Evaluation of Ice Recrystallization Inhibition of Ice-Binding Proteins by Monitoring Specific Ice Crystals.

Ice recrystallization is a phenomenon in which large ice crystals are formed at the expense of smaller ones. The resultant large ice crystals degrade the quality of frozen foods and cryopreserved biomaterials. To minimize freeze damage by controlling the ice recrystallization process, various compounds have been developed, including biological antifreezes, synthetic peptides, glycopeptides, polymers, and small molecules.

The ice-binding site of antifreeze protein irreversibly binds to cell surface for its hypothermic protective function.

Antifreeze proteins (AFPs) are multifunctional polypeptides that adsorb onto ice crystals to inhibit their growth and onto cells to protect them from nonfreezing hypothermic damage. However, the mechanism by which AFP exerts its hypothermic cell protective (HCP) function remains uncertain. Here, we assessed the HCP function of three types of fish-derived AFPs (type I, II, and III AFPs) against human T-lymphoblastic lymphoma by measuring the survival rate (%) of the cells after preservation at 4 °C for 24 h.

The effect of ice-binding proteins on the cryopreservation of .

Ice-binding proteins (IBPs) are capable of binding ice crystals and inhibiting their growth. IBPs have also been reported to stabilize cell membranes under non-freezing conditions. The effects of IBPs help to reduce cold- and freezing-induced damage to cells and tissues in cryopreservation.

Regulating Antifreeze Activity through Water: Latent Functions of the Sugars of Antifreeze Glycoprotein Revealed by Total Chemical Synthesis.

Antifreeze glycoprotein (AFGP), which inhibits the freezing of water, is highly O-glycosylated with a disaccharide, d-Galβ1-3-d-GalNAcα (GalGalNAc). To elucidate the function of the sugar residues for antifreeze activity at the molecular level, we conducted a total chemical synthesis of partially sugar deleted AFGP derivatives, and unnatural forms of AFGPs incorporating glucose (Glc)-type sugars instead of galactose (Gal)-type sugars. These elaborated AFGP derivatives demonstrated that the stereochemistry of each sugar residue on AFGPs precisely correlates with the antifreeze activity.

Visualizing Intramolecular Dynamics of Membrane Proteins.

Membrane proteins play important roles in biological functions, with accompanying allosteric structure changes. Understanding intramolecular dynamics helps elucidate catalytic mechanisms and develop new drugs. In contrast to the various technologies for structural analysis, methods for analyzing intramolecular dynamics are limited.

A mutation to a fish ice-binding protein synthesized in transgenic Caenorhabditis elegans modulates its cold tolerance.

A cryoprotectant known as ice-binding protein (IBP) is thought to facilitate the cold survival of plants, insects, and fungi. Here, we prepared a genetically modified Caenorhabditis elegans strain to synthesize fish-derived IBPs in its body wall muscles and examined whether the antifreeze activity modification of this IBP by point mutation affects the cold tolerance of this worm. We chose a 65-residue IBP identified from notched-fin eelpout, for which the replacement of the 20th alanine residue (A20) modifies its antifreeze activity.

Dynamic motions of ice-binding proteins in living using diffracted X-ray blinking and tracking.

The dynamic properties of protein molecules are involved in the relationship between their structure and function. Time-resolved X-ray observation enables capturing the structures of biomolecules with picometre-scale precision. However, this technique has yet to be implemented in living animals.

Subzero Nonfreezing Hypothermia with Insect Antifreeze Protein Dramatically Improves Survival Rate of Mammalian Cells.

Cells for therapeutic use are often preserved at +4 °C, and the storage period is generally limited to 2-3 days. Here, we report that the survival rate (%) of mammalian cells is improved to 10-20 days when they are preserved with a subzero supercooled solution containing the antifreeze protein (AFP), for which an ability to stabilize both supercooled water and cell membrane integrity has been postulated. We chose adherent rat insulinoma (RIN-5F) cells as the preservation target, which were immersed into -5 °C-, -2 °C-, or +4 °C-chilled "unfrozen" solution of Euro-Collins or University of Washington (UW) containing the AFP sample obtained from insect or fish.

Laboratory diffracted x-ray blinking to monitor picometer motions of protein molecules and application to crystalline materials.

In recent years, real-time observations of molecules have been required to understand their behavior and function. To date, we have reported two different time-resolved observation methods: diffracted x-ray tracking and diffracted x-ray blinking (DXB). The former monitors the motion of diffracted spots derived from nanocrystals labeled onto target molecules, and the latter measures the fluctuation of the diffraction intensity that is highly correlated with the target molecular motion.

Discovery of Hyperactive Antifreeze Protein from Phylogenetically Distant Beetles Questions Its Evolutionary Origin.

