Molecular trees: the rational design of a self-assembling protein scaffold

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dc.contributor.advisor Squire, C en
dc.contributor.advisor Young, P en
dc.contributor.author Kerridge, Robert en
dc.date.accessioned 2017-08-14T00:32:43Z en
dc.date.issued 2017 en
dc.identifier.uri http://hdl.handle.net/2292/35064 en
dc.description Full Text is available to authenticated members of The University of Auckland only. en
dc.description.abstract There are a number of techniques available to a synthetic biologist that enable the joining together of proteins, however, until recently there were few tools that could covalently link multiple proteins together into larger assemblies. The discovery of intramolecular cross-links within the adhesin stalk domains of many Grampositive bacteria, has sparked the development of so called ‘molecular superglues’, that can be engineered from these domains to perform protein ligation. Our lab has developed a handful of protein ligation tools by splitting ester bond cross-link domains into a ‘split-ester’ domains. In previous work, these split-ester domains have demonstrated selective peptide capture and predictable multi-protein self-assembly. Within this MSc project, a handful of these split-ester domains were recombinantly engineered with the aim of developing a large supramolecular scaffold, capable of selectively capturing metabolic enzymes and ultimately – creating an assembly that mimics a naturally occurring metabolon. Here we introduce the foundations of a programable protein scaffold, capable of selectively ligating proteins together to form a predictable branching ‘tree-like’ structure. The completely assembled tree is comprised of nine proteins that are covalently secured to the scaffold by spontaneously forming ester bond cross-links. We have confirmed the presence of the complete scaffold by SDS-PAGE, and have shown it has successfully captured four GFP cargo proteins as a proof of concept. A scaffold with a single branch was investigated by small-angle X-ray scattering (SAXS) revealing a folded and stable structure with dimensions that complement the clustering of cargo-proteins or enzymes. SAXS analysis of the scaffolds components was also performed and this illustrated the dynamic folding event that takes place when a single split-ester domain captures a peptide or cargo protein. Additionally, throughout this project we have expanded the molecular superglue toolkit available to synthetic biologists. We have also gained deeper insights into the general structural properties of Gram-positive ester bond adhesin domains, that dictate whether an engineered domain will work better as a standalone protein ligation tool or as part of a cohort of self-assembling domains. These findings may guide the future discovery of more molecular superglues and provide greater control over orchestrating the assembly of supramolecular structures. en
dc.publisher ResearchSpace@Auckland en
dc.relation.ispartof Masters Thesis - University of Auckland en
dc.relation.isreferencedby UoA99265017214002091 en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher. en
dc.rights Restricted Item. Available to authenticated members of The University of Auckland. en
dc.rights Restricted Item. Full Text is available to authenticated members of The University of Auckland only. en
dc.rights.uri https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm en
dc.rights.uri http://creativecommons.org/licenses/by-nc-sa/3.0/nz/ en
dc.title Molecular trees: the rational design of a self-assembling protein scaffold en
dc.type Thesis en
thesis.degree.discipline Biological Sciences en
thesis.degree.grantor The University of Auckland en
thesis.degree.level Masters en
dc.rights.holder Copyright: The author en
pubs.elements-id 648418 en
pubs.record-created-at-source-date 2017-08-14 en


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