Ice recrystallization is a phenomenon observed as the increase in ice crystal size within an already frozen material. Antifreeze proteins (AFPs), a class of proteins capable of arresting ice crystal growth, are known to inhibit this phenomenon even at sub milli-molar concentrations. A tremendous range in the possible applications of AFPs is hence expected in both medical and industrial fields, while a key determinant of the ice recrystallization inhibition (IRI) is hardly understood. Here, IRI efficiency and ice plane affinity were examined for the wild-type AFPI–III, a defective AFPIII isoform, and a fungal AFP isoform. To simplify the IRI analysis using the formal representation of Ostwald-ripening (r3 = r03 + kt), we monitored specific ice grains exhibiting only uniform growth, for which maximum Feret diameter was measured. The cube of an ice grain’s radius (r3) increased proportionately with time (t), and its slope gave the recrystallization rate (k). There was a significant difference in the IRI efficiency between the samples, and the fungal AFP possessing the activity with the smallest amount (0.27 μM) exhibited an affinity to multiple ice planes. These results suggest that the IRI efficiency is maximized when AFPs bind to a whole set of ice planes.

The concentration of a protein is highly related to its biochemical properties, and is a key determinant for its biotechnological applications. Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) are structurally diverse macromolecules that are capable of binding to embryonic ice crystals below 0 °C, making them useful as protectants of ice-block formation. In this study, we examined the maximal solubility of native AFP I–III and AFGP with distilled water, and evaluated concentration dependence of their ice-binding property. Approximately 400 mg/mL (AFP I), 200 mg/mL (AFP II), 100 mg/mL (AFP III), and >1800 mg/mL (AFGP) of the maximal solubility were estimated, and among them AFGP’s solubility is much higher compared with that of ordinary proteins, such as serum albumin (~500 mg/mL). The samples also exhibited unexpectedly high thermal hysteresis values (2–3 °C) at 50–200 mg/mL. Furthermore, the analysis of fluorescence-based ice plane affinity showed that AFP II binds to multiple ice planes in a concentration-dependent manner, for which an oligomerization mechanism was hypothesized. The difference of concentration dependence between AFPs and AFGPs may provide a new clue to help us understand the ice-binding function of these proteins.

Many microbes that survive in cold environments are known to secrete ice-binding proteins (IBPs). The structure–function relationship of these proteins remains unclear. A microbial IBP denoted AnpIBP was recently isolated from a cold-adapted fungus, Antarctomyces psychrotrophicus. The present study identified an orbital illumination (prism ring) on a globular single ice crystal when soaked in a solution of fluorescent AnpIBP, suggesting that AnpIBP binds to specific water molecules located in the ice prism planes. In order to examine this unique ice-binding mechanism, we carried out X-ray structural analysis and mutational experiments. It appeared that AnpIBP is made of 6-ladder β-helices with a triangular cross section that accompanies an “ice-like” water network on the ice-binding site. The network, however, does not exist in a defective mutant. AnpIBP has a row of four unique hollows on the IBS, where the distance between the hollows (14.7 Å) is complementary to the oxygen atom spacing of the prism ring. These results suggest the structure of AnpIBP is fine-tuned to merge with the ice–water interface of an ice crystal through its polygonal water network and is then bound to a specific set of water molecules constructing the prism ring to effectively halt the growth of ice.

Ice-binding proteins (IBPs) are capable of binding ice crystals and inhibiting their growth at freezing temperatures. IBPs are also thought to stabilize the cell membrane at non-freezing temperatures near 0 °C. These two effects have been assumed to reduce cold- and freezing-induced damage to cells and tissues. However, knowledge regarding the effects of IBP on the living animals is limited. Here, we characterized the relationship between the IBP effects and the physiological role by using the nematode Caenorhabditis elegans . The expression of fish (NfeIBPs)- and fungus-derived IBPs (AnpIBPs and TisIBP8) in C. elegans improved its survival rate during exposure to 0 and −2 °C (cold shock) and −5 °C (freezing). The observed cold tolerance of C. elegans after cold shock is attributable to the stabilization of cell-membrane lipids with IBPs, and the freezing tolerance at −5 °C can be attributed to the inhibition of ice-crystal growth by the IBPs. Significantly, the survival rate of C. elegans at −5 °C was improved by expression of wild-type AnpIBP and maximized by that of TisIBP8, whereas it was lowered when a defective AnpIBP mutant was expressed. These results suggest that the ice-binding ability of IBP has a good correlation with the survival rate of C. elegans during freezing.

Beetle hyperactive antifreeze protein (AFP) has a unique ability to maintain a supercooling state of its body fluids, however, less is known about its origination. Here, we found that a popular stag beetle Dorcus hopei binodulosus (Dhb) synthesizes at least 6 isoforms of hyperactive AFP (DhbAFP). Cold-acclimated Dhb larvae tolerated −5 °C chilled storage for 24 h and fully recovered after warming, suggesting that DhbAFP facilitates overwintering of this beetle. A DhbAFP isoform (~10 kDa) appeared to consist of 6−8 tandem repeats of a 12-residue consensus sequence (TCTxSxNCxxAx), which exhibited 3 °C of high freezing point depression and the ability of binding to an entire surface of a single ice crystal. Significantly, these properties as well as DNA sequences including the untranslated region, signal peptide region, and an AFP-encoding region of Dhb are highly similar to those identified for a known hyperactive AFP (TmAFP) from the beetle Tenebrio molitor (Tm). Progenitor of Dhb and Tm was branched off approximately 300 million years ago, so no known evolution mechanism hardly explains the retainment of the DNA sequence for such a lo­ng divergence period. Existence of unrevealed gene transfer mechanism will be hypothesized between these two phylogenetically distant beetles to acquire this type of hyperactive AFP.