PhD Research Project: Protein structure and function determination using x-ray crystallography

The function of an enzyme or protein is largely determined by its three-dimensional structure. The overall aim of our work is to solve protein structures using X-ray crystallography, and in combination with other techniques, probe the relationship between structure and function. In particular, we are interested in protein-ligand and protein-protein interactions; features which are central to enzyme and cell function. In several projects we are using a structure-based approach to designing drugs and inhibitors.

Recent published work includes: bacterial nitroreductase, an enzyme with potential as a novel pro-drug anti-cancer therapy; transhydrogenase, a membrane-bound ion pump from bacteria, parasites and human-heart mitochondria; family 2 inorganic pyrophosphatases from several sources.

Nitroreductase: E. coli nitroreductase catalyses the reduction of aromatic nitrates. It can be used to activate the pro-drug CB-1954, an aromatic nitrate, to a highly toxic drug that quickly leads to cell death. We have recently solved the structure of nitroreductase with bound nicotinic acid (a mimic of the NAD(H) head group). In collaboration with Dr E. I Hyde, we are trying to produce engineered nitroreductases with improved catalytic properties for CB-1954. Protein crystallography is being used to analyse mutant enzyme:inhibitor complexes and guide further mutagenesis and drug design.

Transhydrogenase: in collaboration with Prof. J. B. Jackson, we are studying transhydrogenase, a tripartite membrane protein, to determine how it couples hydride transfer between NAD(H) and NADP(H) to the translocation of protons across a bilipid membrane. We are using protein crystallography to determine the structures of various protein:nucleotide complexes. We have recently published the crystal structure of an asymmetric complex of three transhydrogenase domains with bound nucleotides.

Family 2 Inorganic Pyrophosphatases: We have recently solved the structures of two members of the recently discovered “Family 2 Inorganic Pyrophosphatases”. These enzymes are very different from the well-characterised pyrophosphatases, and are a sub-class of the emerging family of “DHH phosphoesterases”. In collaboration with Dr T. W. Young, we are probing the structure and function of Family 2 pyrophosphatases using a combination of protein crystallography, mutagenesis and kinetics. The enzymes catalyse the hydrolysis of inorganic pyrophosphatase, a reaction, which is exploited by cells to drive DNA, RNA and polypeptide biosynthesis. Due to their novel properties and the essential nature of the reaction they catalyse, family 2 pyrophosphatases have been proposed as a target for antimicrobial inhibitor design.

Students will be trained in protein purification and production, molecular biology, protein crystallisation, protein crystallography.
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To find out more about studying for a PhD at the University of Birmingham, including full details of the research undertaken in each school, the funding opportunities for each subject, and guidance on making your application, you can now order your copy of the new Doctoral Research Prospectus, at: http://www.birmingham.ac.uk/students/drp.aspx
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Please find additional funding text below. For further funding details, please see the ‘Funding’ section.
The School of Biosciences offers a number of UK Research Council (e.g. BBSRC, NERC) PhD studentships each year. Fully funded research council studentships are normally only available to UK nationals (or EU nationals resident in the UK) but part-funded studentships may be available to EU applicants resident outside of the UK. The deadline for applications for research council studentships is 31 January each year.

Each year we also have a number of fully funded Darwin Trust Scholarships. These are provided by the Darwin Trust of Edinburgh and are for non-UK students wishing to undertake a PhD in the general area of Molecular Microbiology. The deadline for this scheme is also 31 January each year.

Funding Notes

All applicants should indicate in their applications how they intend to fund their studies. We have a thriving community of international PhD students and encourage applications at any time from students able to find their own funding or who wish to apply for their own funding (e.g. Commonwealth Scholarship, Islamic Development Bank).

The postgraduate funding database provides further information on funding opportunities available http://www.birmingham.ac.uk/postgraduate/funding/FundingFilter.aspx and further information is also available on the School of Biosciences website http://www.birmingham.ac.uk/schools/biosciences/courses/postgraduate/phd.aspx

References

Singh, A., Venning, J. D., Quirk, P. G., Van Boxel, G. I., Rodrigues, D. J., White, S. A., Jackson, J. B. (2003) Interactions between Transhydrogenase and Thio-nicotinamide Analogues of NAD(H) and NADP(H) Underline the Importance of Nucleotide Conformational Changes in Coupling to Proton Translocation. J. Biol. Chem., 278, 33208-33216.

van Boxel, G. I., Quirk, P. G., Cotton, N. P., White, S. A., Jackson, J. B. (2003) Glutamine 132 in the NAD(H)-binding component of proton-translocating transhydrogenase tethers the nucleotides before hydride transfer. Biochemistry, 42, 1217-1226.

Jackson, J. B., White, S. A., Quirk, P. G. and Venning, J. D. (2002) The alternating site, binding change mechanism for proton translocation by transhydrogenase. Biochemistry, 41, 4173-4185.

Ahn, S., Milner, A. J., Futterer, K., Konopka, M., Ilias, M., Young, T. W. & White, S. A. (2001) The open and closed structures of the type-C inorganic pyrophosphatases from Bacillus subtilis and Streptococcus gordonii. J. Mol. Biol. 313, 797-811.

Lovering, A. L., Hyde, E. I., Searle, P. F. and White, S. A. (2001) The structure of Escherichia coli nitroreductase complexed with nicotinic acid: three crystal forms at 1.7 Å, 1.8 Å and 2.4 Å resolution. J. Mol. Biol., 309, 203-213

Venning, J. D., Rodrigues, D. J., Weston, C. J., Cotton, N. P. J., Quirk, P. G., Errington, N., Finet, S., White, S. A. and Jackson, J. B. (2001) The heterotrimer of the membrane-peripheral components of transhydrogenase and the alternating-site mechanism of proton translocation. J. Biol. Chem., 276, 30678-30685.

Cotton, N. P. J., White, S. A., Peake, S. J., McSweeney, S. and Jackson, J. B. (2001). The Crystal Structure of an Asymmetric Complex of the Two Nucleotide Binding Components of Proton-Translocating Transhydrogenase. Structure 9, 165-176

White, S. A. Peake, S. J., McSweeney, S., Leonard, G., Cotton, N. P. J. and Jackson, J. B. (2000). The high resolution structure of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase from human-heart mitochondria. Structure, 8, 1-12