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Title: Rational design, construction and characterisation of coiled-coil peptide nanotubes
Author: Burgess, Natasha Claire
ISNI:       0000 0004 6056 7279
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2016
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The Woolfson lab designed the first example of a Self-Assembling peptide Fibre system (SAFs), using a "sticky ended" parallel, heterodimeri~ coiled-coil building block; that is, two complementary α-helices that come together in a staggered fashion leaving an overhang to promote fibre growth. Other groups have since used similar strategies to direct fibre formation. In contrast, few α-helix-based peptide nanotubes have been reported; the main example presented so far is from Conticello's laboratory, who remodelled a known staggered coiled-coil heptamer in a "lock-washer" mechanism for nanotube formation. All of these strategies have required the construction of bespoke building blocks. The goal of this thesis was to expand the possibilities in this area and devise a general and modular method for producing peptide-based fibres and nanotubes. As well as advancing peptide self-assembly, an ability to create nanotubes with defined and mutable internal channels could provide new biomaterials for nanotechnological, engineering and medical applications. This thesis establishes that blunt-ended α-helical barrels-namely a-helices assembled around a central, open and accessible channel-can be redesigned and used as building blocks to create stable fibrous systems, with the added benefit of large and modifiable internal surface areas. The α-helical components of a coiled-coil toolkit, recently published by the Woolfson group, were taken and the termini of the peptides redesigned to promote longitudinal self-association of these coiled-coil bundles and barrels. These components were used to successfully create a range of peptide fibres, based on trimeric and tetrameric building blocks, and nanotubes, from pentameric, hexameric and heptameric blocks. This demonstrates that the design strategy of promoting blunt-ended assembly of standard building blocks is general and systematic. The fibrous structures formed have been characterised using a combination of transmission electron microscopy, X-ray fibre diffraction and circular dichroism spectroscopy. Furthermore, CC-Hex-T (the original hexameric building block) was found to assemble into highly ordered peptide nanotube fibres when heated and cooled. When imaged by electron microscopy, these fibres have clear and persistent longitudinal and lateral striations that originate from the regular periodicity and repeating lattice, demonstrating the precise alignment of the peptide building blocks. The solved molecular structure indicates that the overall symmetry of the peptide building block is the likely driving force to create paracrystalline order in the supramolecular assembly. One of our original goals was to construct single-peptide nanotubes suitable for adding function, and for applications in biotechnology. As a starting point for this, two strategies for preventing fibre thickening with the CC-Hex-T building block have been devised: the first is a self-associating, non-covalent method; and the second, a covalent approach using native chemical ligation. Both methods yield single nanotubes. Moreover, these can passively encapsulate long, thin molecules, such as organic dyes. Finally, the internal modification of the a-helical barrels with cysteine residues has been investigated, although results are only preliminary. If successful, this would allow selective metallation along the centre of the nanotubes, with the goal of creating high-aspect ratio nanowires. The principle advantage of the general nanotube design developed through this thesis is that it allows a range of constructs to be made and investigated. Nanotubes can be made from a selection of initial building blocks, and the internal channel size (currently 5 - 7 A) can be adjusted to match the desired application.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available