| Abstract |
Development of novel biomaterials based on peptides and proteins, is a rapidly developing field of nanotechnology. A detailed and comprehensive understanding of protein folding would provide important information for the development of novel bio-inspired materials as the rational design of biomaterials can be developed through exploitation of the self-assembly properties of suitable protein-based scaffold.
In this PhD thesis, the elucidation of the protein folding of a 4-α-helical bundle protein is followed by the exploitation of the use this recurrent protein motif as building block for biomaterials. The small, homodimeric Rop protein that is a paradigm of a canonical 4-α-helical bundle, is used as a model to this research.
Approaching the folding problem of Rop, the first goal of this PhD research was to elucidate the role of the loops and structural intermediates in folding of Rop protein. Based on A31P, one of the extensively studied loop-mutants of Rop with drastic structural and physicochemical changes, three new mutants, with proline and glycine residues in the loop region were produced. Their structure and biophysical properties were studied confirming the role of turn residues in folding pathways and indicating that the effects of the loop region on a specific folding pathway are complex, being both sequence- and position- dependent.
The development of novel topologies and the extreme malleability of the Rop structure, demonstrated by the above studies, make this protein an attractive candidate for the development of novel biomaterials based on the 4-α-helix bundles which defines the second goal of this thesis. Based on RM6, a Rop mutant with a deletion of Rop residues, that is completely reorganized forming a homotetrameric bundle consisting almost entirely of straight α-helices with remarkable stability, two new Rop mutants have been produced that self-assemble to filament-like structure with nano- to micrometer features. The RM6-based bio-buildings block were, in addition, decorated with attachment of two selected mutants of the I-CreI meganuclease producing fibers that not only retain the activity of the attach endonuclease but also promote the creation of I-CreI heterodimer, that exhibit new
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DNA specificities and creates a basis for the use of these molecules as tools for genome modifications and gene therapy.
Concluding, the results of the present PhD thesis, significantly contribute to the elucidation of the folding pathway of 4-α-helical bundles and to an improved capacity for rational development of protein based fibers. To our knowledge, Rop-based fibers represent the first functionalizable fibers based on a complete natural protein. The Rop-based scaffolds offer a novel class of biomaterials due to their easy and cheap production, remarkable stability, plasticity and potential functionalization.
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Δημιουργία βιοϋλικών στηριζόμενη σε ειδικές αλληλεπιδράσεις στοιχείων δευτεροταγούς δομής πρωτεϊνών.
