Doctor of Science, Hokkaido University, Dec. 1992

Antifreeze protein

Structure

NMR

X-ray

Ice nucleation Protein

Structural biology

AFP

INP

Life Science, Structural biochemistry

May 2020, Japanese Society for Cryobiology and Cryotechnology, Society Award
Functional analysis of antifreeze protein toward its practical applications
Grounds of the award http://square.umin.ac.jp/jscc/en/about/awardL.html, 9839186;9839187;530929;9839188

2000, 研究優秀者表彰 通商産業省工業技術院 工業技術院長賞
Japan

2000, 研究優秀者表彰 北海道工業技術研究所 所長賞
Japan

2000, "AIST President Award" for excellence for work regarding NMR analysis of the structure-function-relationship of low-temparture biomolecules (Koji Kajiwara, Ph.D.).

2000, Director Award" for excellence for work regarding low-temperature-structure determination of apo-troponin C from Hokkaido National Industrial Research Institute (HNIRI) (Tatsuo Katsura, Ph.D.).

1996, John Baldo 学術奨励賞（University of Alberta, CA)

1996, "John Baldo Award" for excellence for work regarding apo- and Ca2+-bound troponin C from University of Alberta (Prof. John Baldo and Brian D. Sykes), Canada.

Hyperactive ice‐binding proteins stabilize cell membranes and improve resistance to dehydration stress in Caenorhabditis elegans
Daiki Shimose; Kotaro Ozaki; Ryohei Kuriyama; Yuka Ikemoto; Kazuhiro Mio; Yuji C. Sasaki; Tatsuya Arai; Sakae Tsuda; Yoichi Shinkai; Masahiro Kuramochi
FEBS Open Bio, 21 May 2026
English, Scientific journal,
Ice‐binding proteins (IBPs) are known to modulate ice growth and promote freezing tolerance and have recently attracted attention due to their cell‐protective functions under freezing or low‐temperature conditions. In this study, we demonstrate that IBP expression improves resistance to dehydration stress in
Caenorhabditis elegans
. After 30 min of drying, transgenic worms expressing a high‐activity fungal IBP (TisIBP8) showed a modest but significant increase in survival compared with wild‐type worms. Furthermore, imaging revealed reduced muscle‐cell damage in the TisIBP8‐expressing worms while synchrotron radiation infrared microspectroscopy showed membrane structural changes during dehydration were attenuated by TisIBP8 expression. These findings suggest that IBPs exert protective effects by stabilizing cell membranes independent of ice binding, therefore broadening the potential applications of IBPs for biological preservation under nonfreezing stress conditions.

Fluctuation of Time-Resolved X-ray Diffraction Reveals the Rotational Dynamics of Nanoparticles in Polymer Materials
Tatsuya Arai; Masahiro Kuramochi; Kazuhiro Mio; Hiroshi Sekiguchi; Sakae Tsuda; Daisuke Sasaki; Tomoyasu Aizawa; Yuji C. Sasaki
Nano Letters, American Chemical Society (ACS), 14 Sep. 2025, [Peer-reviewed]
English, Scientific journal

Effects of Trehalose and Antifreeze Glycoproteins on Long-Term Storage of Cryopreserved Trehalose-Transporter Expressing Cells and on Ice Recrystallization
Tsutomu Uchida; Tsubasa Hohana; Sumire Matsuo; Sakae Tsuda; Takahiro Kikawada
Crystal Growth & Design, 24, 18, 7549, 7560, American Chemical Society (ACS), 04 Sep. 2024, [Peer-reviewed]
Scientific journal

Evaluation of Ice Recrystallization Inhibition of Ice-Binding Proteins by Monitoring Specific Ice Crystals.
Anika T Rahman; Yasushi Ohyama; Sakae Tsuda; Hidemasa Kondo
Methods in molecular biology (Clifton, N.J.), 2730, 93, 100, 2024, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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. To compare their efficiency, evaluation methods of ice recrystallization inhibition are important. This chapter describes a practical protocol to quantify the inhibition efficiency by observing specific ice crystals exhibiting uniform growth.

The ice-binding site of antifreeze protein irreversibly binds to cell surface for its hypothermic protective function
Yue Yang; Akari Yamauchi; Sakae Tsuda; Masahiro Kuramochi; Kazuhiro Mio; Yuji C. Sasaki; Tatsuya Arai
Biochemical and Biophysical Research Communications, Nov. 2023, [Peer-reviewed]
Scientific journal

The effect of ice-binding proteins on the cryopreservation of Caenorhabditis elegans
Masahiro Kuramochi; Tatsuya Arai; Kazuhiro Mio; Sakae Tsuda; Yuji C Sasaki
microPublication Biology, Apr. 2023, [Peer-reviewed]

