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Identifier 000429527
Title Design of protein and peptide materials for use in nanotechnological applications
Alternative Title Σχεδιασμός πρωτεϊνικών και πεπτιδικών υλικών για χρήση σε νανοτεχνολογικές εφαρμογές
Author Κοκοτίδου, Χρυσούλα
Thesis advisor Μητράκη, Άννα
Reviewer Βελώνια, Καλλιόπη
Χαλεπάκης , Γεώργιος
Χατζηνικολαϊδου, Μαρία
Κρετσόβαλη, Ανδρονίκη
Ταμάμης, Φανούριος
Οικονομίδου, Βασιλική
Abstract Protein and peptide materials with a defined conformation are increasingly used in a wide area of applications. Advantages that characterize proteinaceous biomaterials are their inherent biocompatibility, biodegradability and flexibility of their design and fabrication. The present thesis is focused on the design and study of such biomaterials using as model system a natural fibrous protein the adenovirus fiber. The Adenovirus fiber protein is a homotrimer consisting of an N-terminal tail, a long shaft, and a C-terminal knob region that is responsible for high-affinity receptor binding. The fiber adopts a trimeric nanorod conformation of 30nm length. This protein with specific modifications can be used as an efficient carrier in the area of gene transfer and therapy. In the present thesis a series of constructs were designed having as template the Ad2 fiber protein and inserting functionalities through molecular cloning techniques. These constructs were based on the fibrous shaft segment (residues 61-392) while the globular head of the fiber was replaced by the small (27 aa) trimerization domain of the bacteriophage T4 fibritin, termed “foldon”. Moreover, the protein constructs were modified with metal binding sites, a Histag tail for improving purification, a biotinylation site for streptavidin-biotin conjugation of molecules and cysteine residues at exposed positions for linkage of molecules through disulfide bonds. The chimeric proteins were rendered more stable and were targeted for potential use as delivery agents and gene therapy applications. The shaft segment consists of pseudo amino acid sequence repeats, of which the sequence GAITIG was previously identified as a minimal building block that self-assembles into amyloid-type fibrils. Amyloid fibrils, derived from the studied adenovirus sequences and from a common sequence to Alzheimer’s Aβ peptide and HIV-1 V3 loop, due to their intrinsic mechanical properties are excellent candidates for use as scaffolds. By applying computational methods, the peptides can be rationally designed through mutation of regions amenable to modification aiming at the fabrication of biomaterials with ‘on demand’ functionalities. An essential part of this PhD study was focused on the experimental study of two cell penetrating peptides that were rationally and computationally designed by the group of Asst. Prof. Phanourios Tamamis at Texas A&M University. The peptide sequences comprised natural beta-sheet cores that can self-assemble into amyloid fibrils. The peptides were designed to contain positively charged and aromatic residues exposed at key positions in order to additionally promote DNA condensation and cell internalization. The results demonstrate that these designer peptide fibrils can efficiently enter mammalian cells while carrying packaged luciferase encoding plasmid DNA and act as a protein expression enhancer. Interestingly, the peptides exhibited strong antimicrobial activity against the enterobacterium Escherichia coli. In another aspect of this study instead of exploiting amyloid fibrillization advantages, we sought to inhibit or hinder the process of amyloid fibril formation. The GAIPIG sequence was inspired from the beta-sheet key determinants found in the Ad2 fiber shaft, the Alzheimer’s alpha beta peptide and HIV-1 V3 loop. This peptide despite its similarity with amyloid forming sequences, contains the beta breaker residue (proline) and as a result fails to self-assemble into amyloid fibrils as verified by X-ray diffraction and electron microscopy studies. The structural and experimental information provided in this study could serve as the basis for structure-based design of potential inhibitors of amyloid formation.
Language English
Issue date 2020
Collection   Faculty/Department--Faculty of Sciences and Engineering--Department of Materials Science and Technology--Doctoral theses
  Type of Work--Doctoral theses
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