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Structural analysis of DARPin mutants and biochemical characterization of ABC transporters and SGT1


Kramer, Michaela A. Structural analysis of DARPin mutants and biochemical characterization of ABC transporters and SGT1. 2010, University of Zurich, Faculty of Science.

Abstract

My PhD thesis focuses on the structure analysis of proteins by X-ray crystallography, namely bacterial membrane proteins from the family of ABC transporters, the inflammasome adaptor protein SGT1 and Designed Ankyrin Repeat Proteins. These three topics will be summarized below.
Several ABC-transporters of Escherichia coli have been selected for recombinant expression and purification. By screening of several constructs and expression conditions the ABC-transporters YojI, MclB and HlyB were expressed. The purification protocol for the microcin exporter YojI was optimized and it was shown that YojI forms a stable dimer. This protein was used for functional assays. The obtained results form the basis for an increased understanding of the function of ABC-transporters.
The human protein SGT1 was expressed and characterized by biophysical methods. This protein is involved in the pathogen defence reactionin the mammalian immunesystem and its planhomologue is involved in the hypersensitivity response. Since the full length protein SGT1 did not yield crystals, we used limited proteolysis to obtain fragments that might be amendable for rystallization. Several fragments were obtained, cloned and overexpressed. For the central CS-domain an X-ray structures became available during my PhD thesis, for the N-terminal TPR domain structures of homologues were elucidated. The C-terminal SGS-domain revealed a limited degree of folding. The gained information is useful for further immuno-assays in order to determine interaction partners.
Selected Designed Ankyrin Repeat Proteins (DARPins) were structurally analysed. DARPins are extremely stable repeat proteins having a vast field of application as inhibitors and as crystallization aids. Previous studies have shown that the C-terminal capping repeat is limiting the stablility of these molecules. Therefore the group of Prof. A. Plückthun designed new C-terminal caps that showed improved thermal stability. To analyse the structural determinants for this improved thermal stability the DARPins NI1C mutant 4, NI3C mutant 5 and NI3C mutant 6 were crystallized. The crystal structures revealed an improved packing between the C-terminal capping repeat and the last internal repeat explaining the improved thermal stabilityof the new design.

My PhD thesis focuses on the structure analysis of proteins by X-ray crystallography, namely bacterial membrane proteins from the family of ABC transporters, the inflammasome adaptor protein SGT1 and Designed Ankyrin Repeat Proteins. These three topics will be summarized below.
Several ABC-transporters of Escherichia coli have been selected for recombinant expression and purification. By screening of several constructs and expression conditions the ABC-transporters YojI, MclB and HlyB were expressed. The purification protocol for the microcin exporter YojI was optimized and it was shown that YojI forms a stable dimer. This protein was used for functional assays. The obtained results form the basis for an increased understanding of the function of ABC-transporters.
The human protein SGT1 was expressed and characterized by biophysical methods. This protein is involved in the pathogen defence reactionin the mammalian immunesystem and its planhomologue is involved in the hypersensitivity response. Since the full length protein SGT1 did not yield crystals, we used limited proteolysis to obtain fragments that might be amendable for rystallization. Several fragments were obtained, cloned and overexpressed. For the central CS-domain an X-ray structures became available during my PhD thesis, for the N-terminal TPR domain structures of homologues were elucidated. The C-terminal SGS-domain revealed a limited degree of folding. The gained information is useful for further immuno-assays in order to determine interaction partners.
Selected Designed Ankyrin Repeat Proteins (DARPins) were structurally analysed. DARPins are extremely stable repeat proteins having a vast field of application as inhibitors and as crystallization aids. Previous studies have shown that the C-terminal capping repeat is limiting the stablility of these molecules. Therefore the group of Prof. A. Plückthun designed new C-terminal caps that showed improved thermal stability. To analyse the structural determinants for this improved thermal stability the DARPins NI1C mutant 4, NI3C mutant 5 and NI3C mutant 6 were crystallized. The crystal structures revealed an improved packing between the C-terminal capping repeat and the last internal repeat explaining the improved thermal stabilityof the new design.

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Additional indexing

Item Type:Dissertation
Referees:Grütter Markus G
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2 December 2010
Deposited On:14 Jan 2010 13:14
Last Modified:25 Apr 2016 08:14
Additional Information:Structural analysis of DARPin mutants and biochemical characterization of ABC transporters and SGT1 / von Michaela Andrea Kramer. - Zürich, 2010
Related URLs:http://www.recherche-portal.ch/ZAD:default_scope:ebi01_prod006290598 (Library Catalogue)
Permanent URL: http://doi.org/10.5167/uzh-26736

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