昆虫由来不凍タンパク質の氷結晶結合機能と細胞保護機能
彩加林 山内; 栄 津田
低温科学, 20 Mar. 2023, [Peer-reviewed], [Last author]
Japanese, Scientific journal

不凍タンパク質の構造と機能
栄 津田; 達也 新井
低温科学, 20 Mar. 2023, [Peer-reviewed], [Lead author]
Japanese, Scientific journal

Regulating Antifreeze Activity through Water: Latent Functions of the Sugars of Antifreeze Glycoprotein Revealed by Total Chemical Synthesis
Ryo Okamoto; Ryo Orii; Hiroyuki Shibata; Yuta Maki; Sakae Tsuda; Yasuhiro Kajihara
Chemistry – A European Journal, 29, 21, e202203553, Mar. 2023, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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. A hydrogen-deuterium exchange experiment using synthetic AFGPs revealed a different dynamic behavior of water around sugar residues depending on the sugar structures. These results indicate that sugar residues on AFGP form a unique dynamic water phase that disturbs the absorbance of water molecules onto the ice surface, thereby inhibiting freezing.

Visualizing Intramolecular Dynamics of Membrane Proteins
Tatsunari Ohkubo; Takaaki Shiina; Kayoko Kawaguchi; Daisuke Sasaki; Rena Inamasu; Yue Yang; Zhuoqi Li; Keizaburo Taninaka; Masaki Sakaguchi; Shoko Fujimura; Hiroshi Sekiguchi; Masahiro Kuramochi; Tatsuya Arai; Sakae Tsuda; Yuji C. Sasaki; Kazuhiro Mio
International Journal of Molecular Sciences, 23, 23, 14539, 14539, MDPI AG, 22 Nov. 2022, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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. Single-molecule measurements using optical microscopy have been widely used for kinetic analysis. Recently, improvements in detectors and image analysis technology have made it possible to use single-molecule determination methods using X-rays and electron beams, such as diffracted X-ray tracking (DXT), X-ray free electron laser (XFEL) imaging, and cryo-electron microscopy (cryo-EM). High-speed atomic force microscopy (HS-AFM) is a scanning probe microscope that can capture the structural dynamics of biomolecules in real time at the single-molecule level. Time-resolved techniques also facilitate an understanding of real-time intramolecular processes during chemical reactions. In this review, recent advances in membrane protein dynamics visualization techniques were presented.

A mutation to a fish ice-binding protein synthesized in transgenic Caenorhabditis elegans modulates its cold tolerance.
Masahiro Kuramochi; Shumiao Zhu; Chiaki Takanashi; Yue Yang; Tatsuya Arai; Yoichi Shinkai; Motomichi Doi; Kazuhiro Mio; Sakae Tsuda; Yuji C Sasaki
Biochemical and biophysical research communications, 628, 98, 103, Elsevier {BV}, 05 Nov. 2022, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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. These mutant proteins are denoted A20L, A20G, A20T, A20V, and A20I along with the wild-type (WT) protein. We evaluated the survival rate (%) of the transgenic C. elegans that synthesized each IBP mutant following 24 h of preservation at -5, +2, and +5 °C. Significantly, a dramatic improvement in the survival rate was detected for the worms synthesizing the activity-enhanced mutants (A20T and A20I), especially at +2 °C. In contrast, the rate was not improved by the expression of the defective mutants (A20L, A20G, WT and A20V). The survival rate (%) probably correlates with the antifreeze activity of the IBP. These data suggest that IBP protects the cell membrane by employing its ice-binding mechanism, which ultimately improves the cold tolerance of an IBP-containing animal.

Adsorption of ice-binding proteins onto whole ice crystal surfaces does not necessarily confer a high thermal hysteresis activity.
Tatsuya Arai; Akari Yamauchi; Yue Yang; Shiv Mohan Singh; Yuji C Sasaki; Sakae Tsuda
Scientific reports, 12, 1, 15443, 15443, Springer Science and Business Media {LLC}, 14 Sep. 2022, [Peer-reviewed], [International Magazine]
English, Scientific journal, Many psychrophilic microorganisms synthesize ice-binding proteins (IBPs) to survive the cold. The functions of IBPs are evaluated by the effect of the proteins on the nonequilibrium water freezing-point depression, which is called "thermal hysteresis (TH)", and the inhibitory effect of the proteins on the growth of larger ice crystals, which is called "ice recrystallization inhibition (IRI)". To obtain mechanical insight into the two activities, we developed a modified method of ice affinity purification and extracted two new IBP isoforms from Psychromyces glacialis, an Arctic glacier fungus. One isoform was found to be an approximately 25 kDa protein (PsgIBP_S), while the other is a 28 kDa larger protein (PsgIBP_L) that forms an intermolecular dimer. Their TH activities were less than 1 °C at millimolar concentrations, implying that both isoforms are moderately active but not hyperactive IBP species. It further appeared that both isoforms exhibit high IRI activity even at submicromolar concentrations. Furthermore, the isoforms can bind to the whole surface of a hemispherical single ice crystal, although such ice-binding was generally observed for hyperactive IBP species. These results suggest that the binding ability of IBPs to whole ice crystal surfaces is deficient for hyperactivity but is crucial for significant IRI activity.

