Dokument: The Escherichia Haemolysin transporter - a paradigm for Type I secretion

Titel:	The Escherichia Haemolysin transporter - a paradigm for Type I secretion
URL für Lesezeichen:	https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=9870
URN (NBN):	urn:nbn:de:hbz:061-20090121-110905-8
Kollektion:	Dissertationen
Sprache:	Englisch
Dokumententyp:	Wissenschaftliche Abschlussarbeiten » Dissertation
Medientyp:	Text
Autor:	Jenewein, Stefan [Autor]
Dateien:	[Dateien anzeigen] Adobe PDF [Details] 10,19 MB in einer Datei [ZIP-Datei erzeugen] Dateien vom 11.12.2008 / geändert 15.12.2008
Dewey Dezimal-Klassifikation:	500 Naturwissenschaften und Mathematik » 540 Chemie
Beschreibung:	The haemolysin export system of Escherichia coli is comprised of HlyB (an ATP-binding cassette (ABC) transporter), the membrane fusion protein HlyD, and the outer membrane channel TolC. It transports the 108 kDa toxin HlyA in a single step across the inner and outer membrane and constitutes the paradigm for Type I secretion. Knowledge of the detailed mechanism of the ATPase reaction, which is the basis of transport in any particular ABC transporter is of main mechanistic interest. In this work the nucleotide binding domain (NBD) of HlyB was used to characterise the hydrolysis of the nucleotide. Nucleotide binding to the NBD was extensively characterized by fluorescence methods. An environmental sensitive mutant was generated to measure nucleotide binding. Quenching this amino acid allowed the determination of the affinity constants for nucleotides. Using Trinitrophenol(TNP)-derivatised nucleotides the data obtained for the mutant could be verified for the wildtype HlyB-NBD through competition experiments. Since TNP derivatised nucleotides exhibited a 50-100 fold higher affinity, the binding mode was characterized further by mutational and structural analysis showing that the increase of affinity is caused by flexible water-mediated interactions. To address the reaction mechanism of ATP hydrolysis recent structural work was supported in this work by extensive biochemical characterisation. As part of the presented work, it could be shown that cooperative ATP hydrolysis in the HlyB-NBD follows substrate assisted catalysis (SAC). A broad screen including different plasmids, strains and growth conditions was performed to attempt the overexpression of the inner membrane components. While overexpression failed in the homologous host E. coli, expression was successful in Lactococcus lactis. The ATPase activity of full-length HlyB was strictly dependent on the substrate and showed an induction in the presence of the C-terminal secretion sequence of HlyA. In contrast to many other ABC transporters, this is one of the first examples where the ATPase activity of an ABC transporter is strictly coupled rather than just stimulated. Exploiting the capability of secreting heterologous fusion proteins by the Type I machinery a vector pair was designed encoding on one hand for the inner membrane complex and one the other hand for any gene of interest fused to a C-terminal 23 kDa fragment of HlyA. Since secretion failed for a number of fusion proteins, folding kinetics of the MalE-HlyA fusion was selectively changed. It could be clearly shown that mutations resulting in slower folding of the maltose binding protein MalE increased the secretion efficiency. This work is the first report in which a fusion protein was selectively converted into an appropriate substrate of a Type I machinery. The knowledge of how fusion proteins can be modified in order to be secreted will certainly help (i) choosing suitable targets for Type I secretion in biomedical and biotechnical applications and (ii) understanding the nature of Type I substrates and the secretion process itself.
Rechtliche Vermerke:	Aus patentrechtlichen Gründen bis 31.07.2009 zurückgestellt
Lizenz:	Urheberrechtsschutz
Fachbereich / Einrichtung:	Mathematisch- Naturwissenschaftliche Fakultät » WE Chemie » Biochemie
Dokument erstellt am:	03.08.2009
Dateien geändert am:	15.12.2008
Datum der Promotion:	04.12.0008