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.

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.

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.

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.

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.

Multiple binding modes of a moderate ice-binding protein from a polar microalga.

Ice-binding proteins (IBPs) produced by cold-tolerant organisms interact with ice and strongly control crystal growth. The molecular basis for the different magnitudes of activity displayed by various IBPs (moderate and hyperactive) has not yet been clarified. Previous studies questioned whether the moderate activity of some IBPs relies on their weaker binding modus to the ice surface, compared to hyperactive IBPs, rather than relying on binding only to selected faces of the ice crystal.

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.

Concentration-dependent oligomerization of an alpha-helical antifreeze polypeptide makes it hyperactive.

A supersoluble 40-residue type I antifreeze protein (AFP) was discovered in a righteye flounder, the barfin plaice (bp). Unlike all other AFPs characterized to date, bpAFP transitions from moderately-active to hyperactive with increasing concentration. At sub-mM concentrations, bpAFP bound to pyramidal planes of ice to shape it into a bi-pyramidal hexagonal trapezohedron, similarly to the other moderately-active AFPs.

Hydrophobic ice-binding sites confer hyperactivity of an antifreeze protein from a snow mold fungus.

Snow mold fungus, Typhula ishikariensis, secretes seven antifreeze protein isoforms (denoted TisAFPs) that assist in the survival of the mold under snow cover. Here, the X-ray crystal structure of a hyperactive isoform, TisAFP8, at 1.0 Å resolution is presented.

Hyperactive antifreeze protein from an Antarctic sea ice bacterium Colwellia sp. has a compound ice-binding site without repetitive sequences.

Unlabelled: 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.

Annealing condition influences thermal hysteresis of fungal type ice-binding proteins.

The Antarctic sea ice diatom Navicular glaciei produced ice-binding protein (NagIBP) that is similar to the antifreeze protein (TisAFP) from snow mold Typhula ishikariensis. In the thermal hysteresis range of NagIBP, ice growth was completely inhibited. At the freezing point, the ice grew in a burst to 6 direction perdicular to the c-axis of ice crystal.

Cold adaptation of fungi obtained from soil and lake sediment in the Skarvsnes ice-free area, Antarctica.

A total of 71 isolates were collected from lake sediment and soil surrounding lakes in the Skarvsnes area, Antarctica. Based on ITS region sequence similarity, these isolates were classified to 10 genera. Twenty-three isolates were categorized as ascomycetous fungi from five genera (Embellisia, Phoma, Geomyces, Tetracladium or Thelebolus) and 48 isolates were categorized as basidiomycetous fungi in five genera (Mrakia, Cryptococcus, Dioszegia, Rhodotorula or Leucosporidium).

Ice-binding site of snow mold fungus antifreeze protein deviates from structural regularity and high conservation.

Antifreeze proteins (AFPs) are found in organisms ranging from fish to bacteria, where they serve different functions to facilitate survival of their host. AFPs that protect freeze-intolerant fish and insects from internal ice growth bind to ice using a regular array of well-conserved residues/motifs. Less is known about the role of AFPs in freeze-tolerant species, which might be to beneficially alter the structure of ice in or around the host.

Contribution of asparagine residues to the stabilization of a proteinaceous antigen-antibody complex, HyHEL-10-hen egg white lysozyme.

Many germ line antibodies have asparagine residues at specific sites to achieve specific antigen recognition. To study the role of asparagine residues in the stabilization of antigen-antibody complexes, we examined the interaction between hen egg white lysozyme (HEL) and the corresponding HyHEL-10 variable domain fragment (Fv). We introduced Ala and Asp substitutions into the Fv side chains of L-Asn-31, L-Asn-32, and L-Asn-92, which interact directly with residues in HEL via hydrogen bonding in the wild-type Fv-HEL complex, and we investigated the interactions between these mutant antibodies and HEL.

Comparison of functional properties of two fungal antifreeze proteins from Antarctomyces psychrotrophicus and Typhula ishikariensis.

Antifreeze proteins are structurally diverse polypeptides that have thermal hysteresis activity and have been discovered in many cold-adapted organisms. Of these, fungal antifreeze protein has been purified and partially characterized only in a species of psychrophilic basidiomycete, Typhula ishikariensis. Here we report a new fungal antifreeze protein from another psychrophile, Antarctomyces psychrotrophicus.

The crystal structure of a xyloglucan-specific endo-beta-1,4-glucanase from Geotrichum sp. M128 xyloglucanase reveals a key amino acid residue for substrate specificity.

Geotrichum sp. M128 possesses two xyloglucan-specific glycoside hydrolases belonging to family 74, xyloglucan-specific endo-beta-1,4-glucanase (XEG) and oligoxyloglucan reducing-end-specific cellobiohydrolase (OXG-RCBH). Despite their similar amino acid sequences (48% identity), their modes of action and substrate specificities are distinct.

Crystal structure and enhanced activity of a cutinase-like enzyme from Cryptococcus sp. strain S-2.

The structural and enzymatic characteristics of a cutinase-like enzyme (CLE) from Cryptococcus sp. strain S-2, which exhibits remote homology to a lipolytic enzyme and a cutinase from the fungus Fusarium solani (FS cutinase), were compared to investigate the unique substrate specificity of CLE. The crystal structure of CLE was solved to a 1.

Crystal structure and mutational analysis of Ca2+-independent type II antifreeze protein from longsnout poacher, Brachyopsis rostratus.

We recently found that longsnout poacher (Brachyosis rostratus) produces a Ca(2+)-independent type II antifreeze protein (lpAFP) and succeeded in expressing recombinant lpAFP using Phichia pastoris. Here, we report, for the first time, the X-ray crystal structure of lpAFP at 1.34 A resolution.

Critical contribution of VH-VL interaction to reshaping of an antibody: the case of humanization of anti-lysozyme antibody, HyHEL-10.

To clarify the effects of humanizing a murine antibody on its specificity and affinity for its target, we examined the interaction between hen egg white lysozyme (HEL) and its antibody, HyHEL-10 variable domain fragment (Fv). We selected a human antibody framework sequence with high homology, grafted sequences of six complementarity-determining regions of murine HyHEL-10 onto the framework, and investigated the interactions between the mutant Fvs and HEL. Isothermal titration calorimetry indicated that the humanization led to 10-fold reduced affinity of the antibody for its target, due to an unfavorable entropy change.

Thermodynamic consequences of mutations in vernier zone residues of a humanized anti-human epidermal growth factor receptor murine antibody, 528.

To investigate the role of Vernier zone residues, which are comprised in the framework regions and underlie the complementarity-determining regions (CDRs) of antibodies, in the specific, high affinity interactions of antibodies with their targets, we focused on the variable domain fragment of murine anti-human epidermal growth factor receptor antibody 528 (m528Fv). Grafting of the CDRs of m528Fv onto a selected framework region of human antibodies, referred to as humanization, reduced the antibody's affinity for its target by a factor of 1/40. The reduction in affinity was due to a substantial reduction in the negative enthalpy change associated with binding.

The structural basis for the exo-mode of action in GH74 oligoxyloglucan reducing end-specific cellobiohydrolase.

Oligoxyloglucan reducing end-specific cellobiohydrolase (OXG-RCBH) is a unique exo-beta-1,4-glucanase that belongs to glycoside hydrolase family 74. The enzyme recognizes the reducing end of xyloglucan oligosaccharides and releases two glucosyl residue segments from the reducing end of the main chain. Previously, we reported that OXG-RCBH consists of two seven-bladed beta-propeller domains.