Dynamic motions of ice-binding proteins in living Caenorhabditis elegans using diffracted X-ray blinking and tracking
Masahiro Kuramochi; Yige Dong; Yue Yang; Tatsuya Arai; Rio Okada; Yoichi Shinkai; Motomichi Doi; Kouki Aoyama; Hiroshi Sekiguchi; Kazuhiro Mio; Sakae Tsuda; Yuji C. Sasaki
Biochemistry and Biophysics Reports, 29, 101224, 101224, Elsevier {BV}, Mar. 2022, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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. Here, we examined diffracted X-ray blinking (DXB) and diffracted X-ray tracking (DXT) to observe the dynamics of a protein located on intestinal cells in adult Caenorhabditis elegans. This in vivo tissue-specific DXB was examined at temperatures from 20 °C to -10 °C for a recombinant ice-binding protein from Antarctomyces psychrotrophicus (AnpIBP) connected with the cells through a transmembrane CD4 protein equipped with a glycine-serine linker. AnpIBP inhibits ice growth at subzero temperatures by binding to ice crystals. We found that the rotational motion of AnpIBP decreases at -10 °C. In contrast, the motion of the AnpIBP mutant, which has a defective ice-binding ability, did not decrease at -10 °C. The twisting and tilting motional speeds of AnpIBPs measured above 5 °C by DXT were always higher than those of the defective AnpIBP mutant. These results suggest that wild-type AnpIBP is highly mobile in solution, and it is halted at subzero temperatures through ice binding. DXB and DXT allow for exploring protein behaviour in live animals with subnano resolution precision.

Subzero Nonfreezing Hypothermia with Insect Antifreeze Protein Dramatically Improves Survival Rate of Mammalian Cells
Akari Yamauchi; Ai Miura; Hidemasa Kondo; Tatsuya Arai; Yuji C. SASAKI; Sakae Tsuda
International Journal of Molecular Sciences, 22, 23, Nov. 2021, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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. Our results show that the survival rate of the cells preserved with the solution containing insect AFP was always higher than that of the fish AFP solution. A combination of the -5 °C-supercooling and insect AFP gave the best preservation result, namely, UW solution containing insect AFP kept 53% of the cells alive, even after 20 days of preservation at -5 °C. The insect AFP locates highly organized ice-like waters on its molecular surface. Such waters may bind to semiclathrate waters constructing both embryonic ice crystals and a membrane-water interface in the supercooled solution, thereby protecting the cells from damage due to chilling.

Crystal waters on the nine polyproline type II helical bundle springtail antifreeze protein from Granisotoma rainieri match the ice lattice.
Connor L Scholl; Sakae Tsuda; Laurie A Graham; Peter L Davies
The FEBS journal, 288, 14, 4332, 4347, Jul. 2021, [Peer-reviewed], [International Magazine]
English, Scientific journal, A springtail (Collembola) identified as Granisotoma rainieri was collected from snow in Hokkaido, Japan, in late winter when nighttime temperatures were below zero. Extracts of these arthropods showed antifreeze activity by shaping ice crystals and stopping their growth. The glycine-rich proteins responsible for this freezing point depression were isolated by ice-affinity purification and had principal masses of ~ 6.9 and 9.6 kDa. We identified a transcript for a 9.6-kDa component and produced it as a His-tagged recombinant protein for structural analysis. Its crystal structure was solved to a resolution of 1.21 Å and revealed a polyproline type II helical bundle, similar to the six-helix Hypogastrura harveyi AFP, but with nine helices organized into two layers held together by an extensive network of hydrogen bonds. One of the layers is flat, regular, and hydrophobic and likely serves as the ice-binding side. Although this surface makes close protein-protein contacts with its symmetry mate in the crystal, it has bound chains of waters present that resemble those on the basal and primary prism planes of ice. Molecular dynamic simulations indicate most of these crystal waters would preferentially occupy these sites if exposed to bulk solvent in the absence of the symmetry mate. These prepositioned waters lend further support to the ice-binding mechanism in which AFPs organize ice-like waters on one surface to adsorb to ice. DATABASES: Structural data are available in the Protein Data Bank under the accession number 7JJV. Transcript data are available in GenBank under accession numbers MT780727, MT780728, MT780729, MT780730, MT780731 and MT985982.

