Understanding iron acquisition within a bacterial iron-megastore

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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.