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Title: Comparisons of solution structures of free and IL-1β bound Fab suggests a model for B cell receptor signalling
Author: Hall, Catherine Jane
ISNI:       0000 0004 2744 2714
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2010
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Therapeutic antibodies are an important and growing class of biotherapeutics with the potential to treat a range of major human diseases. Structural knowledge of the complexes formed between antibodies and target proteins would provide valuable information about the mode of action of the therapeutic and potentially reveal a structural mechanism underlying antigen induced B cell receptor signalling. Often the Fab fragment of the antibody is used in therapeutics, however, there are currently no NMR-based structures available for Fab or Fab/antigen complexes, presumably due to the difficulties of producing suitable samples and obtaining NMR data for large proteins and complexes. The work described here illustrates the development of approaches that have made it possible to obtain high quality NMR-based structures for the gIC8 Fab alone and in complex with its antigen, IL-1β, including the development of approaches that allow the measurement of reliable RDC data. Expression and purification of a [superscript 15]N/[superscript 13]C/[superscript 2]H-labelled gIC8 Fab fragment allowed for collection of triple resonance data for the free Fab and over 90% of the visible backbone resonances to be assigned. A refined homology model of the free gIC8 Fab and a docked and refined structure for the gIC8 Fab/IL-1β complex were obtained using experimental NMR restraints. Comparisons made between the free and bound Fab structures highlight small antigen induced changes in domain orientation, which suggest a mechanism for B cell receptor signalling. The work reported here shows that NMR spectroscopy can be used as a tool to obtain detailed structural data for large Fab and Fab-target protein complexes and provide an improved understanding of how antibodies interact with their target proteins.
Supervisor: Carr, Mark Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available