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    Structure-Function Studies of a Truncated Ice-Nucleation Protein

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    Date
    2015-10-03
    Author
    Andrin, Evan J. C.
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    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.
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    http://hdl.handle.net/1974/13748
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    • Biochemistry Graduate Theses (July 2007 - Sept 2016)
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