Antifreeze proteins (AFPs) protect organisms from freezing damage at subzero temperatures. They do this by adsorbing to the surface of nascent ice crystals to block further ice growth. The key property of AFPs is to be soluble in liquid water but bind irreversibly to water in the solid state. Hypotheses for the mechanism by which AFPs recognize and bind ice have gone through several radical revisions without a consensus emerging. The remarkable diversity of independently evolved AFP structures, the multiple ice planes bound by AFPs, and uncertainty about the location of the ice-binding site(s) have all added to the difficulty of deducing a unified mechanism of AFP action. The central thesis of my research is that the characterization of additional AFPs will elucidate rather than obfuscate the mechanism of action.
To this end I have advanced knowledge about three hyperactive AFPs. A reliable protocol to express and purify a sufficient quantity of type I hyperactive AFP was developed for further characterization studies. Initial crystallization trials using the recombinant material have produced consistent crystals for diffraction and resolution. A model of the recently discovered snow flea AFP was generated via de novo methods. The folding scheme is polyproline type II helices stacked into anti-parallel sheets, which was to our knowledge previously unobserved in monomeric proteins. The model was subsequently confirmed to be within 1 Å accuracy by X-ray crystallography performed by another group. I have also screened several insects for antifreeze activity. By using mass-spectrometry sequencing and a cDNA library, novel AFPs (3 kDa and 8kDa) were discovered from overwintering inchworms. The translated proteins were subsequently de novo modelled.
After a thorough analysis of the literature, I reason that conflicting results from various AFP studies can be resolved. The hydrogen-bond ice-binding hypothesis was re-introduced to work coherently with elements of the hydrophobic ice-binding theory. We have proposed a unifying mechanism termed “anchored clathrate water,” which is supported by the water bonding on ice-binding surfaces reported both in in silico and in NMR studies. The new data I have obtained have further reinforced and expanded the hypothesis. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2011-04-15 14:54:55.315
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/7474 |
| Date | 17 September 2012 |
| Creators | Lin, Feng-Hsu Nelson |
| Contributors | Queen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.)) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. |
| Relation | Canadian theses |
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