IgA is not only a major serum immunoglobulin (Ig) in humans but is also the predominant antibody class found in external secretions. The mucosal surfaces of the lung, GI and GU tracts, bathed by these secretions, represent a major site of exposure. As the chief antibody at these sites, IgA thus forms a critical first line of defence against many invading pathogens. Serum IgA is mainly monomeric (2 Fab arms and 1 Fc region), while secretory IgA is predominantly dimeric, comprised of two IgA monomers, J chain, and secretory component linked by disulphide bridges. Our recent data, and those of others, indicate a unique functional role for polymeric forms of human IgA, in part mediated by polymeric-specific cellular receptors. Human IgA has two isotypes, IgA1 and IgA2, found in both serum and secretions. Despite the abundance and importance of IgA, surprisingly little is known about the structures of the different IgA forms and their relationship to their roles in immunity. We have recently determined the first averaged solution structure for serum IgA1 using a novel technique based on constrained automated neutron and X-ray scattering curve modelling and homology modelling. A T-shaped structure was revealed in which the glycosylated hinges holds the two Fab arms apart from Fc by a high separation of 17 mm. This is quite different from IgG. It is consistent with both the unique functional properties of the IgA1 hinge, and the distinct location in the Fc region of the key interaction site for the myeloid Fca receptor (FcaR). The success of the IgA1 project acts as a springboard for further novel determinations for the Fab and Fc arrangement in monomeric IgA2, the arrangement of IgA monomers and J chain in dimeric IgA1 and IgA2, and the domain arrangement of secretory component when free (recombinant) and in association with IgA1 dimers. Unique expression systems and isolation techniques to provide both natural and recombinant forms of the above molecules are already established. Structures will be determined using neutron and X-ray scattering and analytical ultracentrifugation, all in conjunction with bioinformatics strategies to general IgA homology models and fit these to the data. The structural models will be tested/corroborated by controls based on cleavage fragments of IgA1, comparisons with human IgG1-4 subclasses, and electron microscopy. We will also attempt crystallisation trials. The structural data, combined with functional data on the interaction of IgA with cellular receptors and proteases, will allow a detailed understanding of IgA function for the first time, and will aid the national development of novel immunotherapy and vaccine strategies.

I will explore human population genetic variation in tetratricopeptide repeat (TPR) domains. TPRs are ubiquitous structural motifs that provide a platform for mediating protein-protein interactions and scaffolding multiprotein complexes. TPR-containing proteins are involved in many fundamental biological processes, including transcription, cell cycle progression, and neurodevelopment. TPRs are thought to act individually in mediating substrate interactions. Furthermore, mutations in TPR domains have been associated with many human diseases. For example mutations in O-linked β-N-acetylglucosaminylation (O-GlcNAc) transferase (OGT) cause X-linked intellectual disability (XLID). The importance of TPRs to the interactome is supported by past experimental work and the prevalence of TPR-associated diseases. I will apply sequence and structural analysis to TPR domains proteome-wide, in concert with a recently developed method to integrate human population variation with multiple sequence alignments. The novelty in applying variants from population data would unravel functionally relevant residues that establish the TPR domain architecture and its multifaceted role in protein-protein interactions. I will bring further insight into protein evolution of TPRs and dissecting diseases associated with them, establishing the basis for future research on other solenoid domains and protein systems.

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Developing future therapies, the first of EPSRC's Healthcare Technologies Grand Challenges, is essential to keep the National Health Service sustainable. Currently, an estimated 70% of the UK's healthcare expenditure goes towards the management of chronic diseases. Regenerative medicine is expected to significantly reduce costs as it can turn chronic, degenerative, diseases into curable conditions. Realising this potential requires the right tools to study the biological development process as it progresses from the single cell to the complex structure of entire organs. The invention of the optical microscope, and in particular the phase contrast microscope, made it possible to highlight the fine features of living cells with unprecedented clarity. However, cells isolated on a microscope slide often do not behave as tissue in its natural, three-dimensional, environment. The recent development of the planar illumination light-sheet microscope enabled the visualisation of the intact, fluorescently-labelled, organisms during development. While light-sheet microscopy is highly successful for transparent zebrafish or chemically cleared tissue, many tissues are too opaque to be studied beyond the first layer of cells. A prime example is the chick embryo in its early stages of development. The complex collective behaviour of the cells in the initial layer can be studied in exquisite detail, yet as soon as the primitive streak forms, to grow the embryo in the third dimension, we lack the tools to keep track of the cell migration and differentiation. The images are too blurred. Correlative refractive index light-sheet microscopy aims to make the invisible visible. It is based on the realization that the turbidity of biological samples is due to the same refractive index variations that yield structural information in phase-contrast microscopy. The distribution of optical properties within the specimen not only contains valuable structural information for the biologist, it also enables the adaptive wavefront correction needed for high resolution fluorescence imaging. By developing a hybrid instrument that maps the optical property distribution in parallel with the fluorescence image, this proposal will enable high-resolution deep-tissue imaging in turbid biological specimen. A direct view into the inner workings of the biological development process is essential to develop effective regenerative-medicine therapies.