| Abstract |
Development of novel biomaterials is a rapidly developing field of nanotechnology. A detailed and comprehensive understanding of protein folding will greatly contribute to the development of new biomaterials. Rop (Repressor Of Primer) is a protein that has been mutated many times in order to give an answer at the “folding problem”. It is the paradigm of a canonical 4-α-helical bundle and is involved in the regulation of copy number of the ColE1 plasmids of E.coli. Like all 4-α-helical bundles, it exhibits a ‘heptad pattern’ in its sequence, that is disturbed only in the loop region which is formed by three amino acids. Several studies has demonstrate the role of turn, in the folding process and the stability of proteins, although the precise nature of this role is not clearly understand.
Rop A31P, one of the extensivly studied loop-mutants of Rop results in drastic structural changes. In this study, we try to further explore the role of loops in the folding pathway, through the design of Rop mutant D30P/A31G. Spectroscopic and thermodynamic studies were carried out and protein crystallization and structure determination were set up. Results of this work suggest that this mutant has a wt Rop-like structure which is different from the A31P structure.
In summary, proline is a crucial amino acid but drastic structural effects by Pro have been so far observed only at the crucial 31 position (A31P); a glycine at position 31 may contribute to a less stable structure, as it makes less tight bonds with both helices. This confirms the role of turn residues in the choice of folding pathway and the deservation that the effects of the loop region on the folding pathway are complex and both sequence- and position- dependent.
In this study, there is also an effort to design two Rop mutants, in order to use them as building blocks for nano-structures – biomaterials. These mutants are soluble, as it is indicated by a small-scale purification. Further characterisation including Gel Filtration is necessary for concluding their oligomerization state. Electron microscopy and circular dichroism will demonstrate the possible self-assembly of these mutants, with respect to different conditions, like pH and time.
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Χρήση της πρωτεΐνης Rop ως νέο βιοϋλικό και δομική ανάλυση της RopPG με στόχο την κατανόηση σχέσεων δομής-αλληλουχίας
