SignificanceThis study expands our knowledge of protein hydration, which is highly related to the macromolecular antifreeze property of proteins. We examined a polypentagonal network formation of waters for a series of artificial variants of a 65-residue ice-binding protein. The polypentagonal waters were created solely on the surface of an activity-improved variant, which appeared to contain two sets of water clusters exhibiting a perfect position match to the waters constructing the first prism and pyramidal ice planes. These data suggest that a minute structural change in a protein organizes the surface waters into a polypentagonal arrangement, which merges with the intrinsically disordered ice surface and freezes to specific ice crystal planes.
Various microbes, including fungi and bacteria, that live in cold environments produce ice‐binding proteins (IBPs) that protect them from freezing. Ascomycota and Basidiomycota are two major phyla of fungi, and Antarctomyces psychrotrophicus is currently designated as the sole ascomycete that produces IBP (AnpIBP). However, its complete amino acid sequence, ice‐binding property, and evolutionary history have not yet been clarified. Here, we determined the peptide sequences of three new AnpIBP isoforms by total cDNA analysis and compared them with those of other microbial IBPs. The AnpIBP isoforms and ascomycete‐putative IBPs were found to be phylogenetically close to the bacterial ones but far from the basidiomycete ones, which is supported by the higher sequence identities to bacterial IBPs than basidiomycete IBPs, although ascomycetes are phylogenetically distant from bacteria. In addition, two of the isoforms of AnpIBP share low sequence identity and are not close in the phylogenetic tree. It is hence presumable that these two AnpIBP isoforms were independently acquired from different bacteria through horizontal gene transfer (HGT), which implies that ascomycetes and bacteria frequently exchange their IBP genes. The non‐colligative freezing‐point depression ability of AnpIBP was not very high, whereas it exhibited significant abilities of ice recrystallization inhibition, ice shaping, and cryo‐protection against freeze–thaw cycles even at submicromolar concentrations. These results suggest that HGT is crucial for the cold‐adaptive evolution of ascomycetes, and their IBPs offer freeze resistance to organisms to enable them to inhabit the icy environments of Antarctica.
Databases
Nucleotide sequence data are available in the DDBJ database under the accession numbers , , for AnpIBP1a, AnpIBP1b, AnpIBP2, respectively.
Antifreeze glycoprotein (AFGP) is an O-glycoprotein that displays antifreeze activity through depression of the freezing point of water. GalNAc is a core sugar structure of AFGP, and contributes to induce antifreeze activity of this glycoprotein. However, the general functional role that this sugar plays at the molecular level is still unknown. To elucidate this, it is essential to determine the relationship between structure and activity of O-GalNAcylated AFGP using homogeneous glycoproteins. Thus, the total synthesis of homogeneous O-GalNAcylated AFGP was conducted by using a unique peptide derivative: peptidyl-N-pivaloylguanidine. It was found that peptidyl-N-pivaloylguanidine is an "unreactive" peptide in peptide coupling reactions but is interconvertible with a "reactive" peptide-α-thioester by means of a simple treatment under buffer condition at pH=7 to 8. The unique switchable reactivity of peptidyl-N-pivaloylguanidine enabled an efficient sequential peptide coupling strategy. By using this strategy, various lengths of homogeneous O-GalNAcylated AFGP were synthesized, including one that was 120 amino acids in length, with 40 O-GalNAcylation sites. The structural analysis by circular dichroism spectroscopy and evaluation of the antifreeze activity of the synthetic AFGP(GalNAc)s revealed that the simple O-glycosylation with GalNAc is essential for both structural and functional basis of AFGP to exhibit antifreeze activity.
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