Structure-Function Studies of a Truncated Ice-Nucleation Protein
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Authors
Andrin, Evan J. C.
Date
2015-10-03
Type
thesis
Language
eng
Keyword
Antifreeze , Ice Nucleation
Alternative Title
Abstract
Bacterial ice-nucleation proteins (INPs) initiate water freezing at high subzero temperatures. Despite having an opposite function to antifreeze proteins (AFPs), INPs and AFPs both have significant similarities in sequence and structure. Based on these common features, I hypothesize that INPs also organize water molecules on their surface into ice-like geometries, as suggested for AFPs. If this is true, an undefined structural characteristic must differentiate ice nucleation. While AFPs are generally small (<40 kDa) monomeric proteins in vivo, INPs form large (>1 MDa) aggregates on the outer-membrane surface. Therefore, I propose that the distinguishing feature of ice nucleation is a relatively large water-organizing template.
To test this hypothesis, I set out to engineer an AFP from a small INP fragment. A 26.5-kDa recombinant protein was designed by joining two sections of the Pseudomonas syringae InaV INP, one encoding P141-A220 and the other encoding G1021-E1196. CD spectroscopy of this recombinant protein indicated it had β-rich secondary structure that was increased by detergent. The construct shaped ice into a hexagonal bipyramid at high concentrations, but evaluation of antifreeze activity was complicated by trace AFP impurities leaching from shared equipment. The truncated INP in a zwitterionic detergent did show small but significant potentiation of ice nucleation. Taken together, these results are inconclusive about the relationships between INPs and AFPs but point the way to more definitive studies.
Description
Thesis (Master, Biochemistry) -- Queen's University, 2015-10-01 11:27:59.622
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ProQuest PhD and Master's Theses International Dissemination Agreement
Intellectual Property Guidelines at Queen's University
Copying and Preserving Your Thesis
Creative Commons - Attribution - CC BY
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.