Please use this identifier to cite or link to this item:
https://hdl.handle.net/2440/92810
Full metadata record
DC Field | Value | Language |
---|---|---|
dc.contributor.advisor | Abell, Andrew David | en |
dc.contributor.advisor | Pukala, Tara Louise | en |
dc.contributor.advisor | Forbes, Briony Evelyn | en |
dc.contributor.advisor | Scanlon, Denis B. | en |
dc.contributor.author | Cottam, Jade Misty | en |
dc.date.issued | 2014 | en |
dc.identifier.uri | http://hdl.handle.net/2440/92810 | - |
dc.description.abstract | Insulin-like growth factor II (IGF-II) is a unique regulatory peptide containing 67 residues and three disulfide bonds. It binds with high affinity to three receptors, the insulin receptor (IR), the type 1 insulin-like growth factor receptor (IGF-1R) and the We 2 insulin-like growth factor receptor (IGF-2R). Binding of IGF-II to these receptors signals mitogenic responses, such as cell proliferation, differentiation and migration. The interactions of IGF-II with the IR and IGF-1R have recently been identified as potential therapeutic targets for the treatment of cancer. Thus, an increased understanding of the interactions of IGF-II with the IGF-1R and the IR-A is required for the improved design and development of potential anticancer therapeutics. A crystal structure of IGF-II bound to either the IGF-1R or the IR-A has not been reported. Thus, the precise location of IGF-II within the receptor binding pocket remains undefined. A fluorescence resonance energy transfer (FRET) approach was proposed to investigate the binding location and orientation of IGF-II within the IGF-1R. Two fluorescent IGF-II analogues, the Fl9Cou IGF-II and F28Cou IGF-II proteins, were synthesised for use in the desired FRET studies. These FRET experiments first required the synthesis of an appropriate coumarin-based probe for incorporation into IGF-II. The synthesis of a range of fluorescent coumaryl amino acids is described in Chapter 2, and an analysis of the spectroscopic properties of these coumaryl amino acids is also detailed. Site-specific incorporation of the coumarin-based probe into IGF-II was then undertaken. Three complementary methods were used for the preparation of the desired fluorescent IGFII analogues. Chapter 3 describes the use of the nonsense suppression methodology for the expression of the novel Fl9Cou IGF-II protein. This was followed by an improved chemical synthesis of the Fl9Cou IGF-II protein using a linear solid phase peptide synthesis (SPPS) approach and is detailed in Chapter 4. A robust native chemical ligation approach was developed in Chapter 5, which allowed for the facile incorporation of the coumarin-based probe at various locations within the IGF-II protein. Chapter 5 also details the synthesis of the native IGF-II, Fl9Cou IGF-II and F28Cou IGF-II proteins. The biological activity of the resultant IGF-II analogues was evaluated by competition binding assays. The fluorescent IGFII analogues bind with low nanomolar affinity to the IR and IGF-1R, and as such were deemed suitable for use in the desired FRET-based experiments. The FRET-based investigation into the binding interactions of the native IGF-II, Fl9Cou IGFII and F28Cou IGF-II proteins to the IGF-1R is described in Chapter 6. FRET interactions were observed for both the Fl9Cou IGF-II and F28Cou IGF-II proteins. The results show the fluorophore binds in close proximity to Trp residues within the IGF-1R receptor and suggest the location of IGF-II binding within the IGF-1R is consistent with what is proposed in the literature. These experiments provide a basis for further investigations for determining the precise binding location and orientation of IGF-II within the IGF-1R. | en |
dc.subject | IGF-II; IGF-1R; IR-A; coumarin; fluorescence; FRET | en |
dc.title | A study on the interactions of synthetic IGF-II analogues with the type 1 IGF and insulin receptors. | en |
dc.type | Thesis | en |
dc.contributor.school | School of Chemistry and Physics | en |
dc.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 | en |
dc.description.dissertation | Thesis (Ph.D.) -- University of Adelaide, School of Chemistry and Physics, 2014 | en |
Appears in Collections: | Research Theses |
Files in This Item:
File | Description | Size | Format | |
---|---|---|---|---|
01front.pdf | 1.18 MB | Adobe PDF | View/Open | |
02whole.pdf | 20.23 MB | Adobe PDF | View/Open | |
Permissions Restricted Access | Library staff access only | 288.5 kB | Adobe PDF | View/Open |
Restricted Restricted Access | Library staff access only | 52.69 MB | Adobe PDF | View/Open |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.