Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.14/192059
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- Title
- Structural characterisation of yeast Lsm protein complexes
- Related
- Australasian Digital Theses
- Publisher
- Australia : Macquarie University
- Date
- 2011
- Author/Creator
- Moll, Jens Mark
- Description
- Thesis (PhD)--Macquarie University, Faculty of Science, Dept. of Chemistry & Biomolecular Sciences, 2011.
- Description
- Bibliography: p. 179-195.
- Description
- Introduction -- Materials and methods -- Solution behaviour of Lsm polyproteins -- Biophysical characterisation of Lsm complexes and their RNA interactions -- Crystallographic studies of Lsm polyproteins -- Conclusion.
- Description
- Lsm proteins are a family of RNA chaperones present in all kingdoms of life. Members of this protein family organise into ring-shaped quaternary structures and engage in the processing, sorting and regulation of a variety of RNA species. In archaea and bacteria, homomeric complexes of six or seven proteins are functional, whilst discrete heteromeric complexes of seven distinct Lsm proteins occur in eukaryotes. Eukaryotic Lsm assemblies modulate according to cellular localisation and RNA target, demonstrating that specific functionalities may exist for individual Lsm proteins. -- In this study, I utilised Lsm polyproteins to pursue structural and functional studies of mixed Lsm rings, as well as to probe their quaternary dynamics. My work focused on two polyprotein forms, fusions of yeast Lsm[2+3] and Lsm[4+1]. Size exclusion chromatography in conjunction with static light scattering detects the formation of stable tetra- and octameric complexes, suggesting the formation of single and stacked tetrameric rings. Elevated populations of octamers are favoured at low ionic strength, indicating electrostatically-mediated packing of Lsm tetramers. A ring morphology for both tetrameric and octameric assemblies is confirmed by small angle X-ray scattering, estimating toroid dimensions to be 75 Å x 50 Å. -- The simplified Lsm polyprotein complexes provide excellent probes of specific Lsm affinities for RNA sequences. Differential affinities of Lsm polyprotein towards U-rich G5U10 and U10 oligonucleotides are detected by surface plasmon resonance and isothermal titration calorimetry. The highest affinity for these oligonucleotides are observed for Lsm[2+3] (KD= 34 ± 15 nM), possibly due to specific basic residues within the linker used to fuse Lsm2 and Lsm3 domains. Isolated Lsm polyprotein complexes were subjected to crystallographic studies, resulting in regular crystalline forms of Lsm[4+1] in three distinct morphologies. Subsequently, native datasets were collected and processed to 3 Å resolution. Extensive phasing attempts using molecular replacement have been made, but have not so far yielded a solution. This data will serve to solve the first crystal structure of a heteromeric Lsm protein complex at atomic resolution upon collection of a suitable heavy atom dataset, however, despite extensive screening, only weakly diffracting crystals were obtained from L-selenomethionine derivatives of the protein to date. -- The results obtained from simplified Lsm complexes aid the understanding of natural Lsm assemblies in vivo. It is the dynamic reorganisation of Lsm complexes that likely contributes to the hurdle of obtaining quality diffracting crystals for Lsm complexes. My biophysical studies have, however, confirmed the likelihood of ring-shaped morphology of mixed Lsm rings in vivo, as well as differential affinities for RNA by their separate Lsm components.
- Description
- Mode of access: World Wide Web.
- Description
- 1 online resource (xiii, 195 p.) ill. (some col.)
- Subject Keyword
- RNA-protein interactions
- Subject Keyword
- Nucleoproteins
- Subject Keyword
- Recombinant proteins
- Subject Keyword
- Protein-protein interactions
- Subject Keyword
- Complex compounds
- Subject Keyword
- Lsm
- Subject Keyword
- Sm like
- Subject Keyword
- mRNA degradation
- Subject Keyword
- macromolecular complexes
- Resource Type
- Thesis PhD
- Organisation
- Macquarie University. Dept. of Chemistry and Biomolecular Sciences
- Identifier
- http://hdl.handle.net/1959.14/192059
- Identifier
- 1736391
- Language
- eng
- Rights
- Copyright disclaimer: http://www.copyright.mq.edu.au
- Rights
- Copyright Jens Mark Moll 2011.
- Full Text
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