Laboratory diffracted x-ray blinking to monitor picometer motions of protein molecules and application to crystalline materials
Tatsuya Arai; Rena Inamasu; Hiroki Yamaguchi; Daisuke Sasaki; Ayana Sato-Tomita; Hiroshi Sekiguchi; Kazuhiro Mio; Sakae Tsuda; Masahiro Kuramochi; Yuji C. Sasaki
Structural Dynamics, 8, 4, 044302, 044302, AIP Publishing, Jul. 2021, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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. However, these reports use a synchrotron x-ray source because of its high average flux, resulting in a high time resolution. Here, we used a laboratory x-ray source and DXB to measure the internal molecular dynamics of three different systems. The samples studied were bovine serum albumin (BSA) pinned onto a substrate, antifreeze protein (AFP) crystallized as a single crystal, and poly{2-(perfluorooctyl)ethyl acrylate} (PC8FA) polymer between polyimide sheets. It was found that not only BSA but also AFP and PC8FA molecules move in the systems. In addition, the molecular motion of AFP molecules was observed to increase with decreasing temperature. The rotational diffusion coefficients (DR) of BSA, AFP, and PC8FA were estimated to be 0.73 pm2/s, 0.65 pm2/s, and 3.29 pm2/s, respectively. Surprisingly, the DR of the PC8FA polymer was found to be the highest among the three samples. This is the first report that measures the molecular motion of a single protein crystal and polymer by using DXB with a laboratory x-ray source. This technique can be applied to any kind of crystal and crystalline polymer and provides atomic-order molecular information.

Discovery of hyperactive antifreeze protein from a stag beetle Dorcus hopei binodulosus
Sakae Tsuda
Kon_chu-to-Sizen, 23 Jun. 2021, [Peer-reviewed], [Domestic magazines]
Japanese, Scientific journal

Partial characterization of an antifreeze protein (CRY-c) from Cryobacterium psychrotolerans MLB-29 of Arctic glacier cryoconite
Purnima Singh; Sakae Tsuda; Shiv Mohan Singh; Sukanta Mondal; Utpal Roy
Polar Science, 28, 100661, 100661, Elsevier {BV}, Jun. 2021, [Peer-reviewed]
Scientific journal

Functional analysis of antifreeze protein for its technological application
Sakae Tsuda
Cryobiology and Cryotechnology, 15 Apr. 2021, [Peer-reviewed], [Domestic magazines]
Japanese, Scientific journal

Characterization of microbial antifreeze protein with intermediate activity suggests that a bound-water network is essential for hyperactivity.
Sakae Tsuda
Scientific reports, 11, 1, 5971, 5971, Springer Science and Business Media {LLC}, 16 Mar. 2021, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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. TisAFP7 exhibited intermediate activity, with the ability to bind the basal plane, compared with a hyperactive isoform TisAFP8 and a moderately active isoform TisAFP6. Analysis of the TisAFP7 crystal structure revealed a bound-water network arranged in a zigzag pattern on the surface of the protein's ice-binding site (IBS). While the three AFP isoforms shared the water network pattern, the network on TisAFP7 IBS was not extensive, which was likely related to its intermediate activity. Analysis of the TisAFP7 crystal structure also revealed the presence of additional water molecules that form a ring-like network surrounding the hydrophobic side chain of a crucial IBS phenylalanine, which might be responsible for the increased adsorption of AFP molecule onto the basal plane. Based on these observations, we propose that the extended water network and hydrophobic hydration at IBS together determine the TisAFP activity.

Discovery of Hyperactive Antifreeze Protein from Phylogenetically Distant Beetles Questions Its Evolutionary Origin
Tatsuya Arai; Akari Yamauchi; Ai Miura; Hidemasa Kondo; Yoshiyuki Nishimiya; Yuji SASAKI; Sakae Tsuda
International Journal of Molecular Sciences, 22, 7, 3637, 3637, {MDPI} {AG}, Mar. 2021, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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 lo-ng divergence period. Existence of unrevealed gene transfer mechanism will be hypothesized between these two phylogenetically distant beetles to acquire this type of hyperactive AFP.

Effect of Ice Recrystallization Phenomenon on Long-Term Storage of Cryopreserved Cells
Uchida Tsutomu; Hohana Tsubasa; Kikawada Takahiro; Tsuda Sakae; Yamazaki Kenji; Gohara Kazutoshi
Summaries of JSSI and JSSE Joint Conference on Snow and Ice Research, 2021, 72, 72, The Japanese Society of Snow and Ice / Japan Society for Snow Engineering, 2021
Japanese

An Ice-Binding Protein from an Antarctic Ascomycete Is Fine-Tuned to Bind to Specific Water Molecules Located in the Ice Prism Planes
Akari Yamauchi; Tatsuya Arai; Hidemasa Kondo; Yuji C. Sasaki; Sakae Tsuda
Biomolecules, 10, 5, 759, 759, {MDPI} {AG}, 13 May 2020, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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.

