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Identifier

000348526

Title

Μελέτη της HRPA από το τύπου ΙΙΙ εκκριτικό σύστημα του φυτοπαθογόνου Pseudomonas syringae Pv. phaseolicola.

Alternative Title

Study of HRPA from type III secration system of Pseudomonas syringae Pv. phaseolicola.

Author

Γκουγκουλιά, Ειρήνη

Thesis advisor

Κοκκινίδης, Μιχαήλ

Abstract

The subject of this master thesis is the study of two deleted mutants of the HrpA protein from the plant pathogenic bacterium Pseudomonas syringae pv. phaseolicola. HrpA, which is the pilin of the Pseudomonas syringae, is a small hydrophilic protein. Taking under consideration that the carboxy-terminal part of the protein is responsible for its polymerization and also the amphipathic profile of the carboxy-predicted α-helix of the HrpA from the P. syringae pv. phaseolicola, two constructs with the last 8 and 18 residues deleted were produced, in order to avoid any spontaneous polymerization, which could prevent crystallization. Both of the mutant proteins were overexpressed and purified by affinity chromatography and size exclusion chromatography. Moreover, study of the secondary structure of both mutants took place under different conditions of temperature, concentration and pH with CD. According to the CD data analysis, there is not high predicted percentage of α-helix (10 %), while the predicted percentages of β-strand and random coil are similar (20-30%) under different conditions, which is contrary to PSIPRED results. In addition to this, thermal denaturation experiments took place for both mutant proteins, which are just the opposite of the molten globule hypothesis for protein folding. Primary structural studies were attempted with the method of SAXS, but the original data were not sufficient to give 3D models of the mutant proteins. Finally, crystallization was attempted with different methods and under various conditions without the desired diffracted crystals.

Physical description

74 σ. : εικ. ; 30 εκ.

Language

Greek

Subject

Pilus

TTSS

Issue date

2009-07-24

Collection

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

Type of Work--Post-graduate theses

Views

375

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