Structure, function and engineering of biotechnologically important proteins

Al-Maslookhi, Husam 2019. Structure, function and engineering of biotechnologically important proteins. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

A knowledge of the molecular structure of proteins is a key part of understanding protein function and rational protein engineering. The insecticidal toxin Vip1Ac1 (vegetative insecticidal protein 1) is a pore-forming toxin, which has potential to act as a biopesticide. First, its molecular structure and function needs to be understood, and how this might contribute to specificity for certain insects. The molecular structure of the monomer Vip1Ac1 (~82 kDa) at 1.47 Å resolution has been solved by X-ray diffraction. The overall fold of Vip1Ac1 is similar to the anthrax protective antigen (PA), which is a receptor binding/translocation component of anthrax toxin. The high similarity amongst the first 3 domains of both Vip1Ac1 and PA in terms of folding, Ca+2 binding residues important motifs and functions, indicates that they have the same mode of action, while the differences in domain 4 (receptor-binding domain) explain the specificity of each toxin towards different targets. Activation of Vip1Ac1 by trypsin led to removal of an ~22 kDa N-terminal fragment (Vip1Ac122) and self-association of the ~60 kDa C-terminal fragment (Vip1Ac160) into a ring-shaped oligomer as observed in electron microscopy. To probe complex formation further, a reactive azide group at specific surface residues (Y358 and Y536) was introduced by codon reprogramming and allowed labelling of Vip1Ac1 with fluorescent dye via Click chemistry. This was used to monitor assembly on artificial membranes. The oligomeric state showed binding and the ability to form pores in these membranes. The restricted functional groups in natural amino acids restricts standard protein engineering. Introduction of new functional groups by incorporation of non-canonical amino acids (ncAAs) expands the protein engineering diversity. Variants of the Venus fluorescent protein with new fluorescent properties (switched off or on), and a heterodimer (sfGFP+Venus) with new spectra properties differ than each monomeric units were generated by incorporation of ncAAs with new functional groups (azide and alkyne) that allow photoactivation and Click chemistry reactions for dimerisation.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Biosciences
Date of First Compliant Deposit:	11 December 2019
Last Modified:	20 Oct 2020 01:38
URI:	https://orca.cardiff.ac.uk/id/eprint/127471

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