Award details

Novel haem proteins from bacterial pathogens: structural and biophysical studies

Reference	BB/D52252X/1
Principal Investigator / Supervisor	Professor Jonas Emsley
Co-Investigators / Co-Supervisors
Institution	University of Nottingham
Department	Sch of Biomedical Sciences
Funding type	Research
Value (£)	180,833
Status	Completed
Type	Research Grant
Start date	17/10/2005
End date	16/10/2008
Duration	36 months

Abstract

Haem is an iron-porphyrin complex associated with many proteins and found in virtually every life form. Haem proteins display diverse biological functions such as transport, storage and control of gene expression. In the past decade a striking number of haem proteins have had their structures elucidated, revealing an unexpected range of different fold architectures. This type of molecules has also been a research focus because the huge chemical versatility of haem means that it should be possible to achieve new activities through design and engineering experiments. The proposed research will involve i) structural and biochemical work on three haem carrier proteins, key components of nutrient uptake systems in bacteria, and ii) capture assays using haem-affinity chromatography to discover novel haem proteins in a group of selected diverse bacterial species. In the first part of the project, haem carriers HemS, HemT and IsdE will be studied. We have already carried out a great deal of preliminary work and obtained diffraction quality crystals of HemS and IsdE have been cloned and purification procedures have been worked out. One aim will be the analysis of the structure of these proteins in complex with haem using X-ray crystallography. Efforts towards the study of the apo-forms will also be made. Mutagenesis experiments will be carried out and mutants will be analysed with UV-vis spectroscopy and NMR to study structure-function relationships. This research strand will provide new knowledge on bacterial haem carriers which are still largely unexplored. In the second part of the project, diverse bacterial proteomes will be probed to discover new haem proteins. Haem-agarose resin will be used according to well-established haem-affinity chromatography protocols. With this haem proteomics approach, it will be possible to assign a function to unknown proteins in many organisms the genomes of which have been sequenced. Binders will be separated and identified by techniquessuch as peptide mass fingerprinting. Diverse bacterial species will be targeted including pathogens (eg. Staphylococcus, Helicobacter, etc.) and extremophiles (eg. Thermotoga, Halobacterium, etc.) Proteins of medical and biotechnological significance may thus be found.

Summary

Iron is essential to humans, animals, plants and bacteria. Many bacteria, including those that cause disease, possess sophisticated mechanisms to steal iron from the body and use it for their own needs. In our body, the largest source of iron is from a compound called haem, which is contained in the protein haemoglobin in the blood. Iron and haem give blood its red colour. Particular protein molecules are made by the bacteria to take haem away from our body, process it and use its iron. These haem-binding proteins are unique to bacteria, and no similar molecules are found in animals or humans. Our research will explore, in a number of ways, how these molecules work. In the first instance, information about the structure of the haem carrier proteins will be obtained using a technique that relies of the diffraction of X-ray by crystals. If we look at the structure of the molecule bound to the haem group, we can understand how the binding is achieved and what interactions hold the two together. At the same time, in another part of the work we will make changes to specific sites of the protein molecule to see how these structural elements contribute to the binding. With this work we will increase the understanding of how bacteria quire haem and iron, an important biological process also relevant to medicine. We will generate new knowledge on the systems used by bacteria to take up nutrients, such as iron. We will study the way in which proteins bind, carry and release smaller molecules. The results of the research will add data to an important group of proteins, the haem proteins, which are found in virtually every living organism. The proposed work will also involve experiments to discover new haem proteins in bacterial species. Bacteria make up a huge group of living organisms. Even though many people do not think bacteria are very different from each other, they actually are chemically the most diverse life forms on Earth. While some are pathogenic, that is they invade a host organism, for example humans, and cause disease, many are responsible for extremely important processes, such as making yoghourt, cheese etc. and some are found in very special conditions of temperature (volcanoes) and salt (Dead Sea). Bacteria have, therefore, widely different metabolic reactions and possess very different proteins and enzymes many of which are still unknown. We will identify new proteins using an already established method in a novel way. The method is based on linking the haem group to a solid, gel-like material; mixtures of proteins from bacterial cells will be passed through this material. Those proteins which bind haem will remain attached to the gel-like resin, while all others will pass through. The bound proteins can then be separated and analysed. This is a very powerful way to discover new proteins, some of which will have significance for medicine and biotechnology.

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search