Molecular evidence of intertidal habitats selecting for repeated ice-binding protein evolution in invertebrates

ABSTRACTIce-binding proteins (IBPs) have evolved independently in multiple taxonomic groups to improve their survival of sub-zero temperatures. Intertidal invertebrates in temperate and polar regions frequently encounter sub-zero temperatures, yet there is little information on IBPs in these organisms. We hypothesized that there are far more ice-binding proteins than are currently known and that the occurrence of freezing in the intertidal zone selects for these proteins. We compiled a list of genome-sequenced invertebrates across multiple habitats and a list of known IBP sequences and used BLAST to identify a wide array of putative IBPs in those invertebrates. We found that the probability of an invertebrate species having an ice-binding protein was significantly greater in intertidal species as compared to those primarily found in open ocean or freshwater habitats. These intertidal IBPs had high sequence similarity to fish and tick antifreeze glycoproteins and fish type II antifreeze proteins. Previously established classifiers based on machine learning techniques further predicted ice-binding activity in the majority of our newly identified putative IBPs. We investigated the potential evolutionary origin of one putative IBP from the hard-shelled mussel Mytilus coruscus and suggest that it arose through gene duplication and neofunctionalization. We show that IBPs likely readily evolve in response to freezing risk, that there is an array of uncharacterized ice binding proteins and highlight the need for broader laboratory-based surveys of the diversity of ice binding activity across diverse taxonomic and ecological groups.Summary statementIntertidal invertebrates have a disproportionate number of putative ice-binding proteins relative to other habitats. These putative proteins are highly similar to antifreeze glycoproteins and type II antifreeze proteins from fish.

Designing and studying a mutant form of the ice-binding protein from Choristoneura fumiferana

Ice-binding proteins are expressed in the cells of some organisms, helping them to survive extremely low temperatures. One of the problems in study of such proteins is the difficulty of isolation and purification. For example, eight cysteine residues in cfAFP from Choristoneura fumiferana (the eastern spruce budworm) form intermolecular bridges during the overexpression of this protein. This impedes the process of the protein purification dramatically.In this work we designed a mutant form of ice-binding protein cfAFP, which is much more easy to isolate that the wild-type protein. The mutant form named mIBP83 did not lose the ability to bind to ice surface. Besides, observation of the processes of freezing and melting of ice in presence of mIBP83 showed that this protein affects the process of ice melting, increasing its melting temperature, and at least does not decrease the freezing temperature.

An ice-binding protein from an Arctic population of American dunegrass, Leymus mollis

Several cold-hardy grasses have been shown to have ice-binding proteins (IBPs) that protect against freeze-thaw injury. Here, we looked for IBP activity in an Alaskan coastal grass, Leymus mollis (Pooidae), that had not previously been examined. Rhizome tissue had strong ice-structuring and ice recrystallization inhibiting (IRI) activities, indicating the probable presence of IBPs. The gene sequence of an IBP was obtained. The sequence encoded a 118-amino acid IRI domain composed of eight repeats and that was 80% identical to the IRI domain of the IBP of perennial ryegrass Lolium perenne. The predicted 3D structure of the IRI domain had eight beta-roll coils like those in L. perenne IBP

An ice-binding protein from an Arctic grass, Leymus mollis

Several cold-hardy grasses have been shown to have ice-binding proteins (IBPs) that protect against freeze-thaw injury. Here, we looked for IBP activity in an Alaskan coastal grass that had not previously been examined, Leymus mollis (Pooidae). Rhizome tissue had strong ice-structuring and ice recrystallization inhibiting (IRI) activities, indicating the probable presence of IBPs. The gene sequence of an IBP was obtained. The sequence encoded a 118-amino acid IRI domain that contained eight repeats. A 3D structure of the IRI domain was predicted from the structure of the IRI domain of the perennial ryegrass Lolium perenne. The predicted structure appeared to have the same eight beta-roll coils found in the L. perenne IBP.

An Ice-Binding Protein from an Antarctic Ascomycete Is Fine-Tuned to Bind to Specific Water Molecules Located in the Ice Prism Planes

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.