226 research outputs found
Solid-phase synthesis of homodimeric peptides: preparation of covalently-linked dimers of amyloid beta peptide
No full textCovalently cross-linked homodimeric Aβ peptides have been prepared by solid-phase peptide synthesis by exploiting ‘site–site interactions’, and exhibit substantially increased oligomerisation and fibrillisation properties compared with the corresponding monomers.W. Mei Kok, Denis B. Scanlon, John A. Karas, Luke A. Miles, Deborah J. Tew, Michael W. Parker, Kevin J. Barnham and Craig A. Hutto
Electron transfer through alpha-peptides attached to vertically aligned carbon nanotube arrays: a mechanistic transition
No full textThis article is part of the Artificial photosynthesis web themed issueThe mechanism of electron transfer in α-aminoisobutyric (Aib) homoligomers is defined by the extent of secondary structure, rather than just chain length. Helical structures (Aib units ≥3) undergo an electron hopping mechanism, while shorter disordered sequences (Aib units <3) undergo an electron superexchange mechanism.Jingxian Yu, Ondrej Zvarec, David M. Huang, Mark A. Bissett, Denis B. Scanlon, Joe G. Shapter and Andrew D. Abel
Total synthesis of the antifungal depsipeptide petriellin A
No full textPublication Date (Web): July 8, 2011We report the solid-phase total synthesis of the antifungal highly modified cyclic depsipeptide petriellin A. The synthesis confirms earlier reports on the absolute configuration of the natural product. The solid-phase approach resulted in a protected linear precursor, which was cleaved from the solid support prior to cyclization and final deprotection. Use of advanced coupling agents for several hindered amides was a feature of the synthesis. The natural product was prepared in overall 5% yield.Marianne M. Sleebs, Denis Scanlon, John Karas, Rani Maharani, and Andrew B. Hughe
Functional fibrils derived from the peptide TTR1-cycloRGDfK that target cell adhesion and spreading
No full textPeptide self-assembly offers a route for the production of fibrous nanomaterials with advanced bioactive properties that promote specific cell interactions. In this study the peptide TTR1-cycloRGDfK was designed to form amyloid-like fibrils that display the functional cyclic RGDfK pentapeptide ligand to target mammalian cell surface α(V)β₃ integrin receptors. The TTR₁₀₅₋₁₁₅ (or TTR1) sequence was used as the self-assembling domain. Once assembled, TTR1-cycloRGDfK fibrils display a characteristic cross-β core structure by X-ray fibre diffraction that was preserved following dehydration. Thin films of fibrils were characterised by infrared synchrotron mapping, scanning electron microscopy and atomic force microscopy. Cell adhesion and spreading were promoted on thin films of TTR1-cycloRGDfK fibrils via specific interactions with the cyclic RGDfK ligand. Low levels of non-specific interactions were also observed between cells and non-functionalised fibrils. TTR1-cycloRGDfK fibrils are an advance on bioactive fibrils previously designed to interact with a range of RGD binding integrins and our findings show that the assembly of amyloid-like fibrils based on the TTR1 sequence is robust and can be directed to form materials with specific properties.Marie N. Bongiovanni, Denis B. Scanlon, Sally L. Gra
Synthesis of peptide sequences derived from fibril-forming proteins
No full textAlso published as a book chapter: Protein Folding, Misfolding, and Disease: Methods and Protocols, 2011 / A. F. Hill, K. J. Barnham, S. P. Bottomley & R. Cappai (eds.), Ch.3 pp.29-43The pathogenesis of a large number of diseases, including Alzheimer's Disease, Parkinson's Disease, and Creutzfeldt-Jakob Disease (CJD), is associated with protein aggregation and the formation of amyloid, fibrillar deposits. Peptide fragments of amyloid-forming proteins have been found to form fibrils in their own right and have become important tools for unlocking the mechanism of amyloid fibril formation and the pathogenesis of amyloid diseases. The synthesis and purification of peptide sequences derived from amyloid fibril-forming proteins can be extremely challenging. The synthesis may not proceed well, generating a very low quality crude product which can be difficult to purify. Even clean crude peptides can be difficult to purify, as they are often insoluble or form fibrils rapidly in solution. This chapter presents methods to recognise and to overcome the difficulties associated with the synthesis, and purification of fibril-forming peptides, illustrating the points with three synthetic examples.Denis B. Scanlon and John A. Kara
Total chemical synthesis of an intra-A-chain cystathionine human insulin analogue with enhanced thermal stability
