Understanding iron acquisition within a bacterial iron-megastore

了解细菌铁储存库内的铁获取

• 批准号: BB/N005570/1
• 负责人: Jon Marles-Wright
• 金额: $ 60.56万
• 依托单位: Newcastle University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FN005570%2F1
• 关键词: Understanding; iron; acquisition; within; bacterial

Iron is essential for the function of ten per cent of all enzyme families, and is responsible for the ability of haemoglobin to transport gasses in the blood. Iron is highly reactive with oxygen, which is very useful in the case of haemoglobin, but this is dangerous when the iron is in solution as it produces damaging free radicals. These free radicals can damage proteins and DNA in cells, and in serious cases this can cause mutations in the DNA and even kill cells. To help manage the balance between the demand for iron and the danger of free iron, cells have evolved different types of iron stores. These iron stores are called ferritins and all living organisms have at least one type of ferritin. Deletion of the ferritin genes is lethal in mammals and it significantly reduces the fitness of bacteria.Ferritins are remarkable proteins whose form is essential for their function. They form small cages that enclose a central cavity where iron can be safely stored away from the rest of the cell. The ferritin cage is made up of multiple copies of a single protein, each with an active site that can safely catalyse the reaction of iron with oxygen to produce an iron mineral that is stored within the cavity. Different organisms have different types of ferritin with different sizes and thus capacity for storing iron. Some ferritins can even protect DNA from damage by directly binding to it and wrapping it around their shell. We have started the study of a new family of ferritin proteins from bacteria and archaea that do not have a cage-like structure and instead look like ring-doughnuts. These doughnut ferritins are usually found within a cage, formed by another protein, that is twice as large as any other ferritin cage studied so far; this combination is known as a bacterial nanocompartment, or encapsulin. To be able to store iron ferritins absolutely have to have a cage-like structure, so this new arrangement of a doughnut-ferritin inside a cage protein is particularly interesting. We do not know how these proteins work together to sequester iron. In this project we will use structural biology methods, such as X-ray crystallography, mass spectrometry, and electron microscopy, coupled with metal analysis and biochemistry, to investigate the structure and function of this new iron storage system. The remarkable ability of the encapsulin protein cages to bind and enclose their specific cargo protein has great potential to be exploited in biotechnology. Proteins and drug molecules that are toxic to bacterial cells could be produced and safely stored in these cages and only released when separated from the cells. To be able to fully understand the mechanism by which proteins are captured by the cage, we will reconstitute the encapsulin shell with enzymes and fluorescent proteins that are not normally found within it. Using experiments to separate proteins still left in solution from those within the cage, we will be able to measure the ability of the cage to bind to proteins of various sizes and properties. This information will allow us to make genetic systems for the production of these in bacteria for use in biotechnology.Finally, we will study the metal binding ability of the new ferritin protein to determine how strongly it binds to iron and whether other metals can bind to the protein. We will change the amino acids present in the metal-binding site to alter the specificity of the protein to explore the potential for using this protein as a sensor for heavy metals, or for the production of metal nanoparticles that could be used as contrast agents in medical imaging. The knowledge gained from this programme of work will give us a comprehensive understanding of this new system for iron storage and detoxification that we can use to begin to engineer nanocompartment systems. This work will lay a foundation for applications of nanocompartments in the healthcare and industrial biotechnology industries.

