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Structural and Biochemical Studies of Cell-Grown Cry11Ba Insecticidal Protein Crystals

Abstract

Nature self-assembles protein structures for various functions, including: storage, protection, and fortification. These self-assemblies range from filaments to full three-dimensional crystals and are pervasive across the tree of life. They include the granules present in immune system cells, the packing of hormones in the pancreas, the storage of proteins in plants, carboxysomes, viruses, and cell-grown crystals in microbes. To accomplish this, a better understanding of how certain organisms are able to naturally self-assemble macromolecules and how to recreate these in living cells must be achieved. Bacillus thuringiensis subsp. israelenesis’ (Bti) crystalline inclusions are of exceptional interest, since they naturally package a single protein into a crystalline inclusion through a life cycle process called sporulation. Compared to classical macromolecular crystallography that takes a plethora of variables to exhaustion can still yield no crystals. The laborious process could be prevented; however, by better understanding Bt and these crystalline inclusions’ cellular self-assembly process. Cry11Ba is a protein packed into these crystalline (Cry) inclusions and found to be one of the most toxic pesticidal proteins. These crystals are then ingested by their host and switch from their packaged toxin crystal to their inactive protoxin at the high pH within their gut. My interests have been in elucidating the macromolecular structure from in vivo produced crystals, further understanding the ambiguous mode of action to gain better perspective for other δ-endotoxins, and probing the self-assembly of the crystalline inclusions in vivo throughout the sporulation process. I have studied Cry11Ba with structural analysis, solubility & toxicity assays, mutational studies, and imaging capabilities developed in cryo-EM to successfully in solve a de novo structure via in vivo crystalline inclusions, charting the pH sensitivity of the crystals, identifying key residues for stability and toxicity, analyzed the monomeric and multimeric particles in alkaline environments to understand Cry11Ba’s mode of action, and visualized previously unobserved sporulation stages for Bti.

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