Fish-Derived Antifreeze Proteins and Antifreeze Glycoprotein Exhibit a Different Ice-Binding Property with Increasing Concentration
Sakae Tsuda; Akari Yamauchi; N. M.-Mofiz Uddin Khan; Tatsuya Arai; Mahatabuddin S; Ai Miura; Hidemasa Kondo
Biomolecules, 10, 3, 423, 423, {MDPI} {AG}, Mar. 2020, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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.

Expression of Ice-Binding Proteins in Caenorhabditis elegans Improves the Survival Rate upon Cold Shock and during Freezing
Masahiro Kuramochi; Chiaki Takanashi; Akari Yamauchi; Motomichi Doi; Kazuhiro Mio; Sakae Tsuda; Yuji C. Sasaki
Scientific Reports, 9, 1, 6246, 6246, Springer Science and Business Media {LLC}, Dec. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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.

Presence of a basic secretory protein in xylem sap and shoots of poplar in winter and its physicochemical activities against winter environmental conditions
Sakae Tsuda
Journal of Plant Research, 09 Jul. 2019, [Peer-reviewed]
English, Scientific journal

Calcium-Binding Generates the Semi-Clathrate Waters on a Type II Antifreeze Protein to Adsorb onto an Ice Crystal Surface
Tatsuya Arai; Yoshiyuki Nishimiya; Yasushi Ohyama; Hidemasa Kondo; Sakae Tsuda
Biomolecules, 9, 5, 162, 162, {MDPI} {AG}, Apr. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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 Ca2+-dependent species of AFP, however, it has not been clarified how 1 mol of Ca2+-binding is related with the hydration and the ice-binding ability. Here we determined the X-ray crystal structure of a Ca2+-dependent AFP (jsAFP) from Japanese smelt, Hypomesus nipponensis, in both Ca2+-bound and -free states. Their overall structures were closely similar (Root mean square deviation (RMSD) of Cα = 0.31 Å), while they exhibited a significant difference around their Ca2+-binding site. Firstly, the side-chains of four of the five Ca2+-binding residues (Q92, D94 E99, D113, and D114) were oriented to be suitable for ice binding only in the Ca2+-bound state. Second, a Ca2+-binding loop consisting of a segment D94–E99 becomes less flexible by the Ca2+-binding. Third, the Ca2+-binding induces a generation of ice-like clathrate waters around the Ca2+-binding site, which show a perfect position-match to the waters constructing the first prism plane of a single ice crystal. These results suggest that generation of ice-like clathrate waters induced by Ca2+-binding enables the ice-binding of this protein.

Freeze Tolerance in Sculpins (Pisces; Cottoidea) Inhabiting North Pacific and Arctic Oceans: Antifreeze Activity and Gene Sequences of the Antifreeze Protein
Aya Yamazaki; Yoshiyuki Nishimiya; Sakae Tsuda; Koji Togashi; Hiroyuki Munehara
Biomolecules, 9, 4, Apr. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal

Point Mutation of the Ice-Binding Site in Antifreeze Protein Modify the Cold Tolerance in Caenorhabditis Elegans
Kuramochi, Masahiro; Takanashi, Chiaki; Yamauchi, Akari; Doi, Motomichi; Mio, Kazuhiro; Tsuda, Sakae; Sasaki, Yuji C.
Biophysical Journal, 116, 3, 551A, 2019, [Peer-reviewed]
Scientific journal

Ice-binding proteins from the fungus Antarctomyces psychrotrophicus possibly originate from two different bacteria through horizontal gene transfer
Arai, Tatsuya; Fukami, Daichi; Hoshino, Tamotsu; Kondo, Hidemasa; Tsuda, Sakae
Febs Journal, 286, 5, 946, 962, 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal

Ice recrystallization is strongly inhibited when antifreeze proteins bind to multiple ice planes
Rahman, Anika T.; Arai, Tatsuya; Yamauchi, Akari; Miura, Ai; Kondo, Hidemasa; Ohyama, Yasushi; Tsuda, Sakae
Scientific Reports, 9, 1, 2212, 2212, 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal

Unexpected rise of glass transition temperature of ice crystallized from antifreeze protein solution.
Azuma, N; Miyazaki Y; Nakano, M; Tsuda, S
J. Phys. Chem. Lett., 9, 16, 4512, 4515, Aug. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal

Polypentagonal ice-like water networks emerge solely in an activity-improved variant of ice-binding protein
Mahatabuddin S; Fukami, D; Arai,T; Nishimiya, Y; Shimizu, R; Shibazaki, C; Kondo, H; Adachi, M; Tsuda, S
Proc. Natl. Acad. Sci. USA, 115, 21, 5456, 5461, Mar. 2018, [Peer-reviewed]
English, Scientific journal

