Research Interests
2-oxoglutarate oxygenases
I am currently working with Professor Chris Schofield in the Department of Chemistry, using bioinformatics methods to analyse a family of enzymes called 2-oxoglutarate oxygenases. This is a large superfamily of proteins which are involved in a wide range of reactions, such as collagen modification, hypoxic signalling, histone modification and DNA repair. They require oxygen and 2-oxoglutarate as cosubstrates and use a Fe(II) as a cofactor, and have a double stranded beta helix (DSBH) fold with a highly conserved iron binding motif (HXD/E...H). I am looking at how these enzymes evolved from bacteria through to humans, and what the differences are in the different branches of the tree of life.
Specificity of protein:protein interactions
Residues which determine the specificity of protein-protein interactions must be found at the interface between protein binding partners and it has been proposed that compensatory mutations take place during evolution to maintain binding. Many algorithms have been developed to capture this correlated mutation signal but these typically have low accuracy with very high false positive rates. We have developed a new method which detects inter-domain contact sites by considering a protein as a network with nodes of residues and edges of contacts. Structural information available for each separate domain, or each protein in a complex, can be used to define the intra-domain edges and give exposed/buried information for each node. The question then is how to establish inter-domain edges between the intra-domain networks. In these networks, the profiles of nodes, edges and triangles for inter-domain contact sites in a set of multi-domain proteins with known structures were calculated, and shown to be different to those for non contact sites. A score for each residue can therefore be established by examining the degree to which it fits the profiles for inter-domain contact sites. This work has been done in collaboration with Qiang Luo. The method has been tested on the chemotaxis protein complexes of CheA-CheW and CheA-CheY. Based on the results, we have made suggestions of specific residues in these proteins which could be mutated to change the specificity of interaction. Experiments to verify these are currently being undertaken using proteins from Rhodobacter sphaeroides, by George Wadhams, Sonja Pawelczyk and Kathryn Scott in the OCISB. Dr Steven Porter and Jen de Beyer have recently brought in a third test case looking at the interactions of CheB and CheR with the chemotaxis receptors. This work is ongoing and predictions will again be tested on R. sphaeroides proteins.
Our prediction software, i-Patch, is available as a web server here, and to download here.
Chemotaxis
Chemotaxis is the process by which motile bacteria sense their chemical environment and move towards more favourable conditions. Escherichia coli utilises a single sensory pathway (shown on the left below),but little is known about signalling pathways in species with more complex systems (for example Rhodobacter sphaeroides, shown on the right).
To investigate whether chemotaxis pathways in other bacteria follow the E. coli paradigm, we analysed 206 species encoding at least 1 homologue of each of the 5 core chemotaxis proteins (CheA, CheB, CheR, CheW and CheY). 61 species encode more than one of all of these 5 proteins, suggesting they have multiple chemotaxis pathways. Operon information is not available for most bacteria so we developed a novel statistical approach to cluster che genes into putative operons. Using operon-based models, we reconstructed putative chemotaxis pathways for all 206 species. We show that cheA-cheW and cheR-cheB have strong preferences to occur in the same operon as two-gene blocks, which may reflect a functional requirement for co-transcription. However, other che genes, most notably cheY, are more dispersed on the genome. Comparison of our operons with shuffled equivalents demonstrates that specific patterns of genomic location may be a determining factor for the observed in vivo chemotaxis pathways.
We then examined the chemotaxis pathways of Rhodobacter sphaeroides. Here, the PpfA protein is known to be critical for correct partitioning of proteins in the cytoplasmically-localised pathway. We found ppfA in che operons of many species, suggesting that partitioning of cytoplasmic Che protein clusters is common. We also examined the apparently non-typical chemotaxis components, CheA3, CheA4 and CheY6. We found that though variants of CheA proteins are rare, the CheY6 variant may be a common type of CheY, with a significantly disordered C-terminal region which may be functionally significant.
We find that many bacterial species potentially have multiple chemotaxis pathways, with grouping of che genes into operons likely to be a major factor in keeping signalling pathways distinct. Gene order is highly conserved with cheA-cheW and cheR-cheB blocks, perhaps reflecting functional linkage. CheY behaves differently to other Che proteins, both in its genomic location and its putative protein interactions, which should be considered when modelling chemotaxis pathways.