No full textDespite recent advances in the treatment of diabetes mellitus, storage of insulin formulations at 48C is still necessary to minimize chemical degradation. This is problematic in tropical regions where reliable refrigeration is not ubiquitous. Some degradation byproducts are caused by disulfide shuffling of cystine that leads to covalently bonded oligomers. Consequently we examined the utility of the non-reducible cystine isostere, cystathionine, within the A-chain. Reported herein is an efficient method for forming this mimic using simple monomeric building blocks. The intra-A-chain cystathionine insulin analogue was obtained in good overall yield, chemically characterized and demonstrated to possess native binding affinity for the insulin receptor isoform B. It was also shown to possess significantly enhanced thermal stability indicating potential application to next-generation insulin analogues.John A. Karas, Nitin A. Patil, Julien Tailhades, Marc-Antoine Sani, Denis B. Scanlon, Briony E. Forbes, James Gardiner, Frances Separovic, John D. Wade, and Mohammed Akhter Hossai
Bacterial expression and purification of active hematopoietic cell kinase
No full textSrc family kinases (SFKs) are traditionally purified from eukaryotic expression systems. These expression systems can be costly, yield heterogeneously phosphorylated protein samples and present difficulties when metabolic labeling is required for structural studies. Therefore, many attempts have been made to develop bacterial purification systems for SFKs. So far, high-yield bacterial expression systems have only been achieved for SFK kinase domains or for inactive mutants of constructs containing the regulatory SH3 and SH2 domains, but not for their active forms. Herein described is a bacterial expression system for the wild type, active SFK Hck containing SH3, SH2 and kinase domains. Hck plays an important role in phagocyte function as well as the etiology of chronic myeloid leukemia as Hck is an interaction partner of Bcr-Abl. Structural studies of Hck are essential to fully understand the signaling processes involved in host defense and leukemogenesis. Successful bacterial expression of Hck was possible by a dual strategy: (1) co-expression with YopH phosphatase in order to control host toxicity, and (2) expression in a bacterial strain that is RNase E deficient, which dramatically increased overall expression levels. The expressed Hck construct is unphosphorylated and appears to be in an open conformation. Bacterially expressed Hck is capable of autophosphorylation, phosphorylates substrate at rates comparable to insect cell expressed Hck, and can be inhibited by staurosporine and Csk.Romana Kristelly, Theresa Wenli Qiu, Natalie J. Gunn, Denis B. Scanlon, Terrence D. Mulher
‐Helical PIP‐Box Mimetic Targeting PCNA, the Human Sliding Clamp
No full textThe human sliding clamp (PCNA) controls access to DNA for many proteins involved in DNA replication and repair. Proteins are recruited to the PCNA surface by means of a short, conserved peptide motif known as the PCNA-interacting protein box (PIP-box). Inhibitors of these essential protein-protein interactions may be useful as cancer therapeutics by disrupting DNA replication and repair in these highly proliferative cells. PIP-box peptide mimetics have been identified as a potentially rapid route to potent PCNA inhibitors. Here we describe the rational design and synthesis of the first PCNA peptidomimetic ligands, based on the high affinity PIP-box sequence from the natural PCNA inhibitor p21. These mimetics incorporate covalent i,i+4 side-chain/side-chain lactam linkages of different lengths, designed to constrain the peptides into the 310-helical structure required for PCNA binding. NMR studies confirmed that while the unmodified p21 peptide had little defined structure in solution, mimetic ACR2 pre-organised into 310-helical structure prior to interaction with PCNA. ACR2 displayed higher affinity binding than most known PIP-box peptides, and retains the native PCNA binding mode, as observed in the co-crystal structure of ACR2 bound to PCNA. This study offers a promising new strategy for PCNA inhibitor design for use as anti-cancer therapeutics.Kate L. Wegener, Amy E. McGrath, Nicholas E. Dixon, Aaron J. Oakley, Denis B. Scanlon, Andrew D. Abell, John B. Brunin
Chemical synthesis of a fluorescent IGF-II analogue
No full textInsulin-like growth factor II (IGF-II) is a protein with high structural and sequence similarity to insulin. Unlike insulin, it binds both the type 1 IGF receptor and the exon 11- isoform of the insulin receptor with high affinity. The overexpression and up regulation of IGF-II has been associated with the progression of various forms of cancer. The exact binding mechanism of IGF-II to its high affinity receptors is still not completely understood. Herein we describe the successful synthesis of a novel fluorescent IGF-II protein (F19Cou IGF-II), where residue 19 (phenylalanine) has been replaced by a fluorescent chromophore (coumaryl glycine). This novel coumaryl IGF-II analogue will be a useful tool for analysing the receptor interaction mechanisms in future studies.Jade M. Cottam, Denis B. Scanlon, John A. Karas, Antonio N. Calabrese, Tara L. Pukala, Briony E. Forbes, John C. Wallace, Andrew D. Abel
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