Understanding iron acquisition within a bacterial iron-megastore

铁是所有酶家族中10%的功能所必需的，并负责血红蛋白在血液中运输气体的能力。铁与氧气高度反应，这对血红蛋白非常有用，但当铁在溶液中时，这是危险的，因为它会产生破坏性的自由基。这些自由基可以破坏细胞中的蛋白质和DNA，在严重的情况下，这可能导致DNA突变，甚至杀死细胞。为了帮助平衡对铁的需求和游离铁的危险，细胞进化出了不同类型的铁储存。这些铁存储被称为铁蛋白，所有活着的有机体都至少有一种铁蛋白。在哺乳动物中，铁蛋白基因的缺失是致命的，它显著降低了细菌的适合度。铁蛋白是一种重要的蛋白质，其形式对其功能至关重要。它们形成一个小笼子，包围着一个中央空腔，在那里铁可以安全地储存在远离细胞其余部分的地方。铁蛋白笼子由单一蛋白质的多个副本组成，每个副本都有一个活性部位，可以安全地催化铁与氧气的反应，产生储存在腔内的铁矿物。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与DNA结合并将其包裹在外壳上来保护DNA免受损害。我们已经开始研究来自细菌和古菌的铁蛋白新家族，它们没有笼状结构，而是看起来像环状甜甜圈。这些甜甜圈铁蛋白通常是在另一种蛋白质形成的笼子里发现的，这种蛋白质是目前研究的任何其他铁蛋白笼子的两倍；这种组合被称为细菌纳米隔间，或胶囊。为了能够储存铁蛋白，铁蛋白绝对必须具有笼状结构，所以这种在笼状蛋白内的甜甜圈-铁蛋白的新安排特别有趣。我们不知道这些蛋白质是如何共同作用来隔离铁的。在这个项目中，我们将使用结构生物学方法，如X射线结晶学、质谱学和电子显微镜，结合金属分析和生物化学，来研究这个新的铁存储系统的结构和功能。被包裹的蛋白笼具有结合和包裹其特定货物蛋白的显著能力，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被生产出来，并安全地储存在这些笼子里，只有在与细胞分离时才会释放出来。为了能够充分了解蛋白质被笼子捕获的机制，我们将用正常情况下不在其内发现的酶和荧光蛋白重建囊壳。通过实验将仍留在溶液中的蛋白质从笼子中分离出来，我们将能够测量笼子与不同大小和性质的蛋白质结合的能力。这些信息将使我们能够在细菌中制造用于生物技术的基因系统。最后，我们将研究新铁蛋白的金属结合能力，以确定它与铁结合的强度，以及其他金属是否可以与蛋白质结合。我们将改变金属结合部位的氨基酸，以改变蛋白质的特异性，以探索将该蛋白质用作重金属传感器的潜力，或用于生产可用作医学成像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对这一新的铁储存和解毒系统有一个全面的了解，我们可以利用它开始设计纳米隔间系统。这项工作将为纳米隔间在医疗保健和工业生物技术行业中的应用奠定基础。

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.