Current research
I am currently employed by the Medical Research Council (MRC) in the Laboratory of Molecular Biology, Cambridge. My research involves the study of protein-protein binding by way of using small isotopically-labelled linker molecules. These linker molecules bind between residues that are within range of each other and then the cross-linked protein complex is digested and analysed by mass spectrometry. The interactions we investigate are important for understanding and developing new cures for a wide range of diseases including cancer.
Figure 1. Cartoon image outlining the method of protein cross-linking. A protein sample is cross-linked with a homobifunctional reagent that links residues within a certain distance of each other. The protein sample is then digested into small peptides and the cross-linked fragments are detected by mass spectrometry. These peptides are then fragmented to provide an amino acid sequence detailing where in the protein the cross-linkers occurred and thereby we can work out the structure of the protein and how multiple proteins interact.
Published Research
Presentations
- Development of arginine and guanine specific isotopically-labelled linkers for the characterisation of binding interactions by mass spectrometry
Poster presentation at 59th ASMS Conference on Mass Spectrometry and Allied Topics, 2011, Colorado Convention Center, Denver, CO, USA.
- Cross-linking in mass spectrometry
Oral presentation at Proteomics Methods Forum, 2010, Department of Biochemistry, University of Cambridge.
- Cross-linking in mass spectrometry
Oral presentation at mini-symposium on Biophysics, 2010, MRC Laboratory of Molecular Biology.
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Sdo1/Efl1 interaction site mapped by photocrosslinking
Poster presentation at a one-day memorial symposium for Dr. Joe Spencer, 2010, University of Cambridge.
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Investigation into the mechanism of phenolic couplings during the biosynthesis of glycopeptide antibiotics
Poster presentation at the Zing Conference in Natural Products, 2009, Antigua.
Publications
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Holding AN, Spencer JB. (2008)
Investigation into the mechanism of phenolic couplings during the biosynthesis of glycopeptide antibiotics.
ChemBioChem. 9. 2209-14. [Pubmed]
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O'Hare HM, Huang F, Holding A, Choroba OW, Spencer JB. (2006)
Conversion of hydroxyphenylpyruvate dioxygenases into hydroxymandelate synthases by directed evolution.
FEBS Lett. 580. 3445-50. [Pubmed]
PhD thesis
Studies on the biosynthesis of non-ribosomal peptides
Abstract: The problem of bacterial resistance is of growing concern within the medical community. Eventually, even with responsible use of antibiotics, new compounds will be required to bypass the resistance that bacteria have acquired. Thus the expansion of knowledge and understanding of antibiotics is key in the development of new compounds in the fight against infection. One attractive starting point for the development of new compounds are those natural products generated by non-ribosomal peptide synthetases (NRPS), which include a range of clinically relevant glycopeptide antibiotics.
Several aspects of the biosynthesis of glycopeptide antibiotics were examined: first, the investigation to identify, by the use of directed evolution if 4-hydroxymandelic acid synthase (HmaS) from the gene cluster of the antibiotic chloroeremomycin may have evolved from its homologue 4-hydroxyphenylpyruvate dioxygenase (HppD). The summation of this work is published in FEBS Lett. 2006; 580:3445. Following on from this work was an investigation into the hypothesis that HmaS catalyses the turnover of the non-natural phenylpyruvic acid to produce a product with an inverted chiral centre compared to that of the natural substrate due to differences in substrate binding. Results showed that, while not the major product, the inverted product was detected via chiral GCMS. Secondly, it is shown that all three cytochrome P450 enzymes (OxyA-C) that catalyse the sequential formation of three essential oxidative cross-links within the chloroeremomycin molecule do so with the retention of the oxygen atom on the peptide backbone and without the incorporation of oxygen in the air. This portion of the work is published in ChemBioChem 2008; 9:2209. The final part of the study was the development of a high-throughput screening method for NRPS A-domains, with the aim of both rapid characterisation and directed evolution of novel substrate specificity. This led to the identification that the amino acid loaded by the first A-domain of the teicoplanin NRPS was shown to load the d-amino acid in preference to the l-amino acid. This is in contrast to the equivalent domain in the chloroeremomycin gene cluster that loads the l-amino acid.
Download: Studies on the biosynthesis of non-ribosomal peptides.