Use this URL to cite or link to this record in EThOS:
Title: Design, characterisation and functional evaluation of a ɑ-helical barrels
Author: Burton, Anthony J.
ISNI:       0000 0004 5918 334X
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2015
Availability of Full Text:
Access from EThOS:
The de novo design of active biomolecules, where both structure and function are constructed from first principles, requires the precise positioning of chemical functionality within stable and thoroughly characterised scaffolds. The first hexameric coiled coil, CC-Hex, is a homo-hexameric peptide assembly with a contiguous central channel of ~6 A in diameter, which presents clear opportunities for its use as a scaffold for the installation· of catalytic function. In Chapter 3, the reactivity of cysteine (Cys) residues installed within the lumen of CC-Hex with small-molecule alkylating agents is investigated. Comparison of the rates of reaction with iodoacetamide and unfolding of the assembly shows that the former proceeds largely via the assembled channel, rather than via the unfolded state. Furthermore, iodoacetate does not react, suggesting selectivity for uncharged reagents by the hydrophobic lumen of the assembly. Further mutations to the CC-Hex sequence, however, result in largely unfolded peptides. To address this issue, a computational design approach to higher-order coiled coils - the a-helical barrels - is described in Chapter 4, with the aim of delivering highly stable and mutable assemblies. This treats the two interfaces on either side of each helix of the barrel as heterodimer-like interfaces. This allows one million sequences to be generated and scored for the ability to form a-helical barrels. This process is used to generate 22 sequences for synthesis and experimental validation. All of these are a-helical, and, as determined by analytical ultracentrifugation, 8 match the computationally predicted oligomeric state. X-ray crystal structures reveal the first de novo pentameric and bluntended heptameric (CC-Hept) barrels, as well as further hexameric architectures. An iterative approach is described to install a Cys-His-Glu catalytic triad into CC-Hept in Chapter 5. All of the peptide designs are a-helical and heptameric in solution, and X-ray crystal structures are obtained for each of the iterations. For each mutant, hydrolase activity is assessed using p-nitrophenyl acetate (PNPA) as the substrate. Enhanced hydrolysis rates over baseline are observed for the Cys-His dyad peptide and particularly for the Cys-His-Glu triad peptide. Moreover, an increase in catalytic efficiency is observed when homocysteine is installed as the nucleophile, consistent with the increased conformational flexibility of the side-chain thiol. In Chapter 6, metal binding to the a-helical barrels is described, with up to 18 HgII ions binding to multiple Cys residues installed within a lengthened hexameric coiled coil. This once more highlights the mutability of the a-helical barrels and presents their use as monomers for metallated peptide nanotubes and as scaffolds for bioremediation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available