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Dr. David J. Clarke

Chancellor's Fellow
School of Chemistry, University of Edinburgh.
Group Leader - Biomolecular Mass Spectrometry.




































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Biography

1996 - 2001 Masters Degree Chemistry (MChem) - University of Edinburgh
2001 – 2005 PhD in Biological Chemistry - University of Edinburgh
Thesis: Structural and Functional Studies of Protein Complexes
2005 - 2007 PDRA, University of Edinburgh, Joint School of Chemistry and MRC Human Genetic Unit, Edinburgh.
2006 Teaching Fellow, University of Edinburgh, School of Chemistry.
2007 – 2012 RASOR Post-Doctoral Research Fellow, SIRCAMS. University of Edinburgh, School of Chemistry
2013 Senior PDRA, School of Chemistry. University of Edinburgh
2014 - present Chancellor's Fellow, School of Chemistry. University of Edinburgh

Research Interests

My research focuses on utilizing high resolution mass spectrometry to study protein structure and function. This work utilises a range of MS-based techniques including native MS, top-down fragmentation, ion mobility MS and hydrogen/deuterium exchange techniques.

A Mass Spectrometry Platform for the Analysis of Protein Redox Modifications.

The intracellular redox environment is a highly compartmentalised and regulated state function which varies considerably in differing subcellular location and during cell cycle, cell differentiation and cell death. Disregulation of cellular redox potential, particularly to pro-oxidative states, is implicated in the initiation and proliferation of several disease states (e.g. cardiovascular disease, neurodegenerative disease and cancer). Flux in cellular redox potential can directly influence protein structure by altering the oxidation state of redox-sensitive cysteine (Cys) residues. There is an emerging realisation that these reversible redox modifications (RMs) are used in many proteins as molecular switches to regulate their function or activity (e.g. tyrosine phosphatases, Redox Factor-1, and the NRF2/KEAP1 system). Therefore, investigating the molecular details and the structural/functional consequences of RMs in susceptible proteins is crucial in understanding their biochemical regulation. Similarly, influencing a protein’s function by targeted chemical modification of specific Cys residues has been proposed as a potential strategy to manipulation cellular pathways in disease treatment. We are developing new methodologies and apply a platform of modern MS-based techniques to (i) comprehensively describe the chemistry of specific Cys RMs; (ii) accurately quantify their redox-midpoint potentials; (iii) determine the structural consequences resulting from specific RMs; and ultimately, (iv) link our structural findings to protein function.

MS applications in Enzymology.

We are also interested in using these MS based techniques in order to elucidate enzyme mechanisms. We have performed studies on a series of cysteine dependant peroxidases, known as bacterioferratin co- migratory proteins (BCPs), enzymes which play a crucial role in protecting bacteria from the reactive oxygen species. Covalently-bound protein intermediates within the enzyme pathway have been directly detected, or chemically trapped and detected, by FT-ICR MS; allowing us to ‘map’ the reaction pathways of several BCPs.

We also have a long standing collaboration with the Campopiano Research Group in studying the mechanistic details of pyridoxal 5’-phosphate (PLP) dependent enzymes. In a related project we are developing methodologies for integrating quench-flow techniques with FT-ICR MS. We have applied this to directly study the appearance of covalently bound enzyme intermediates by high resolution MS in a time resolved manner. This instrumentation allows us to measure pre-steady-state kinetics at low millisecond temporal resolution without the use of spectrophotometric active substrates. Furthermore fragmentation of captured intermediates using top-down methodologies has been performed to determinate the active site within the enzymes studied.

Characterisation on Protein-Ligand and Protein-Protein Interactions.

We are currently using a mass spectrometry based approach to analyse non-covalent protein-ligand and protein-protein complexes. This work is focused on using FT-ICR MS and top-down fragmentation in order to locate binding site and binding interfaces within protein complexes.

In addition, we are currently exploring the utility of combining hydrogen/deuterium exchange (HDX) with top down fragmentation in order to characterise protein dynamics and structure.

Recent Research Articles

Full Publication List Available on Google Scholar

1. D. J. Clarke*, J. Scotcher*, C. L. Mackay, T. Hupp, P. J. Sadler, and P. R. R. Langridge-Smith.
Redox Regulation of Tumour Suppressor Protein p53: Identification of the Sites of Hydrogen Peroxide Oxidation and Glutathionylation.
Chemical Science, 2013, 4, 1257-1269.

2. E. Jurneczko, F. Cruickshank, M. Porrini, D. J. Clarke, I. D. G. Campuzano, M. Morris, P. V. Nikolova, and P. E. Barran.
Probing the conformational diversity of cancer-associated mutations in p53 by Ion Mobility-Mass Spectrometry.
Angewandte Chemie Int. Ed., 2013, 52, 4370-4374.

3. D. J. Clarke*, J. M. Wadsworth*, S. A. McMahon, J. P. Lowther, A. E. Beattie, H. Broughton, T. M. Dunn, J. H. Naismith, and D. J. Campopiano.
The chemical basis of serine palmitoyltransferase inhibition by myriocin.
J. Am. Chem. Soc., 2013, 38, 14276.

4. A. E. Beattie, D. J. Clarke, J. M. Wadsworth, J. Lowther, H. Sin and D. J. Campopiano.
Reconstitution of the pyridoxal 5’-phosphate (PLP) dependent enzyme serine palmitoyltransferase (SPT) with pyridoxal reveals a crucial role for the phosphate during catalysis.
Chem. Comm., 2013, 49, 7058-7060.

5. V. Mallikarjun, D. J. Clarke and C. J. Campbell.
Cellular Redox Potential and the Biomolecular Electrochemical Series: A Systems Hypothesis.
Free Radical Biology & Medicine, 2012, 53, 280-288.

6. D. J. Clarke, E. Murray, T. Hupp, C. L. Mackay, and P. R. R. Langridge-Smith.
Mapping a Non-covalent Protein-Peptide Interface by Top-Down FT-ICR Mass Spectrometry using Electron Capture Dissociation.
J. Am. Soc. Mass Spec. 2011, 22 ,1432-1440.



Contact

Address
Dr. David J. Clarke
School of Chemistry
King's Buildings
University of Edinburgh
West Mains Road
Edinburgh
Scotland
EH9 3JJ

e.mail
dave.clarke@ed.ac.uk

Telephone
Office +44(0)131 651 3048
Facsimile
+44(0)131 650 4743