Unexpected Rise of Glass Transition Temperature of Ice Crystallized from Antifreeze Protein Solution.
Azuma, Nobuaki; Miyazaki, Yuji; Nakano, Motohiro; Tsuda, Sakae
The journal of physical chemistry letters, 9, 16, 4512, 4515, 2018, [Peer-reviewed]
Scientific journal

Polypentagonal ice-like water networks emerge solely in an activity-improved variant of ice-binding protein
Mahatabuddin, Sheikh; Fukami, Daichi; Arai, Tatsuya; Nishimiya, Yoshiyuki; Shimizu, Rumi; Shibazaki, Chie; Kondo, Hidemasa; Adachi, Motoyasu; Tsuda, Sakae
Proceedings of the National Academy of Sciences of the United States of America, 115, 21, 5456, 5461, 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal

Functional Analysis of Antifreeze Proteins for Cold Tolerance Behavior and X-Ray Single Molecule Observations in C-elegans
Kuramochi, Masahiro; Takanashi, Chiaki; Sekiguchi, Hiroshi; Doi, Motomichi; Tsuda, Sakae; Sasaki, Yuji C.
Biophysical Journal, 114, 3, 65A, 2018, [Peer-reviewed]
Scientific journal

Applications of Antifreeze Proteins
Sakae Tsuda
Survival Strategies in Extreme Cold and Desiccation, 2018, [Peer-reviewed], [International Magazine]
English

Gene expression of antifreeze protein in relation to historical distributions of Myoxocephalus fish species
Yamazaki, A.; Nishimiya, Y.; Tsuda, S.; Togashi, K.; Munehara, H.
Marine Biology, 165, 11, 2018, [Peer-reviewed]
Scientific journal

Applications of Antifreeze Proteins: Practical Use of the Quality Products from Japanese Fishes
Mahatabuddin, Sheikh; Tsuda, Sakae
Survival Strategies in Extreme Cold and Desiccation: Adaptation Mechanisms and Their Applications, 1081, 321, 327, 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal

タンパク質動態からの不凍機能X線１分子解析
岡田 璃生; 新井 達也; 津田 栄; 佐々木 裕次
低温生物工学会誌, 2017, [Peer-reviewed]
Japanese, Scientific journal

Concentration-dependent oligomerization of an alpha-helical antifreeze polypeptide makes it hyperactive
Mahatabuddin, Sheikh; Hanada, Yuichi; Nishimiya, Yoshiyuki; Miura, Ai; Kondo, Hidemasa; Davies, Peter L.; Tsuda, Sakae
Scientific Reports, 7, 42501, 42501, 2017, [Peer-reviewed], [International Magazine]
English, Scientific journal

Total Synthesis of O-GalNAcylated Antifreeze Glycoprotein using the Switchable Reactivity of Peptidyl-N-pivaloylguanidine
Orii, Ryo; Sakamoto, Noriko; Fukami, Daichi; Tsuda, Sakae; Izumi, Masayuki; Kajihara, Yasuhiro; Okamoto, Ryo
Chemistry-a European Journal, 23, 39, 9253, 9257, 2017, [Peer-reviewed], [International Magazine]
English, Scientific journal

Temperature Dynamics of Single Molecular Antifreeze Protein
Okada, Rio; Arai, Tatsuya; Fukami, Daichi; Matsushita, Yuhuku; Chang, Jae-won; Sekiguchi, Hiroshi; Ohta, Noboru; Mori, Tadashi; Nishijima, Masaki; Miyazawa, Keisuke; Fukuma, Takeshi; Ikezaki, Keigo; Tsuda, Sakae; Sasaki, Yuji C.
Biophysical Journal, 112, 3, 323A, 323A, 2017, [Peer-reviewed]
English, Scientific journal

Hydrophobic ice-binding sites confer hyperactivity of an antifreeze protein from a snow mold fungus
Jing Cheng; Yuichi Hanada; Ai Miura; Sakae Tsuda; Hidemasa Kondo
BIOCHEMICAL JOURNAL, 473, 4011, 4026, Nov. 2016, [Peer-reviewed]
English, Scientific journal

Hydrophobic ice-binding sites confer hyperactivity of an antifreeze protein from a snow mold fungus
Cheng, Jing; Hanada, Yuichi; Miura, Ai; Tsuda, Sakae; Kondo, Hidemasa
Biochemical Journal, 473, 21, 4011, 4026, 2016, [Peer-reviewed]
English, Scientific journal

Critical Ice Shaping Concentration (CISC): A New Parameter to Evaluate the Activity of Antifreeze Proteins.
Sakae Tsuda
Cryobiology and Cryotechnology, 62, 2, 95, 103, 低温生物工学会, 2016, [Peer-reviewed]
English, Scientific journal

