Physical Sciences for Health CDT
Thesis project - "Biomolecular tetris: Elucidating the structural consequences of phosphorylation"
Professor Helen Cooper, School of Biosciences
Dr Iain Styles, School of Computer Sciences
Dr Peter Winn, School of Biosciences
Professor John K. Heath, School of Biosciences
Professor David Russell, Dept. of Chemistry, Texas A&M University
This project will develop mass spectrometry to probe the effect of phosphorylation on the structure of proteins implicated in age-related diseases. Gas phase measurements of model phosphoprotein and phosphopeptide systems will be correlated with liquid phase measurements and computational predictions to elucidate the structure of the biomolecules.
The fragmentation of proteins by tandem mass spectrometry (MS/MS) has long been used to ascertain a protein's amino acid sequence, however, it cannot currently be used to infer any information about the secondary or tertiary structure. The mostly commonly used fragmentation method, collision induced fragmentation (CID), cleaves intra- and inter- molecular bonds in proteins, removing any information about the 3D structure of a protein. Conversely, ECD (electron capture dissociation) and ETD (electron transfer dissociation) are fragmentation methods that have been shown to conserve non-covalent bonds. These methods also retain post-translational modifications of the protein, such as phosphorylation. This makes ECD/ETD suitable methods to investigate salt bridges formed between phosphate groups and basic amino acid side-chains, as both are conserved. These salt-bridges affect the shape of the intact phosphoprotein and its fragments, so by studying the fragment structures we will be able to localise the salt-bridge in the amino-acid sequence and infer information on the 3D shape of the intact phosphoprotein.
Phosphorylation of proteins acts as a biomolecular switch in many signalling cascades, particularly those which regulate the cell cycle, by introducing salt bridges that change the shape of the protein's active site. When phosphorylation becomes dysregulated, cellular processes can be shut down or switched on irreversibly. This causes many diseases, such as Alzheimer's, COPD and rheumatoid arthritis. The likelihood of these proteins mutating increases with biological age as the genome becomes more unstable, so the occurrence of these diseases also increases with age. Phosphoproteins present a possible therapeutic target, so studying their structure and behaviour is of both pharmacological and biomolecular interest To study salt bridges a model system will be developed based on a well-characterised, biologically-relevant phosphoprotein will be developed. This will consist of a number of phosphopeptides derived from the phosphoprotein that each contain a salt-bridge. In addition to using ECD/ETD to fragment proteins and record their mass spectra, ion mobility spectroscopy will be included in the workflow to measure the collision crosssection for peptides and their fragments and give a measure of their shape and size. Structural information on the intact peptides will also be collected by IR and IR-MPD in the gas phase; and by CD, IR and NMR in the liquid phase. This will allow comparisons between the gas and liquid phase structures to be made. Structures and spectra will be modelled by molecular dynamics and DFT simulations, to validate correlations made between experimental data. Finally, the workflow developed to infer peptide structure from MS, IR and computational data will be extended to the intact protein.
In summary, we will develop a mass spectrometry workflow to study the effect of phosphorylation, and the resulting salt-bridges, on the shape and structure of phosphoproteins. This will involve the optimisation of fragmentation conditions for this purpose, introducing new methods to interpret and correlate data from multiple analytical methods, and the development of computational methods to model shape and spectra of peptides and proteins.