Structural analysis of pesticidal proteins and their interactions

Williamson, Lainey 2023. Structural analysis of pesticidal proteins and their interactions. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Due to their high specificity and biocompatibility, pesticidal proteins produced by Bacillus thuringiensis and Lysinibacillus sphaericus have been successfully applied as bioinsecticides for use in the control of agricultural pests and disease vectors. Given their significance, it is critical that we understand the mechanisms by which B. thuringiensis and L. sphaericus exert their pesticidal activity, both to counteract emerging field resistance and develop new bioinsecticides with enhanced potency, stability, and target insect range. In this work, a combination of serial femtosecond crystallography (SFX), single-particle cryogenic electron microscopy (cryoEM), and computational modelling were employed to study the structure and interactions of B. thuringiensis and L. sphaericus pesticidal proteins. Chapter 3 uses SFX to elucidate the Cry8Ba2 structure from crystals produced naturally by B. thuringiensis. The Cry8Ba2 structure represents the first structure of a full-length Cry protein elucidated from natural crystals and provides insight into crystal packing and the biological function of the pro-toxin domains. Chapter 4 builds on this work by utilising SFX to elucidate the structure of Tpp49Aa1 from the binary Cry48/Tpp49 mosquitocidal toxin. Complementary pH mixing studies enabled the early structural changes in crystal dissolution to be investigated. In addition, the interaction of Tpp49Aa1 with its partner protein, Cry48Aa1, was predicted, enabling discussion related to models for mode of action. Finally, insect bioassays were performed, leading to the identification of new target mosquito species. Chapter 5 investigates the interaction of the Tpp2 protein, from the binary Tpp1/Tpp2 mosquitocidal toxin, with its target receptor, Cqm1, using computational modelling. The predicted model was compared with that of the Tpp2Aa2-Cqm1 structure elucidated using single-particle cryoEM. iii Directed by the Tpp2Aa2-Cqm1 structure, mutagenesis studies were performed with the aim of engineering the Tpp1Aa2/Tpp2Aa2 pesticidal protein to broaden the target insect range.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	24 April 2024
Last Modified:	25 Apr 2024 13:33
URI:	https://orca.cardiff.ac.uk/id/eprint/168271

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