Fluorescence microscopy evidence for quasi-permanent attachment of antifreeze proteins to ice surfaces

Natalya Pertaya, Christopher B. Marshall, Carlos L. DiPrinzio, Larry Wilen, Erik S. Thomson, J. S. Wettlaufer, Peter L. Davies, Ido Braslavsky*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

102 Scopus citations

Abstract

Many organisms are protected from freezing by the presence of extracellular antifreeze proteins (AFPs), which bind to ice, modify its morphology, and prevent its further growth. These proteins have a wide range of applications including cryopreservation, frost protection, and as models in biomineralization research. However, understanding their mechanism of action remains an outstanding challenge. While the prevailing adsorption-inhibition hypothesis argues that AFPs must bind irreversibly to ice to arrest its growth, other theories suggest that there is exchange between the bound surface proteins and the free proteins in solution. By conjugating green fluorescence protein (GFP) to a fish AFP (Type III), we observed the binding of the AFP to ice. This was accomplished by monitoring the presence of GFP-AFP on the surface of ice crystals several microns in diameter using fluorescence microscopy. The lack of recovery of fluorescence after photobleaching of the GFP component of the surface-bound GFP-AFP shows that there is no equilibrium surface-solution exchange of GFP-AFP and thus supports the adsorption-inhibition mechanism for this type of AFP. Moreover, our study establishes the utility of fluorescently labeled AFPs as a research tool for investigating the mechanisms underlying the activity of this diverse group of proteins.

Original languageAmerican English
Pages (from-to)3663-3673
Number of pages11
JournalBiophysical Journal
Volume92
Issue number10
DOIs
StatePublished - May 2007
Externally publishedYes

Bibliographical note

Funding Information:
We thank S. Gauthier and R. Milford for their assistance. This study was supported by the Canadian Institutes for Health Research, the Bosack and Kruger Foundation, the National Science Foundation (grant Nos. OPP0440841, OPP0439805, and OPP0135989), the Condensed Matter and Surface Science program at Ohio University, (grant Nos. OPP0440841, OPP0439805, and OPP0135989) the Ohio University's NanoBioTechnology Initiative.

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