Ice Active Proteins from Cold Tolerant Organisms

• Main Author: Clarke, Stephen Russell
• Other Authors: Marshall, Craig, Wharton, David
• Format: Thesis
• Language: English
• Published: University of Otago 2016
• Subjects: Ice-Active; Proteins; Antarctic; Panagrolaimus; recyrstallisation; The Antarctic; Antarc*
• Online Access: http://hdl.handle.net/10523/6366

The Antarctic nematode Panagrolaimus davidi is the only animal known to survive both intracellular and extracellular freezing. Homogenates made from P. davidi freeze at the usual temperature (approximately 0 °C) but are able to maintain the small size of the ice crystals formed i.e. inhibit recrystallization. This ability is thought to be key to their unique ability to survive intracellular freezing as it prevents large ice crystals forming that may damage intracellular structures and membranes. The aim of this research was to further our understanding of how P. davidi achieves this control over the ice crystal structure by identifying the proteins responsible for the recrystallization inhibition. This would have wide ranging implications not only in the basic understanding of freeze tolerance, but huge potential in the biotechnology field e.g. frost resistant crops. Initially the aim was to identify any proteins that bind to ice using a cold finger ice binding technique. This involved freezing a solution very slowly and trapping any ice binding proteins within the ice while all others were excluded. Interestingly no proteins were found to bind to the ice and using a grass extract that is also known to inhibit recrystallization it was found that these proteins could exert their effect on the ice crystal structure without binding to ice. This led to the proposal of a mechanism in which the recrystallization inhibition proteins situate themselves in the liquid phase between the ice crystals and prevent the movement of water molecules between ice crystals, which in turns prevents recrystallisation. This is very different to the mechanism of antifreeze proteins, which bind to the ice and provide an energy barrier to ice growth. To help in the isolation of recrystallisation inhibiting proteins it was necessary to develop a quick reliable screen to detect recrystallization inhibition activity. This was achieved using a optical recrystallometer. This is capable of measuring the intensity of light passing through a sample and we proposed that as the ice crystal structure changed so would the intensity of light passing through a sample. Ultimately it was shown that the change in intensity of light passing through samples with recrystallisation inhibition activity was a good indication of recrystallisation inhibition activity, in that samples with high activity showed very small changes in transmittance, whereas those with low activity showed a large change. An attempt was made to isolate the protein responsible for the recrystallisation inhibition activity. As part of this, several other basic properties were also investigated. It was shown that the activity was sensitive to proteinase K, did not require metal ions, was heat stable and likely required more than one component to be active. Several types of chromatography were not successful at isolating any proteins responsible for this activity and so a proteomic approach was then attempted. It was shown that there was a difference in the recrystallisation inhibition activity of acclimated and non-acclimated P. davidi (known to have different freezing survival rates) and that these differences were also matched with a difference in the protein profile of the samples. Sixty one spots across the two gels were identified as differentially expressed and excised and are awaiting mass spectrometry analysis to identify them.