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Functional and structural characterisation of glycosyltransferases involved in protein mannosylation

Principal Supervisor: Dr Manuela Tosin

Secondary Supervisor(s): Professor Alexander Cameron & Dr Deborah Brotherton

University of Registration: University of Warwick

BBSRC Research Themes:

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Deadline: 4 January, 2024


Project Outline

Glycosylation is one the most abundant post-translational modifications and an evolutionary indicator: indeed the increasing complexity of host-pathogen and organism-environment interactions, amongst others, is reflected in the newly-reported ability of prokaryotes (e.g. bacteria and archaea) to carry out eukaryotic functions, such as glycosylation of proteins and other biomolecules.1-2

Protein mannosylation is vital for healthy organisms3 and is also involved in the pathogenesis of many virulent diseases, such as meningitis and tubercolosis.4 It is carried out in different organisms by highly specific, membrane-bound glycosyltransferases that utilise complex mannosyl donors. This membrane association means there is still a general lack of direct information on the enzyme catalysis and structure.

The aim of this PhD project is to fully elucidate the structure and function of key glycosyltransferases involved in the mannosylation of proteins and small bioactive molecules in bacteria and humans.

The project work will include gene cloning, protein expression in heterologous hosts, protein purification, site-directed mutagenesis and the chemoenzymatic preparation of sugar donors/substrates for the functional and structural characterisation of these enzymes via kinetics, crystallisation and cryoEM.

The results from these experiments will provide crucial insights on mannosylation, with important implications for the understanding of prokaryotic and eukaryotic biology and future drug/therapy design targeting glycosylation machineries.

For further project details please contact .

References

1. M. Abu-Qarn, J. Eichler, N. Sharon, Not just for Eukarya anymore: protein glycosylation in Bacteria and Archaea’, Curr Opin Struct Biol. 2008, 18, 544.

2. M. Lommel, S. Strahl, ‘Protein O-mannosylation: conserved from bacteria to humans’, Glycobiology 2009, 19, 816.

3. C.-F. Liu et al., ‘Bacterial protein O-mannosylating enzyme is crucial for virulence of Mycobacterium tubercolosis’, Proc Natl Acad Sci USA 2013,

112685.

Techniques

  • Molecular biology for gene cloning and site-directed mutagenesis: PCR, digestion of DNA with restriction enzymes, agarose gel electrophoresis, …
  • Protein heterologous expression (e.g. in E. coli, yeast and insect cells) and purification by affinity chromatography, size-exclusion, ion- exchange, hydrophobic interactions, etc.
  • Enzymology: enzyme activity assays with natural and synthetic substrates; enzyme kinetics (UV, HPLC, etc.)
  • Small molecules, DNA and protein characterisation via a range of analytical techniques, including mass spectrometry, fluorescence, circular dichroism, electrophoresis, etc.
  • Chemoenzymatic preparation of carbohydrate donors/substrates
  • Protein X-ray crystallography (Life Sciences): sample preparation, set up of automated crystallisation screening (Mosquito crystallisation robot), crystal growth optimisation, X-ray diffraction data collection and protein structure determination and refinement
  • CryoEM: sample preparation, grid screening and data collection, data processing, structure solution and refinement
  • Bioinformatics: design of PCR primers, analysis of DNA/protein sequences, protein structural modelling and analyses