The majority of proteins interact with other proteins/nucleic acids to form functional complexes that are essential to the proteins cellular role. Solving the structures of these complexes is vital for a full understanding of a proteins function. However, many protein complexes have resisted attempts of structure determination by established methods, making modelling based on experimental data and known structures of individual proteins an attractive alternative. The work presented here describes the in silico testing, experimental validation and application of a technique that uses HN-CH3 NOEs to determine sequence-specific 13C/1H NMR assignments for side-chain methyl groups in proteins, which are generally abundant at protein-protein interfaces. The approach developed relies on the preparation of residually protonated protein samples, avoiding limitations imposed by the molecular weight of larger complexes. Using this approach it was possible to obtain comprehensive assignments for the methyl groups of IL-1β (17 kDa) both in the free form and in complex with a potential therapeutic Fab antibody fragment (a complex of 65 kDa). It was shown that these assignments could be used to identify a number of backbone amide to side chain methyl NOEs across the protein-protein interface. These NOEs provided a significant number of 1H-1H distance restraints that made a substantial difference to the accuracy and reliability of docked structures obtained for the IL-1β/Fab complex by restraint driven docking. This was confirmed by comparison to a crystal structure determined for the complex. The developed approach is both conceptually and experimentally straight-forward and is expected to be generally applicable to a wide range of protein complexes up to a molecular weight of approximately 100 kDa. The use of a homology model as the starting structure for the Fab fragment also demonstrates that this technique is tolerant of some differences in the starting and final structures.

The high abundance of chemical pollutants in the environment represents a genetic risk to humans. The development of reliable and sensitive tests for the analysis of the genetic effects of exposure to chemical mutagens is required. Previous work has shown that expanded simple tandem repeat (ESTR) loci provide a sensitive system for monitoring radiation-induced mutation in the mouse germline. Here, the results of the first systematic study on germline mutation induction at mouse ESTR loci by chemical mutagens are presented. Mutation rates at two ESTR loci were studied in the germline of male mice exposed to two monofunctional alkylating agents, ethyl-nitrosourea (ENU) and isopropyl methanesulfonate (iPMS), as well as to the topoisomerase-II inhibitor, etoposide (ET). Pre-meiotic exposure to alkylating agents resulted in a highly significant increase in ESTR mutation rate, but did not alter post-meiotically exposed cells. Pre-meiotic mutation induction by ENU and iPMS was linear within the interval of doses from 12.5 mg/kg to 25 mg/kg and reached a plateau at higher concentrations. Paternal exposure to etoposide resulted in ESTR mutation induction at meiotic stages but did not affect post- or pre-meiotic cells. The pattern of ESTR mutation induction after pre-meiotic and meiotic exposure to chemical mutagens was similar to that previously obtained by various traditional approaches for monitoring germline mutation in mice. Using microarrays, the analysis of the pattern of changes in gene expression in the testis of male mice exposed to ENU was studied. This analysis revealed that exposure to this chemical mutagen does not result in detectable changes in gene expression. The results of this study show that ESTR loci provide a new and efficient biomonitoring system for assessing the genetic effects of chemical mutagens, capable of detecting increases in mutation rates at very low doses and in small sample sizes.

Antibiotic resistance determinants carried on mobile genetic elements such as integrons are widespread in Acinetobacter baumannii. A total of 76 clinical isolates of A. baumannii were screened for class 1, 2 and 3 integron structures using PCR-based assays. Further chromosome walking and Southern blot-based analyses revealed a significant proportion of incomplete integrons, primarily due to IS element-mediated deletions or truncations at the 5’ end of these integrons. Based on these multiple detection methods, a total of 72 integron structures distributed among 58 isolates were identified. Remarkably, 29 (40.3 %) were either incomplete, or had a truncated integrase gene. These data also revealed that using established PCR-based detection methods alone would yield high rates of false-negative and –positive results. The high frequencies of intI1 and/or 5’ end integron truncations suggest that IntI1 amelioration confers a significant selective advantage. This hypothesis was investigated in E. coli and A. baumannii backgrounds using an IPTG-inducible IntI1-expressing plasmid. Significant fitness costs were observed through growth curve analysis when IntI1 was overexpressed in recA-deficient E. coli lab strains, and also in an A. baumannii strain. The latter was predicted to have a ~1.5-fold higher number of non-canonical IntI1 recombination sites in a typical E. coli strain. The archetypal intI1 repressor, LexA, is not present in the A. baumannii genome, so the role of the closest homologue, ∆UmuDAb, was investigated through knockout studies. ΔumuDAb mutants were created through allelic exchange, and although the mutants showed a significant upregulation of intI1 expression, when the mutants were complemented with a umuDAb-overexpressing plasmid, intI1 expression was also increased, albeit to a lesser extent. This study demonstrates a complex DNA-damage response in A. baumannii, and will aid further investigation for a full understanding of the development and maintenance of antibiotic resistance in this important nosocomial pathogen.

