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Title: The crystallographic analysis of the Bacillus thuringiensis delta-endotoxin Cry1Ac, on its own and in a complex with its receptor ligand
Author: Derbyshire, D. J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2000
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Cry toxins are produced as inactive protoxins and require proteolytic processing (activation) within the midguts of susceptible insect larvae. Activated toxins then bind to high affinity receptor molecules on the midgut epithelium, leading to membrane insertion and pore-formation. This study is the structural determination of the active Cry1Ac toxin and its comparison with Cry1Aa. The success of this study was critically dependent upon the re-development and optimisation of existing activation and purification procedures (following problems with aggregation). Trypsin-activated Cry1Ac was crystallised, on its own and in complex with its high affinity receptor ligand (GalNAc). Diffraction data were collected to 2.36 Å and to 2.95 Å resolution for the 'free toxin' and the 'complex' respectively. The structures were then solved by molecular replacement and a structural analysis undertaken. Differences between the structures of Cry1Ac and Cry1Aa are localised to regions correlated with mutagenesis experiments indicating a role in receptor binding. The most prominent of these structural differences occurs where Cry1Ac contains a protruding loop corresponding to a sequence insertion unique to Cry1Ac. Mutagenesis experiments (performed by collaborators) designed on this basis, confirm the hypothesis that this loop is directly involved in GalNAc binding. The structure of the complex directly identifies the residues interacting with GalNAc. These, do indeed, include residues within this loop, prompting further biochemical analysis and discussion of how Cry toxin specificity is determined.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available