Structural and Functional Characterisation of Nuclear Transport Protein Complexes

Mary Marfori (2011). Structural and Functional Characterisation of Nuclear Transport Protein Complexes PhD Thesis, School of Chemistry & Molecular Bioscience, The University of Queensland.

       
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Author Mary Marfori
Thesis Title Structural and Functional Characterisation of Nuclear Transport Protein Complexes
School, Centre or Institute School of Chemistry & Molecular Bioscience
Institution The University of Queensland
Publication date 2011-06
Thesis type PhD Thesis
Supervisor Bostjan Kobe
Jade Forwood
Thierry Lonhienne
Total pages 193
Total colour pages 44
Total black and white pages 149
Language eng
Subjects 03 Chemical Sciences
Abstract/Summary In eukaryotic cells, the nucleus and cytoplasm are physically separated by the nuclear membrane, segregating nuclear DNA replication and RNA transcription from cytoplasmic protein synthesis. This compartmentalisation therefore requires an efficient mechanism to transport mRNA and proteins between the nucleus and cytoplasm. Bi-directional transport of these molecules occurs through large macromolecular channels, called nuclear pore complexes that traverse the nuclear envelope. Although small molecules can passively diffuse through the nuclear pore channels, proteins >40 kDa require an active transport mechanism for nuclear translocation. Nuclear protein import is a process that delivers protein cargo into the nucleus in a regulated and efficient manner. The majority of nuclear transport pathways are mediated by the β-karyopherin superfamily of proteins, while the small GTPase Ran imparts directionality on the transport cycle. Many aspects of the nuclear transport pathway have been characterised structurally and biochemically. Nonetheless, there are still various interactions that have been identified in past studies that are not adequately described within our current model of protein transport. The objective of this study was to structurally and functionally investigate a range of proteins and protein complexes within the nuclear transport cycle, for which their role in protein import was unclear, or have not been sufficiently structurally characterised. In particular, this study has gained new insights into the specificity determinants of the importin-α and nuclear localisation signal (NLS) interaction, through the structural characterization of the importin-α:high-affinity NLS interaction. Additionally, this work has extended our understanding of importin-β flexibility in both the unliganded state, and in complex with importin-α through the use of small-angle x-ray scattering techniques. Lastly, the structures of importin-β:Ran complexes with and without bound nucleotide were determined using x-ray crystallography. These results have shed light on the Ran GDP-to-GTP exchange activity of importin-β, and have revealed a novel conformation for the C-terminal helix of Ran when in complex with importin-β.
Keyword nuclear transport
importins
RanGTPase
nuclear localisation signals
X-ray Crystallography
Small-angle X-ray Scattering
Additional Notes Colour pages: 25,27,32,36,38,41,43,45,49,51,69,78,81,82,84,85,87,89,91,93,97,100,102,113,115,117,119,120,123,127,130,133,136,147,148,152,154,155,157-159,162,165,167 Landscape pages: 25,28,32,43,49,51,70-77,91,115,126,131,136,165

 
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Created: Thu, 15 Dec 2011, 18:38:35 EST by Miss Mary Marfori on behalf of Library - Information Access Service