Principal Supervisor: Dr Manuela Tosin, Department of Chemistry
Co-supervisor: Prof Alex Cameron, School of Life Sciences, and Dr Józef Lewandowski, Department of Chemistry
PhD project title: Functional and structural characterization of glycosyltransferases involved in bacterial mannosylation
University of Registration: University of Warwick
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 glycosyltransferases that utilise complex mannosyl donors and are generally membrane-bound; therefore 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 function and the structure of key bacterial glycosyltransferases involved in the mannosylation of proteins and small bioactive molecules.
The project work will include gene cloning, enzyme expression and purification, site-directed mutagenesis and the chemoenzymatic preparation of sugar donors/substrates for enzyme functional and structural characterisation via kinetics, crystallisation and NMR spectroscopy investigations.
The results from these experiments will give for the first time a comprehensive picture of bacterial protein mannosylation, with important implications for synthetic biology of protein glycosylation and future drug/therapy design targeting glycosylation machineries.
- M. Abu-Qarn, J. Eichler, N. Sharon, ‘Not just for Eukarya anymore: protein glycosylation in Bacteria and Archaea’, Curr Opin Struct Biol. 2008, 18, 544-50.
- C. Espitita, L. Servin-Gonzales, R. Mancilla, ‘New insights into protein O-mannosylation in actinomycetes’, Mol. BioSyst. 2010, 6, 775-781.
- M. Lommel, S. Strahl, ‘Protein O-mannosylation: conserved from bacteria to humans’, Glycobiology 2009, 19, 816-828.
- C.-F. Liu et al., ‘Bacterial protein O-mannosylating enzyme is crucial for virulence of Mycobacterium tubercolosis’, Proc Natl Acad Sci USA 2013, 110, 6560-6565.
BBSRC Strategic Research Priority: Bioenergy and Industrial biotechnology, Molecules, Cells and Systems
Techniques that will be undertaken during the project:
Molecular biology for gene cloning and site-directed mutagenesis: PCR, digestion of DNA with restriction enzymes, agarose gel electrophoresis, …
Protein expression and purification by affinity chromatography, size-exclusion, ion- exchange, hydrophobic interactions, …
Enzymology: enzyme activity assays with natural and synthetic substrates; enzyme kinetics (UV, HPLC, ..)
Small molecules, DNA and protein characterisation via a range of analytical techniques, including mass spectrometry, fluorescence, circular dichroism, electrophoresis, …
Chemoenzymatic preparation of carbohydrate donors/substrates
Protein X-ray crystallography (at the Warwick Macromolecular Crystallisation Facility, Life Sciences): sample preparation, set up of automated crystallisation screening (using the Honeybee 963 nanodrop crystallisation robot), crystal growth optimisation, X-ray diffraction patterns analyses and protein structure resolution
Protein NMR: spectral assignment in solution and solid state, chemical shift perturbations, structure determination both in solution and solid-state, protein dynamics in solution and solid-state
Bioinformatics: design of PCR primers, analysis of DNA/protein sequences, protein structural modelling and analyses.
Contact: Dr Manuela Tosin, University of Warwick