铁是所有酶家族的10%的功能所必需的，并且负责血红蛋白在血液中运输气体的能力。铁与氧的反应性很强，这对血红蛋白非常有用，但当铁在溶液中时，这是危险的，因为它会产生有害的自由基。这些自由基会破坏细胞中的蛋白质和DNA，在严重的情况下，这会导致DNA突变，甚至杀死细胞。为了帮助管理对铁的需求和游离铁的危险之间的平衡，细胞已经进化出不同类型的铁储存。这些铁储存被称为铁蛋白，所有生物体都至少有一种铁蛋白。铁蛋白基因的缺失在哺乳动物中是致命的，它显著降低了细菌的适应性。铁蛋白是一种重要的蛋白质，其形式对其功能至关重要。它们形成了一个小笼子，里面有一个中央空腔，铁可以安全地储存在那里，远离细胞的其他部分。铁蛋白笼由单个蛋白质的多个拷贝组成，每个拷贝都有一个活性位点，可以安全地催化铁与氧的反应，产生储存在空腔内的铁矿物质。不同的生物体有不同类型的铁蛋白，大小不同，因此储存铁的能力也不同。一些铁蛋白甚至可以通过直接结合DNA并将其包裹在外壳周围来保护DNA免受损伤。我们已经开始研究来自细菌和古细菌的铁蛋白新家族，它们没有笼状结构，而是看起来像环形甜甜圈。这些甜甜圈铁蛋白通常存在于一个由另一种蛋白质形成的笼子里，这个笼子是迄今为止研究的任何其他铁蛋白笼子的两倍大;这种组合被称为细菌纳米区室，或细菌蛋白。为了能够储存铁，铁蛋白绝对必须具有笼状结构，因此这种在笼蛋白内的甜甜圈铁蛋白的新排列特别有趣。我们不知道这些蛋白质如何协同工作来螯合铁。在这个项目中，我们将使用结构生物学方法，如X射线晶体学，质谱法和电子显微镜，结合金属分析和生物化学，研究这种新的铁储存系统的结构和功能。蛋白笼结合和包裹其特定货物蛋白的显着能力在生物技术中具有巨大的开发潜力。对细菌细胞有毒的蛋白质和药物分子可以在这些笼子里安全地产生和储存，只有在与细胞分离时才能释放。为了充分理解蛋白质被笼捕获的机制，我们将用通常不存在于笼中的酶和荧光蛋白来重组蛋白质壳，通过实验将留在溶液中的蛋白质与笼中的蛋白质分离，我们将能够测量笼与各种大小和性质的蛋白质结合的能力。最后，我们将研究新铁蛋白的金属结合能力，以确定它与铁的结合强度，以及其他金属是否能与该蛋白结合。我们将改变金属结合位点中存在的氨基酸，以改变蛋白质的特异性，从而探索使用这种蛋白质作为重金属传感器的潜力，或者用于生产可用作医学成像中的造影剂的金属纳米颗粒。从这项工作计划中获得的知识将使我们全面了解这种新的铁储存和解毒系统，我们可以用它来开始设计纳米隔室系统。这项工作将为纳米隔室在医疗保健和工业生物技术行业的应用奠定基础。

项目成果

Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments

封装铁蛋白的结构表征有助于深入了解细菌纳米室中的铁储存

• DOI: 10.1101/063495
• 发表时间: 2016
• 作者: He D

Dissecting the structural and functional roles of a vicinal iron-binding site in encapsulated ferritins

• DOI: 10.1101/785121
• 发表时间: 2019-09
• 期刊: bioRxiv
• 作者: Cecilia Piergentili;J. Ross;D. He;Kelly J. Gallagher;Will A. Stanley;Laurène Adam;C. Mackay;Kevin J. Waldron;David J. Clarke;J. Marles-Wright

Mass spectrometry reveals the assembly pathway of encapsulated ferritins and highlights a dynamic ferroxidase interface.

质谱分析揭示了封装铁蛋白的组装途径，并突出了动态亚铁氧化酶界面。

• DOI: 10.1039/c9cc08130e
• 发表时间: 2020
• 期刊: Chemical communications (Cambridge, England)
• 作者: Ross J

A User's Guide to Golden Gate Cloning Methods and Standards.

• DOI: 10.1021/acssynbio.2c00355
• 发表时间: 2022-11-18
• 期刊: ACS SYNTHETIC BIOLOGY
• 影响因子: 4.7
• 作者: Bird, Jasmine E.;Marles-Wright, Jon;Giachino, Andrea
• 通讯作者: Giachino, Andrea

Structural evidence for the covalent modification of FabH by 4,5-dichloro-1,2-dithiol-3-one (HR45).

• DOI: 10.1039/c7ob01396e
• 发表时间: 2017-08-02
• 期刊: Organic & biomolecular chemistry
• 影响因子: 3.2
• 作者: Ekström AG;Kelly V;Marles-Wright J;Cockroft SL;Campopiano DJ
• 通讯作者: Campopiano DJ