Chemical synthesis of homogeneous antifreeze glycoprotein having uniform GalNAc modification and its antifreeze activity
Okamoto, Ryo; Orii, Ryo; Izumi, Masayuki; Fukami, Daichi; Tsuda, Sakae; Kajihara, Yasuhiro
Protein Science, 25, 65, 65, 2016, [Peer-reviewed]
English, Scientific journal

Observation of Inhibitory Effect of Antifreeze Protein on Progressive Freeze-Concentration
Arai T; Cheng J; Mahatabuddin S; Kondo H; Tsuda S
Cryobiology and Cryotechnology, 61, 2, 121, 124, Japanese Society for Cryobiology and Cryotechnology, Oct. 2015, [Peer-reviewed]
Japanese, Symposium, A water solution placed in a freezer changes into a polycrystalline state of ice in keeping with the exclusion of the solute. This exclusion is attributed to growth of the ice crystals, as the growth only consumes water molecules, leading to concentration of the excluded solute into a lastly frozen area. This phenomenon was named "progressive freeze-concentration (PFC)", which is sometimes the problem for quality preservation of water-containing materials, such as foods and tissues. Here we examined whether antifreeze protein (AFP) works to prevent PFC, since AFP can bind specifically to ice crystals to inhibit their growth. For this purpose, we froze the AFP solution containing red-colored ink in a house-made PFC device consisting of a glass vessel sandwiched by heat insulating materials. The ink was concentrated near the bottom of a placoid ice prepared in the PFC device without AFP, while it was dispersed with AFP. A level of dispersion was dependent on the AFP concentration and ice crystal shape. These results suggest that AFP possesses inhibitory effect on PFC, which was presumably correlated with the known abilities of AFP, such as ice-shaping, thermal hysteresis, and ice recrystallization inhibition.

Prolonging hypothermic storage (4oC) of bovine embryos with fish antifreeze protein.
Sakae Tsuda
J. Reprod. Dev., 61, 1, 1, 6, The Japanese Society of Animal Reproduction (JSAR), 30 Jan. 2015, [Peer-reviewed], [Domestic magazines]
English, Scientific journal

Prolonging hypothermic storage (4 C) of bovine embryos with fish antifreeze protein
Ideta, Atsushi; Aoyagi, Yoshito; Tsuchiya, Kanami; Nakamura, Yuuki; Hayama, Kou; Shirasawa, Atsushi; Sakaguchi, Kenichiro; Tominaga, Naomi; Nishimiya, Yoshiyuki; Tsuda, Sakae
Journal of Reproduction and Development, 61, 1, 1, 6, 2015, [Peer-reviewed]
English, Scientific journal

3P043 Mutagenesis study of an antifreeze protein isoform from a snow-mold fungus, Typhula ishikariensis(01B. Protein: Structure & Function,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))
Cheng Jing; Hanada Yuichi; Kondo Hidemasa; Tsuda Sakae
Seibutsu Butsuri, 54, 1, S256, The Biophysical Society of Japan General Incorporated Association, 2014
English

2P027 Function and Structure of Antifreeze Protein from Ascomycete(01B. Protein: Structure & Function,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))
Fukami Daichi; Hanada Yuichi; Cheng Jing; Tsuda Sakae; Kondo Hidemasa
Seibutsu Butsuri, 54, 1, S199, The Biophysical Society of Japan General Incorporated Association, 2014
English

1P031 Functional analysis of calcite-binding site of lithostathine(Protein: Structure & Function,Poster,The 52th Annual Meeting of the Biophysical Society of Japan(BSJ2014))
Maho Nara; Yuichi Hanada; Hidemasa Kondo; Sakae Tsuda
Seibutsu Butsuri, 54, 1, S146, The Biophysical Society of Japan General Incorporated Association, 2014
English

1P045 Functional Analysis of a New Type I Antifreeze Protein from Barfin plaice, Liposetta pinnifasciata(01B. Protein : Structure & Function,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))
Mahatabuddin Sheikh; Ishihara Kazunari; Hanada Yuichi; Miura Ai; Kondo Hidemesa; Tsuda Sakae
Seibutsu Butsuri, 54, 1, S148, The Biophysical Society of Japan General Incorporated Association, 2014
English

Annealing condition influences thermal hysteresis of fungal type ice-binding proteins
Xiao, Nan; Hanada, Yuichi; Seki, Haruhiko; Kondo, Hidemasa; Tsuda, Sakae; Hoshino, Tamotsu
Cryobiology, 68, 1, 159, 161, 2014, [Peer-reviewed]
English, Scientific journal

Identification of a Novel LEA Protein Involved in Freezing Tolerance in Wheat
Sasaki, Kentaro; Christov, Nikolai Kirilov; Tsuda, Sakae; Imai, Ryozo
Plant and Cell Physiology, 55, 1, 136, 147, 2014, [Peer-reviewed]
English, Scientific journal

