The main success of my thesis has been to establish the mechanism by which antifreeze
proteins (AFPs) bind irreversibly to ice crystals, and hence prevent their growth. AFPs organize
ice-like water on their ice-binding site, which then merges and freezes with the quasi-liquid layer
of ice. This was revealed from studying the exceptionally large (ca. 1.5-MDa) Ca 2+-dependent
AFP from the Antarctic bacterium Marinomonas primoryensis (MpAFP). The 34-kDa antifreeze-
active region of MpAFP was predicted to fold as a novel Ca 2+-binding β-helix. Site-directed
mutagenesis confirmed the model and demonstrated that its ice-binding site (IBS) consisted of
solvent-exposed Thr and Asx parallel arrays on the Ca 2+-binding turns.
The X-ray crystal
structure of the antifreeze region was solved to a resolution of 1.7 Å. Two of the four molecules
within the unit cell of the crystal had portions of their IBSs freely exposed to solvent. Identical
clathrate-like cages of water molecules were present on each IBS. These waters were organized
by the hydrophobic effect and anchored to the protein via hydrogen bonds. They matched the
spacing of water molecules in an ice lattice, demonstrating that anchored clathrate waters bind
AFPs to ice.
This mechanism was extended to other AFPs including the globular type III AFP from
fishes. Site-directed mutagenesis and a modified ice-etching technique demonstrated this protein
uses a compound ice-binding site, comprised of two flat and relatively hydrophobic surfaces, to
bind at least two planes of ice. Reinvestigation of several crystal structures of type III AFP
identified anchored clathrate waters on the solvent-exposed portion of its compound IBS that
matched the spacing of waters on the primary prism plane of ice.
Ice nucleation proteins (INPs), which can raise the temperature at which ice forms in
solution to just slightly below 0oC, have the opposite effect to AFPs. A novel dimeric β-helical
model was proposed for the INP produced by the bacterium Pseudomonas borealis. Molecular
dynamics simulations showed that INPs are also capable of ordering water molecules into an ice-
like lattice. However, their multimerization brings together sufficient ordered waters to form an
ice nucleus and initiate freezing. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2011-08-08 14:09:05.143
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/6619 |
| Date | 09 August 2011 |
| Creators | GARNHAM, CHRISTOPHER P |
| 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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