E-Locus - Ιδρυματικό Καταθετήριο Πανεπιστημίου Κρήτης

Αρχική Αναζήτηση

Αποτελέσματα - Λεπτομέρειες

Εντολή Αναζήτησης : Συγγραφέας="Κοκκινίδης" Και Συγγραφέας="Μ."

Τρέχουσα Εγγραφή: 6 από 7

[Προσθήκη στο καλάθι]

Κωδικός Πόρου

000352751

Τίτλος

Λειτουργικός χαρακτηρισμός πρωτεινών του εκκριτικού συστήματος τύπου ΙΙΙ του φυτοπαθογόνου βακτηρίου Pseudomonas syringae pv. phaseolicola

Άλλος τίτλος

Functional characterization of type III secretion proteins of the phytopathogenic bacterium Pseudomonas syringae pv. phaseolicola

Συγγραφέας

Μπαστάκη, Μαρίνα

Σύμβουλος διατριβής

Πανόπουλος, Ν.
Κοκκινίδης, Μ

Περίληψη

Type III secretion system (TTSS) is a specialized multiprotein complex which is found in Gram- bacteria and mediates the secretion/translocation of proteins (effectors) directly from the bacterial to the eukaryotic cytoplasm. In phytopathogenic bacteria, the TTSS is essential for pathogenesis and is encoded by a cluster of genes located in the hrp/hrc pathogenicity island. In the present study, the phytopathogenic bacterium Pseudomonas syringae pv. phaseolicola (Psph) was used as a model for the structural and functional characterization of TTSS proteins. The avrPphF operon of Psph encodes a ΤΤSS chaperone/cognate effector protein pair (AvrPphF_ORF1/ORF2). The ORF1-ORF2 complex was detected in vivo, via yeast two-hybrid analysis, as well by co-purification from Escherichia coli. Formation of ORF1-ORF2 complex in vitro requires proper refolding of the proteins which seem to participate in a 2:1 ratio, consistent with a chaperone/substrate complex profile for TTSS secretion. The AvrPphF_ORF1 protein binds in the first 55 N-terminal residues of AvrPphF_ORF2 whereas the 42-55 residue region of the latter protein appear to be critical for this interaction. HrcQ_Β is a conserved component of the TTSS core and its C-terminal region (HrcQ_Β-C, residues 50-128) forms stable tetramers in crystals while the homologous protein (FliN) from Thermotoga maritima forms homodimers. Accordingly, we investigated if the biologically significant unit of HrcQ_Β-C is a tetramer or lower or higher order complexes by constructing four site-directed mutants, aiming at the disruption or the destabilization of the tetrameric association. Three mutants, although still capable of forming homo-tetramers of the same hydrodynamic radius as those of the native protein, displayed a certain degree of heterogeneity of the molecular population and different thermostability compared to the native molecule in solution. The site-directed insertion of tryptophan residues in HrcQ_Β-C, which lacks aromatic aminoacids, allowed the collection of near UV circular dichroism spectra which support the hypothesis that the solution tetramer resembles that in the HrcQ_Β-C crystal. The ΗrpΕ protein, while is not listed among the conserved components of the secretion apparatus, seems to play an important and functionally conserved role in TTSS secretion. In the present study, we investigated the structural characteristics of the protein and demonstrated its ability to self-associate in vivo and in vitro as well as to interact physically with the HrpO and HrcN proteins. The construction of a series of truncated forms of HrpE allowed us to sketch a probable oligomerization pattern in solution and to localize the regions of the molecule which participate in these interactions. In addition, the subcellular localization of HrpE and its effect at the ATPase activity of HrcN, determined in this study, in combination with the data from studies on flagellar or non-flagellar TTSS led to a hypothetical model which outlines the role of these interactions in the function of the secretion apparatus.

Φυσική περιγραφή

219 σ. : εικ. ; 30 εκ.

Γλώσσα

Ελληνικά

Θέμα

Circular dichroism

Gel Filtration chromatography

Protein-protein interactions

Αλληλεπιδράσεις πρωτεινών

Κυκλικός Διχρωισμός

Συστήματα δύο υβριδιών

Χρωματογραφία μοριακής διήθησης