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The Role of Intrinsically Disordered Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2 in Stabilization of Membranes and Cytoskeletal Actin Filaments

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dc.contributor.advisor Harauz, George
dc.contributor.advisor Dutcher, John
dc.contributor.author Rahman, Luna
dc.date.accessioned 2012-05-11T18:43:05Z
dc.date.available 2012-10-31T05:00:10Z
dc.date.copyright 2012-04
dc.date.created 2012-04-27
dc.date.issued 2012-05-11
dc.identifier.uri http://hdl.handle.net/10214/3624
dc.description.abstract The group 2 late embryogenesis abundant (LEA) proteins, also known as the dehydrins, are intrinsically disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. In this work, we study the potential roles that dehydrins may have in stabilizing membranes and actin microfilaments during cold stress. We have cloned and expressed in E. coli two dehydrins from Thellungiella salsuginea, denoted TsDHN-1 (acidic) and TsDHN-2 (basic). These proteins were expressed as SUMO-fusion proteins for in vitro phosphorylation by casein kinase II (CKII), and for structural analysis by CD and Fourier transform infrared (FTIR) spectroscopy. We show using transmission-FTIR spectroscopy that ordered secondary structure is induced and stabilized in these proteins by association with large unilamellar vesicles emulating the lipid compositions of plant plasma and organellar membranes. The increase in secondary structure by membrane association is further facilitated by the presence of Zn2+. Lipid composition and temperature have synergistic effects on the secondary structure. Our single molecule force spectroscopy studies also suggest tertiary folding of both TsDHN-1 and TsDHN-2 induced by association with lipids. From Langmuir-Blodgett monolayer compression studies, and from topographic studies using atomic force microscopy at variable temperature, we conclude that TsDHN-1 stabilizes the membrane at lower temperatures. Finally, we show that the conformations of TsDHN-1 and TsDHN-2 are affected by pH, interactions with cations and membranes, and phosphorylation. Actin assembly by these dehydrins was assessed by sedimentation assays, and viewed by transmission electron and atomic force microscopy. Phosphorylation enabled both dehydrins to polymerize actin filaments, a phenomenon that may occur in the cytosols of plant cells undergoing environmental stress. These results support the hypothesis that dehydrins stabilize plant organellar membranes and/or the cytoskeleton in conditions of stress, and further that phosphorylation may be an important feature of this stabilization. en_US
dc.description.sponsorship NSERC en_US
dc.language.iso en en_US
dc.subject dehydrins en_US
dc.subject late embryogenesis abundant (LEA) en_US
dc.subject cold tolerance en_US
dc.subject Drought tolerance en_US
dc.subject intrinsically disordered protein (IDP) en_US
dc.subject induced folding en_US
dc.subject Poly-proline type II en_US
dc.subject CD spectroscopy en_US
dc.subject FTIR spectroscopy en_US
dc.subject ATR-FTIR spectroscopy en_US
dc.subject peripheral membrane protein en_US
dc.subject Langmuir-Blodgett monolayers en_US
dc.subject supported lipid bilayers en_US
dc.subject atomic force microscopy en_US
dc.subject single molecule force spectroscopy en_US
dc.subject phosphorylation en_US
dc.subject actin polymerization en_US
dc.title The Role of Intrinsically Disordered Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2 in Stabilization of Membranes and Cytoskeletal Actin Filaments en_US
dc.type Thesis en_US
dc.degree.programme Biophysics en_US
dc.degree.name Doctor of Philosophy en_US
dc.degree.department Department of Molecular and Cellular Biology en_US
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