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 () synthesizes at least 6 isoforms of hyperactive AFP (AFP). Cold-acclimated larvae tolerated -5 °C chilled storage for 24 h and fully recovered after warming, suggesting that AFP facilitates overwintering of this beetle.

Characterization of microbial antifreeze protein with intermediate activity suggests that a bound-water network is essential for hyperactivity.

Antifreeze proteins (AFPs) inhibit ice growth by adsorbing onto specific ice planes. Microbial AFPs show diverse antifreeze activity and ice plane specificity, while sharing a common molecular scaffold. To probe the molecular mechanisms responsible for AFP activity, we here characterized the antifreeze activity and crystal structure of TisAFP7 from the snow mold fungus Typhula ishikariensis.

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 IBP was recently isolated from a cold-adapted fungus, .

Fish-Derived Antifreeze Proteins and Antifreeze Glycoprotein Exhibit a Different Ice-Binding Property with Increasing Concentration.

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.

Presence of a basic secretory protein in xylem sap and shoots of poplar in winter and its physicochemical activities against winter environmental conditions.

XSP25, previously shown to be the most abundant hydrophilic protein in xylem sap of Populus nigra in winter, belongs to a secretory protein family in which the arrangement of basic and acidic amino acids is conserved between dicotyledonous and monocotyledonous species. Its gene expression was observed at the same level in roots and shoots under long-day conditions, but highly induced under short-day conditions and at low temperatures in roots, especially in endodermis and xylem parenchyma in the root hair region of Populus trichocarpa, and its protein level was high in dormant buds, but not in roots or branches. Addition of recombinant PtXSP25 protein mitigated the denaturation of lactate dehydrogenase by drying, but showed only a slight effect on that caused by freeze-thaw cycling.

Expression of Ice-Binding Proteins in Caenorhabditis elegans Improves the Survival Rate upon Cold Shock and during Freezing.

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.

Calcium-Binding Generates the Semi-Clathrate Waters on a Type II Antifreeze Protein to Adsorb onto an Ice Crystal Surface.

Hydration is crucial for a function and a ligand recognition of a protein. The hydration shell constructed on an antifreeze protein (AFP) contains many organized waters, through which AFP is thought to bind to specific ice crystal planes. For a Ca-dependent species of AFP, however, it has not been clarified how 1 mol of Ca-binding is related with the hydration and the ice-binding ability.

Freeze Tolerance in Sculpins (Pisces; Cottoidea) Inhabiting North Pacific and Arctic Oceans: Antifreeze Activity and Gene Sequences of the Antifreeze Protein.

Many marine species inhabiting icy seawater produce antifreeze proteins (AFPs) to prevent their body fluids from freezing. The sculpin species of the superfamily Cottoidea are widely found from the Arctic to southern hemisphere, some of which are known to express AFP. Here we clarified DNA sequence encoding type I AFP for 3 species of 2 families (Cottidae and Agonidae) belonging to Cottoidea.

Ice recrystallization is strongly inhibited when antifreeze proteins bind to multiple ice planes.

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.

Ice-binding proteins from the fungus Antarctomyces psychrotrophicus possibly originate from two different bacteria through horizontal gene transfer.

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.

Applications of Antifreeze Proteins: Practical Use of the Quality Products from Japanese Fishes.

Numerous embryonic ice crystals are generated in water at the moment of freezing. These crystals grow and merge together to form an ice block that can be generally observed. Antifreeze protein (AFP) is capable of binding to the embryonic ice crystals, inhibiting such an ice block formation.

Unexpected Rise of Glass Transition Temperature of Ice Crystallized from Antifreeze Protein Solution.

Antifreeze protein (AFP) is known to bind to a single ice crystal composed of hexagonally arranged waters, hexagonal ice. To investigate the effect of the AFP binding to a general ice block that is an assembly of numerous hexagonal ice crystals, thermodynamic properties, dynamics, and the crystal structure of the ice block were examined in the presence of type I AFP (AFP-I). Previously, it was found that hexagonal ice has a glass transition based on the proton ordering in the ice lattice at low temperature.

Polypentagonal ice-like water networks emerge solely in an activity-improved variant of ice-binding protein.

Polypentagonal water networks were recently observed in a protein capable of binding to ice crystals, or ice-binding protein (IBP). To examine such water networks and clarify their role in ice-binding, we determined X-ray crystal structures of a 65-residue defective isoform of a -derived IBP (wild type, WT) and its five single mutants (A20L, A20G, A20T, A20V, and A20I). Polypentagonal water networks composed of ∼50 semiclathrate waters were observed solely on the strongest A20I mutant, which appeared to include a tetrahedral water cluster exhibiting a perfect position match to the [Formula: see text] first prism plane of a single ice crystal.

Total Synthesis of O-GalNAcylated Antifreeze Glycoprotein using the Switchable Reactivity of Peptidyl-N-pivaloylguanidine.

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.