Results - Details
Search command : Author="Κοκκινίδης"
And Author="Μ."
Current Record: 7 of 7
|
|
| Identifier |
uch.biology.phd//2004fadouloglou |
| Title |
Δομικές μελέτες της πρωτεΐνης Ηrcqb από το τύπου ΙΙΙ εκκριτικό σύστημα tης Pseudomonas syringae |
| Alternative Title |
Structural studies of the HRCqb protein from the type III secretion system of Pseudomonas syringae |
|
Author
|
Φαδούλογλου, Βασιλική Ε
|
|
Thesis advisor
|
Κοκκινίδης, Μ.
|
| Abstract |
The subject of this work is the structural study of the HrcQB protein from the plant pathogenic bacterium Pseudomonas syringae pv. phaseolicola. Interestingly, high sequence conservations between the HrcQB and homologues from plant and animal pathogens are concentrated at the carboxy-terminal part (79 resi-dues) where our crystallographic study focused. HrcQB is a conserved com-ponent of the type III secretion apparatus in Gram-negative bacteria with homologous proteins participating in the export mechanism for the flagellum assembly. The method of X-ray crystallography was used for structure deter-mination. Initially, the full length HrcQB protein, overexressed in Escherichia coli cells, was purified by chromatography. The molecule was very sensitive to proteoly-sis even in the presence of protein inhibitors at low temperatures. After the optimization of the purification scheme, stable and pure protein was pro-duced. However, all the crystallization attempts were unsuccessful. Subsequently, we focused on the 79 residues of the C-terminal region (HrcQB-C) where significant similarities with the homologous proteins are observed. The HrcQB-C is a stable molecule and was crystallized with the va-pour diffusion method from MPD and Mg(AcO)2. The crystals were thin plates and most of them exhibited crystal lattice defects that were reflected in the diffraction patterns. All attempts for optimizing the crystals quality or finding another crystal form remained unsuccessful. The bad quality of the majority of the crystals made the solution of the phase problem difficult. We did not find any useful isomorphous heavy atom de-rivative while the Multiple Anomalous Diffraction technique (MAD) was suc-cessful only after the mutation of a leucine in the protein interior to a me-thionine and then substitution of this methionine by selenomethionine. The substituted mutant was isolated and crystallized without significant deviations from the corresponding conditions of the native protein. The structure was determined from three data sets which were collected at three wavelengths around the absorption edge of selenium and was refined to 2.3 ? resolution. HrcQB-C is an elongated, gently curved homotetramer which can be charac-terized as an overwhelmingly beta structure. The four monomers assemble into two tightly bound homodimers which are packed together to form a dimer of dimers. There are three indications which support that the tetramer is the biologically relevant molecule: (i) A relatively large, total surface area (~1200 ?2) is buried upon tetramer formation via dimer-association. (ii) The tetramer is stabilized by several hydrogen bonds. (iii) The Stokes radius of the protein in solution which was estimated by size exclusion chromatography is in excellent agreement with the value calculated from the crystal structure of the whole tetramer. It is worth noting that the distribution of the conserved residues on the molecular surface is markedly asymmetric, with the majority of them being concentrated on the concave side of the tetramer. The striking analogies between HrcQB and its flagellum homologous FliN protein may support the hypothesis that HrcQB participates in the formation of a C-ring like assembly.
|
| Language |
Greek |
| Subject |
Προσδιορισμός τρισδιάστατης διαμόρφωσης πρωτείνης; Κρυσταλλογραφία ακτίνων Χ; Διάγραμμα περίθλασης ακτίνων Χ; Απομόνωση πρωτεϊνών; Κρυστάλλωση πρωτείνης; Εκκριτικό σύστημα τύπου ΙΙΙ; Παθογένεια |
| Issue date |
2004-02-25 |
| Collection
|
School/Department--School of Sciences and Engineering--Department of Biology--Doctoral theses
|
|
|
Type of Work--Doctoral theses
|
| Permanent Link |
https://elocus.lib.uoc.gr//dlib/5/7/0/metadata-dlib-2004fadouloglou.tkl
|
| Views |
215 |
Search
