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Identifier

000397196

Title

Βιοχημικός και βιοφυσικός χαρακτηρισμός των διακριτών δομικών περιοχών της πιθανής απακετυλάσης πολυσακχαρίτων BAO330 από Bacillus anthracis

Alternative Title

Biochemical and biophysical chracterization of the sturctural domains of the putative polycaccharide deacetylases BAO330 from Bacillus anthracis.

Author

Μακράκη, Ελένη Κ.

Thesis advisor

Μπουριώτης, Βασίλης

Abstract

The lipoprotein BA0330 is one of 11 putative polysaccharide deacetylases in Bacillus anthracis. Recently, the structural role of BA0330 in maintaining the integrity of the cell wall of the bacterium was demonstrated. BA0330 interacts with peptidoglycan and contributes to the adaptation of B. anthracis to high salt stress. The structure determination of BA0330 revealed the presence of an N-terminal fibronectin type-3 (FN3) domain and a carboxyterminal catalytic domain (NodB). BA0330 lacked de-N-acetylase activity against common polysaccharide deacetylase substrates. In this study we performed biochemical and biophysical analysis of the distinct structural domains of BA0330. ba0330(fn3) and ba0330(nodb) were expressed in E.coli and recombinant BA0330(FN3) was isolated employing two chromatographic steps (Ni-NTA agarose, Sephacryl S100). Summarizing the results of this study we demonstrated that the N-terminal fibronectin type-3 (FN3) domain, BA0330(FN3) exhibit a high melting point and a completely reversible form after heating at 90^οC and then cooling back to 10^οC. It seems that it can be fold independently from the catalytic domain maintaining mainly β-sheets in the secondary structure. Initial results demonstrated that BA0330(FN3) contributes to the interaction of BA0330 with peptidoglycan. The analysis of BA0330(FN3) by Multi-angle light scattering (MALS) revealed the maintenance of the domain in one population as monomer while no effect of ionic strength on oligomerization was observed. The results of this study provide important information on the role of fibronectin type-3 (FN3) domain in the function of the native BA0330 and contributes to further understanding of the pathophysiology of the pathogenic bacterium Bacillus anthracis.

Language

Greek

Subject

FN3

Fibronectin type-3

Φιμπρονεκτίνη τύπου-3

Issue date

2015-11-20

Collection

School/Department--School of Sciences and Engineering--Department of Biology--Post-graduate theses

Type of Work--Post-graduate theses

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