Doctoral theses
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| Identifier |
000429527 |
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| Title |
Design of protein and peptide materials for use in nanotechnological applications |
| Alternative Title |
Σχεδιασμός πρωτεϊνικών και πεπτιδικών υλικών για χρήση σε νανοτεχνολογικές εφαρμογές |
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Author
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Κοκοτίδου, Χρυσούλα
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Thesis advisor
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Μητράκη, Άννα
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Reviewer
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Βελώνια, Καλλιόπη
Χαλεπάκης , Γεώργιος
Χατζηνικολαϊδου, Μαρία
Κρετσόβαλη, Ανδρονίκη
Ταμάμης, Φανούριος
Οικονομίδου, Βασιλική
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| 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.
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| Language |
English |
| Issue date |
2020 |
| Collection
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School/Department--School of Sciences and Engineering--Department of Materials Science and Technology--Doctoral theses
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Type of Work--Doctoral theses
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| Permanent Link |
https://elocus.lib.uoc.gr//dlib/a/2/a/metadata-dlib-1591009327-823522-17637.tkl
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| Views |
395 |
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