程中的金属化优化或抗菌离子载体的错金属化优化。我们将使用三个不同的系统来测试计算器的三种不同类型的预测。我们之前与所有系统合作过，并获得了支持他们选择这项工作的先前数据。其中一个系统是大肠杆菌，由沃伦设计，可以产生非天然的四吡唑：这些四吡唑包括辅因子F430和维生素B12。后者具有直接的实际意义。我们有证据（未发表）表明，在合成大肠杆菌系统中，用于B12的钴递送蛋白与锌发生错金属化，但当细胞补充多余的钴时，这可以克服。这项工作的第二个目的是操纵这种菌株，为越来越多转向低肉饮食的个人增加维生素B12的供应（植物既不制造也不需要维生素B12），反过来又证明了金属化计算器的优点。

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.

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.

Protein metalation in biology.

• DOI: 10.1016/j.cbpa.2021.102095
• 发表时间: 2022-03
• 期刊: Current opinion in chemical biology
• 影响因子: 7.8
• 作者: Foster AW;Young TR;Chivers PT;Robinson NJ
• 通讯作者: Robinson NJ

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