Antifreeze proteins (AFPs) are structurally diverse macromolecules that bind to ice crystals and inhibit their growth to protect the organism from injuries caused by freezing. An AFP identified from the Antarctic bacterium Colwellia sp. strain SLW05 (ColAFP) is homologous to AFPs from a wide variety of psychrophilic microorganisms. To understand the antifreeze function of ColAFP, we have characterized its antifreeze activity and determined the crystal structure of this protein. The recombinant ColAFP exhibited thermal hysteresis activity of approximately 4°C at a concentration of 0.14 mM, and induced rapid growth of ice crystals in the hexagonal direction. Fluorescence-based ice plane affinity analysis showed that ColAFP binds to multiple planes of ice, including the basal plane. These observations show that ColAFP is a hyperactive AFP. The crystal structure of ColAFP determined at 1.6 A resolution revealed an irregular b-helical structure, similar to known homologs. Mutational and molecular docking studies showed that ColAFP binds to ice through a compound ice-binding site (IBS) located at a flat surface of the b-helix and the adjoining loop region. The IBS of ColAFP lacks the repetitive sequences that are characteristic of hyperactive AFPs. These results suggest that ColAFP exerts antifreeze activity through a compound IBS that differs from the characteristic IBSs shared by other hyperactive AFPs. This study demonstrates a novel method for protection from freezing by AFPs in psychrophilic microorganisms. DatabaseStructural data for ColAFP have been submitted to the Protein Data Bank (PDB) under accession number 3WP9.

Antifreeze proteins (AFPs) are a structurally diverse class of macromolecules that interact with water molecules located in the surface of an ice crystal at temperatures below the melting point of the solution [1]. Interaction of a substantial number of AFPs with the ice surface modifies the shape of the ice crystal, resulting in unique morphologies, such as a hexagonal bipyramid or hexagonal trapezohedron [2]. AFPs inhibit ice growth by adsorbing onto the ice surface (adsorption-inhibition model) [3,4] as the temperature is lowered, and such inhibition becomes insufficient below a certain temperature that is favourable for the initiation of crystal growth. For AFP solutions, this 'ice-growth initiation temperature' (T ini ) is different from the melting point (T m ) of the ice crystal, and the T ini is referred to as the nonequilibrium freezing point (nonequilibrium T f ). The difference between T m and the nonequilibrium T f is defined as thermal hysteresis (TH), and is generally used as a measure of the growth inhibition ability of an AFP [5]. Therefore, determination of the T ini and T m of a seed ice crystal is an essential procedure to evaluate the TH activity of AFP. However, the mechanism of ice binding that alters the TH value remains unclear [6].The TH value of AFPs has been evaluated using a nanolitre osmometer and the 'capillary technique' [5,7]. In the former, submicrolitre volumes of an AFP solution are introduced into an oil droplet, the temperature of which is controlled by a Peltier device. The TH value is determined by observing the growth of an Antifreeze proteins (AFPs) possess a unique ability to bind to a seed ice crystal to inhibit its growth. The strength of this binding has been evaluated by thermal hysteresis (TH). In this study, we examined the dependence of TH on experimental parameters, including cooling rate, annealing time, annealing temperature and the size of the seed ice crystal for an isoform of type III AFP from notched-fin eelpout (nfeAFP8). TH of nfeAFP8 dramatically decreased when using a fast cooling rate (0.20°CAEmin )1 ). It also decreased with increasing seed crystal size under a slow cooling rate (0.01°CAEmin), but such dependence was not detected under the fast cooling rate. TH was enhanced 1.4-and 2.5-fold when ice crystals were annealed for 3 h at 0.05 and 0.25°C below T m , respectively. After annealing for 2 h at 0.25°C below T m , TH activity showed marked dependence on the size of ice crystals. These results suggest that annealing of an ice crystal for 2-3 h significantly increased the TH value of type III AFP. Based on a proposed adsorption-inhibition model, we assume that type III AFP undergoes additional ice binding to the convex ice front over a 2-3 h time scale, which results in the TH dependence on the annealing time.Abbreviations AFGP, antifreeze glycoprotein; AFP, antifreeze protein; nfeAFP8, an isoform of type III AFP from Notched-fin eelpout; TH, thermal hysteresis.

We found that Notched‐fin eelpout, which lives off the north east coast of Japan, expresses an antifreeze protein (AFP). The liver of this fish contains DNAs that encode at least 13 type III AFP isoforms (denoted nfeAFPs). The primary sequences of the nfeAFP isoforms were categorized into SP‐ and QAE‐sephadex binding groups, and the latter were further divided into two subgroups, QAE1 and QAE2 groups. Ice crystals observed in HPLC‐pure nfeAFP fractions are bipyramidal in shape with different ratios of c and a axes, suggesting that all the isoforms are able to bind ice. We expressed five recombinant isoforms of nfeAFP and analyzed the thermal hysteresis (TH) activity of each as a function of protein concentration. We also examined the change in activity on mixing the isoforms. TH was estimated to be 0.60 °C for the QAE1 isoform, 0.11 °C for QAE2, and almost zero for the SP isoforms when the concentrations of these isoforms was standardized to 1.0 mm. Significantly, the TH activity of the SP isoforms showed concentration dependence in the presence of 0.2 mm QAE1, indicating that the less active SP isoform becomes ‘active’ when a small amount of QAE1 is added. In contrast, it does not become active on the addition of another SP isoform. These results suggest that the SP and QAE isoforms of type III AFP have different levels of TH activity, and they accomplish the antifreeze function in a co‐operative manner.

The psychrophilic fungi Coprinus psychromorbidus and Typhula ishikariensis produced unique antifreeze proteins (AFPs) in the extracellular space. Molecular masses of purified fungal AFPs of C. psychromorbidus and T. ishikariensis were approximately 22 and 23 kDa, respectively. Cloned genes of AFPs from T. ishikariensis do not have any similarity with known proteins. Purified fungal AFPs from cultural filtrate of T. ishikariensis and recombinant fungal AFP from methylotrophic yeast formed specific ice crystals resembling "Stone Age knives". These observations indicate that fungal AFPs do not form proper hexagonal ice crystals to inhibit their growth and that fungal AFPs can probably bind to surfaces of ice crystals in an irregular manner.Key words: antifreeze protein, snow mold fungi, Coprinus psychromorbidus, Typhula ishikariensis.

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.