Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/115168
Type: Theses
Title: Biological and structure characterisation of eukaryotic prefoldin
Author: Tran, Denise Phuong
Issue Date: 2018
School/Discipline: School of Physical Sciences
Abstract: Prefoldin is a hexameric protein complex ubiquitously expressed and found to influence the conformation of amyloidogenic peptides. Relatively high degrees of sequence identity and conservation across evolutionary lineages are observed, however differences in binding abilities have been noted between the homologs. This thesis describes work examining the structure of eukaryotic prefoldin and its biological activities with respect to interaction with amyloid β. The structure and biological activities of prefoldin’s individual subunits are also explored. Although many studies have investigated the structure of prokaryotic prefoldin, there is limited information available for eukaryotic prefoldin. Two-dimensional ¹H-¹H and ¹H-¹³C nuclear magnetic resonance (NMR) spectroscopy was utilised to probe the structure of both α and β human prefoldin subunits. The data revealed the highly alpha helical secondary structure of the subunits, which was further verified through far-UV circular dichroism. Further thermal aggregation assays utilising this technique have demonstrated the stability of the prefoldin subunits. The biological effect of prefoldin on the amyloid fibril formation of the Alzheimer’s disease related amyloid β peptide was investigated using a combination of dye-binding assays and cytotoxicity assays. The presence and absence of fibrils was confirmed by transmission electron microscopy. In terms of fibril formation, prefoldin and its subunits prevented in vitro conversion of the amyloid β peptide to amyloid fibrils. In some cases, total inhibition of fibril formation occurred and a 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted on the resultant products. The product was incubated with healthy PC-12 cells and induced cellular death, therefore establishing the cytotoxicity of the resultant oligomeric amyloid β form. Previous investigations into the binding capabilities of prokaryotic prefoldin identified the distal tips as an important structural aspect, interacting with the amyloidogenic peptide. The binding interface of prefoldin subunits 5 and 6 with amyloid β was probed using chemical cross-linking (CXL) experiments. Traditional methods to identify cross-linked peptides are challenging and the results are often ambiguous. In this study, CXL products were analysed by liquid chromatography-ion mobility-mass spectrometry (LC-IM-MS) to investigate the utility of IM in enhancing the CXL analytical workflow. The orthogonal separation of ion mobility enabled the identification of the cross-linked amino acids. The distal end of prefoldin subunit 5 was found to interact with the Nterminus of the amyloid peptide, whereas prefoldin subunit 6 was identified to interact with the peptide in the middle of its sequence. Ion mobility-mass spectrometry (IM-MS) analysis of the eukaryotic prefoldin complex identified the collisional cross section of the intact hexamer. Solution disruption experiments of the intact complex revealed the disengaging sub-complexes, and information on the intersubunit contacts and relative interfacial strengths were obtained. A capillary temperature controller (CTC) was developed to observe the thermal dissociation of the complex using nano-electrospray IM-MS. The combination of these results confirmed a structural aspect common to both mammalian prefoldin and prokaryotic prefoldin, despite the primary sequence differences. The biological assays revealed the ability of prefoldin to prevent the aggregation and amyloid fibril formation of amyloid β, and low resolution MS techniques were able to postulate the arrangement of the subunits and the possible interface interactions of the hexameric complex with the amyloidogenic peptide. This thesis has therefore provided an in-depth investigation of the structural characteristics of eukaryotic prefoldin and its chaperoning capability, therefore implicating a potential role for prefoldin in modulating protein misfolding and aggregation.
Advisor: Pukala, Tara Louise
Booker, Grant William
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Physical Sciences, 2018
Keywords: Eukaryotic prefoldin
amyloid beta (1-40)
biophysical characterisation
protein structure
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at http://www.adelaide.edu.au/legals
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