The Case for Irreversible Binding of Ice-Binding Proteins to Ice

Abstract Microalgal ice-binding proteins (IBPs) in the polar region are poorly understood at the genome-wide level, although they are important for cold adaptation. Through the transcriptome study with the Arctic green alga Chloromonas sp. KNF0032, we identified six Chloromonas IBP genes (CmIBPs), homologous with the previously reported IBPs from Antarctic snow alga CCMP681 and Antarctic Chloromonas sp. They were organized with multiple exon/intron structures and low-temperature-responsive cis-elements in their promoters and abundantly expressed at low temperature. The biological functions of three representative CmIBPs (CmIBP1, CmIBP2 and CmIBP3) were tested using in vitro analysis and transgenic plant system. CmIBP1 had the most effective ice recrystallization inhibition (IRI) activities in both in vitro and transgenic plants, and CmIBP2 and CmIBP3 had followed. All transgenic plants grown under nonacclimated condition were freezing tolerant, and especially 35S::CmIBP1 plants were most effective. After cold acclimation, only 35S::CmIBP2 plants showed slightly increased freezing tolerance. Structurally, the CmIBPs were predicted to have β-solenoid forms with parallel β-sheets and repeated TXT motifs. The repeated TXT structure of CmIBPs appears similar to the AidA domain-containing adhesin-like proteins from methanogens. We have shown that the AidA domain has IRI activity as CmIBPs and phylogenetic analysis also supported that the AidA domains are monophyletic with ice-binding domain of CmIBPs, and these results suggest that CmIBPs are a type of modified adhesins.

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Structure-based classification of ice-binding proteins

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314089451 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1054-C1054

Author(s):

Hackwon Do ◽

Chang Woo Lee ◽

Jun Hyuck Lee

Keyword(s):

Disulfide Bond ◽

Binding Proteins ◽

Structure Refinement ◽

Head Region ◽

Sequence Alignments ◽

Range Interaction ◽

Loop Region ◽

Ice Binding ◽

Structural Identity ◽

Eukaryotic Microorganisms

Since the antifreeze protein (AFP) super family has low structural identity, classification standard of the AFPs is presently ambiguous. Newly identified ice-binding proteins (IBPs), named so after the function of the AFPs, have similar structural identity and function that interact to the ice. Identification and characterization of IBPs from the eukaryotic microorganisms Typhulaishikariensis (TisAFP) and Leucosporidium sp. (LeIBP) revealed that both are glycosylated and have irregular motif on the ice-binding site (IBS). The IBPs share a unique right-handed β-helix, which provides an advantage of broad-range interaction surface. The other IBP encoded by the Antarctic bacterium Flavobacterium frigoris PSI was determined at 2.1-Å resolution to gain insight into its ice-binding mechanism. The structure of FfIBP shows the presence of an intra-molecular disulfide bond in the loop region between α2 and α4 (capping region), unlike that of LeIBP and TisAFP. Electron density for this disulfide bond was seen between Cys107 and Cys124 during the structure refinement process and the Cβ–Cβ distance between Cys107 and Cys124 was 3.9 Å. By sequence alignments and structural comparisons of IBPs, we defined two groups within IBPs, depending on the sequence differences between the α2 and α4 loop regions and the presence of the disulfide bond. In addition, to investigate the effects of the capping region on the activity and stability of FfIBP, we determined the crystal structure and measured the thermal stability of mutants that swapped the capping region of FfIBP and LeIBP (mFfIBP and mLeIBP). In thermal denaturation experiments, it is clear that the capping-head region of FfIBP is more stable than that of LeIBP and is important for the overall stability of IBP, although it is not directly involved in the antifreeze activity.

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Isolation and Characterization of Ice-Binding Proteins from Higher Plants

Methods in Molecular Biology - Plant Cold Acclimation ◽

10.1007/978-1-4939-0844-8_19 ◽

2014 ◽

pp. 255-277 ◽

Cited By ~ 5

Author(s):

Adam J. Middleton ◽

Barbara Vanderbeld ◽

Melissa Bredow ◽

Heather Tomalty ◽

Peter L. Davies ◽

...

Keyword(s):

Binding Proteins ◽

Higher Plants ◽

Isolation And Characterization ◽

Ice Binding

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Ice Nucleation Properties of Ice-binding Proteins from Snow Fleas

Biomolecules ◽

10.3390/biom9100532 ◽

2019 ◽

Vol 9 (10) ◽

pp. 532 ◽

Cited By ~ 2

Author(s):

Akalabya Bissoyi ◽

Naama Reicher ◽

Michael Chasnitsky ◽

Sivan Arad ◽

Thomas Koop ◽

...

Keyword(s):

Binding Proteins ◽

Thermal Hysteresis ◽

Ice Nucleation ◽

Ice Crystals ◽

Nucleation Theory ◽

Classical Nucleation Theory ◽

Ice Binding ◽

Nucleation Activity ◽

Ice Shell ◽

Ice Nucleation Activity

Ice-binding proteins (IBPs) are found in many organisms, such as fish and hexapods, plants, and bacteria that need to cope with low temperatures. Ice nucleation and thermal hysteresis are two attributes of IBPs. While ice nucleation is promoted by large proteins, known as ice nucleating proteins, the smaller IBPs, referred to as antifreeze proteins (AFPs), inhibit the growth of ice crystals by up to several degrees below the melting point, resulting in a thermal hysteresis (TH) gap between melting and ice growth. Recently, we showed that the nucleation capacity of two types of IBPs corresponds to their size, in agreement with classical nucleation theory. Here, we expand this finding to additional IBPs that we isolated from snow fleas (the arthropod Collembola), collected in northern Israel. Chemical analyses using circular dichroism and Fourier-transform infrared spectroscopy data suggest that these IBPs have a similar structure to a previously reported snow flea antifreeze protein. Further experiments reveal that the ice-shell purified proteins have hyperactive antifreeze properties, as determined by nanoliter osmometry, and also exhibit low ice-nucleation activity in accordance with their size.

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