Living inside Sea Ice : Distribution and Functional Characterisation of Antifreeze Proteins in Polar Diatoms
Antifreeze proteins (AFPs) are an important adaptation mechanism for organisms subjected to subzero temperatures. The motivation of this thesis was to elucidate the distribution of AFPs in sea ice diatoms and to study their function. The findings were used to deduce the mechanisms of action and rele...
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| Other Authors: | , , |
| Format: | Doctoral or Postdoctoral Thesis |
| Language: | English |
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Universität Bremen
2011
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| Subjects: | |
| Online Access: | https://media.suub.uni-bremen.de/handle/elib/253 https://nbn-resolving.org/urn:nbn:de:gbv:46-00102440-11 |
| Summary: | Antifreeze proteins (AFPs) are an important adaptation mechanism for organisms subjected to subzero temperatures. The motivation of this thesis was to elucidate the distribution of AFPs in sea ice diatoms and to study their function. The findings were used to deduce the mechanisms of action and relevance in vivo. Diatom isolates were tested in culture experiments for the presence of recrystallisation inhibition activity, as a measure for AFP activity. Seven Arctic and four Antarctic diatom isolates were subjected to a temperature decrease and salinity shift resembling the inclusion of the diatoms into sea ice. All tested polar diatom species showed AFP activity, ten thereof even without being stressed. In three species, AFP activity was furthermore up-regulated by the temperature and salinity shift. Cell numbers and photosynthetic quantum yield indicated that cellular damage caused by the stress was more severe for water column isolates than for isolates from the ice column. The correlation of recrystallisation to the protein concentration for a Fragilariopsis nana Ant cell extract and a recombinant AFP allowed the calculation of AFP concentrations as AFP equivalents. Intracellular concentrations of 0.3 µM to 68.5 µM AFP equivalents confirmed the function of AFP as recrystallisation inhibitor and even come close to the concentrations required for thermal hysteresis. As AFP equivalents made up 0.1% to 5% of the total cell protein, diatoms invest a large amount of energy to produce AFPs. This indicates that AFPs are an important factor for diatom success in sea ice. Further, two isoforms of AFPs from Fragilariopsis nana were heterologously expressed in Escherichia coli, to study their molecular function. Recombinant proteins were deposited in inclusion bodies, but successfully refolded to functionally active proteins with respect to crystal deformation, recrystallisation inhibition and thermal hysteresis. The two isoforms showed different characteristics. One isoform produced a thermal hysteresis of up to 1.53 °C ... |
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