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Angiogenesis is the formation of new blood vessels from existing vasculature. It is a process fundamental to normal development and tissue repair, and is implicated in many pathological conditions. The major pro-angiogenic factor is VEGF, for which the major receptor is VEGFR2. Blood vessels are lined with endothelial cells that express VEGFR2 to detect VEGF in surrounding tissue. This detection mediates cell responses to initiate fOlmation of angiogenic sprouts. VEGFR2 belongs to the family of receptor tyrosine kinases. Ligand binding to the extracellular domain results in receptor dimerisation and autophosphorylation of the intracellular kinase domain. This activates multiple downstream signalling cascades, which in the case of VEGFR2 have four main outcomes: proliferation, migration, permeability and survival. Receptor activation is often followed by internalisation and degradation to downregulate signalling pathways. VEGFR2 has unusual trafficking kinetics for a receptor tyrosine kinase because it constitutively internalises and recycles back to the cell surface in the absence of VEGF. It is distributed such that a significant proportion is localised to an intracellular endosomal storage pool. Moreover, the behaviour of VEGFR2 during angiogenesis depends on the location of the endothelial cell within the growing sprout. The importance of this unusual trafficking in relation to signal transduction is poorly understood. My aim was to elucidate how VEGFR2 trafficking controls angiogenic signalling. I worked to identify sorting proteins required for VEGFR2 trafficking. I investigated proteins that are known to mediate the trafficking of other cargoes. Using an ELISA-based screen, I discovered several potential regulators of VEGFR2, including the motor protein myosin Vb. Myosin Vb traffics organelles along actin filaments and has been described in the recycling of many receptors. I used biochemical methods to establish an essential role for myosin Vb in VEGFR2 recycling in unstimulated conditions. I show that myosin Vb depletion impairs vessel formation in an organotypic angiogenesis assay and disrupts phosphorylation of several kinases activated upon VEGF stimulation in endothelial cells. Furthermore, I show that myosin Vb is required for the polarised distribution of VEGFR2 in endothelial cells to enable chemotactic migration towards VEGF. My data suggest that myosin Vb-dependent constitutive trafficking of inactive VEGFR2 is necessary for angiogenesis.

Membrane shape is dynamic, and cellular membranes adopt different curvatures during fundamental vesicle trafficking events. This is exemplified by the concept of iterative, geometric based sorting. The BAR (Bin/Amphiphysin/Rvs) domain protein superfamily is the largest group of proteins responsible for generating membrane curvature. The work presented in this thesis has examined the cellular function of the BAR domain containing Rho GTPase-activating protein 44 (ARHGAP44; also known as RhoGAP interacting with CIP4 homologs 2 (RICH2)). Recent studies have strongly suggested that ARHGAP44 contributes to intracellular trafficking pathways. In support of this role, work presented here demonstrates that ARHGAP44 functions as a component of the endosomal recycling machinery. ARHGAP44 localises to the Rab4-, Rabll- tubulovesicular recycling compartment, where it facilitates the sorting of endocytic cargo, namely the transferrin receptor and CD317/tetherin, away from the degradation pathway promoting their redelivery to the plasma membrane. Based on its domain organisation, ARHGAP44 would be predicted to link the actin-based cytoskeleton with membrane curvature. I have shown that ARHGAP44 promotes membrane deformation in vitro and in cellulo, and further examination revealed that ARHGAP44-driven membrane tubulation is dependent on its amino-terminal amphipathic helix. Suppression of ARHGAP44 led to an extensive loss of F-actin and an intracellular accumulation of actin into short disorganised bundles, which were frequently found in close proximity to EEA1-positive early endosomes. Rescue experiments revealed that the domain organisation of ARHGAP44 is critical for its ability to regulate endocytic recycling of the transferrin receptor. The activity of ARHGAP44's RhoGAP domain and the hydrophobic composition of its amino-terminal amphipathic helix and conjoined BAR domain were indispensable for this function. In light of these findings, the function of ARHGAP44 was examined during the highly specialised developmental process of erythropoiesis. Data acquired using a model in vitro erythropoiesis culture system demonstrated that ARHGAP44 regulates the intracellular distribution and cell surface homeostasis of the transferrin receptor in maturing erythroblasts.