Antifreeze protein activity in Arctic cryoconite bacteria
Singh, Purnima; Hanada, Yuichi; Singh, Shiv Mohan; Tsuda, Sakae
Fems Microbiology Letters, 351, 1, 14, 22, 2014, [Peer-reviewed]
English, Scientific journal

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity
Basu, Koli; Garnham, Christopher P.; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter
Jove-Journal of Visualized Experiments, 83, e51185, 2014, [Peer-reviewed]
English, Scientific journal

Hyperactive antifreeze protein from an Antarctic sea ice bacterium Colwellia sp has a compound ice-binding site without repetitive sequences
Hanada, Yuichi; Nishimiya, Yoshiyuki; Miura, Ai; Tsuda, Sakae; Kondo, Hidemasa
Febs Journal, 281, 16, 3576, 3590, 2014, [Peer-reviewed]
English, Scientific journal

2P029 Analysis of structure and function of a new type I antifreeze protein from a Japanese fish, Barfin Plaice(01B. Protein: Structure & Function,Poster)
Ishihara Kazunari; Hanada Yuichi; Kondo Hidemasa; Miura Ai; Tsuda Sakae
Seibutsu Butsuri, 53, 1, S163, The Biophysical Society of Japan General Incorporated Association, 2013
Japanese

1P051 Identification of calcite-binding site of lithostathine(01C. Protein:Property,Poster)
Togashi Seiya; Hanada Yuichi; Nara Maho; Tsuda Sakae
Seibutsu Butsuri, 53, 1, S114, The Biophysical Society of Japan General Incorporated Association, 2013
English

Fabrication of Highly Porous Alumina Prepared by Gelation Freezing Route with Antifreeze Protein
Fukushima, Manabu; Tsuda, Sakae; Yoshizawa, Yu-ichi
Journal of the American Ceramic Society, 96, 4, 1029, 1031, 2013, [Peer-reviewed]
English, Scientific journal

NMR structure note: a defective isoform and its activity-improved variant of a type III antifreeze protein from Zoarces elongates Kner
Kumeta, Hiroyuki; Ogura, Kenji; Nishimiya, Yoshiyuki; Miura, Ai; Inagaki, Fuyuhiko; Tsuda, Sakae
Journal of Biomolecular Nmr, 55, 2, 225, 230, 2013, [Peer-reviewed]
English, Scientific journal

Microwave-Assisted Solid-Phase Synthesis of Antifreeze Glycopeptides
Izumi, Ryukou; Matsushita, Takahiko; Fujitani, Naoki; Naruchi, Kentaro; Shimizu, Hiroki; Tsuda, Sakae; Hinou, Hiroshi; Nishimura, Shin-Ichiro
Chemistry-a European Journal, 19, 12, 3913, 3920, 2013, [Peer-reviewed]
English, Scientific journal

Antifreeze Protein Prolongs the Life-Time of Insulinoma Cells during Hypothermic Preservation
Kamijima, Tatsuro; Sakashita, Mami; Miura, Ai; Nishimiya, Yoshiyuki; Tsuda, Sakae
Plos One, 8, 9, e73643, 2013, [Peer-reviewed]
English, Scientific journal

Antifreeze proteins from Japanese organisms: Functional analyses for their general use.
Sakae Tsuda
Cryobiology, 2013, [Peer-reviewed]
Scientific journal

Dependence of freeze-concentration inhibition on antifreeze protein.
Sakae Tsuda
Low Temperature Science, 71, 91, 96, 北海道大学低温科学研究所 = Institute of Low Temperature Science, Hokkaido University, 2013, [Peer-reviewed]
English, Scientific journal, The ability for freeze-concentration inhibition (FCI) was examined for type I antifreeze protein (AFPI)and antifreeze glycoprotein (AFGP)through the observation of the condensation of red-colored ink in an ice block for various concentrations of the proteins. The thermal hysteresis (TH), which indicates the ice growth inhibition strength, and ice-shaping ability were also examined for the two samples. The amount of AFPI and AFGP necessary for FCI was determined to be 0.1 mg/ml and 0.25 mg/ml, respectively. There was no significant difference in the TH of both samples. AFPI and AFGP shaped the ice crystals into hexagonal trapezohedrons and hexagonal bipyramids, respectively, where the former was thinner than the latter. These results suggest that a principle determinant of FCI is the ice-shaping ability, rather than the ice growth inhibition of AFPs.

Artificial dormancy of bovine embryos for a maximum of 7 days using a simple medium.
Sakae Tsuda
Reproduction, Fertility and Development, 2013, [Peer-reviewed]
Scientific journal

Cold adaptation of fungi obtained from soil and lake sediment in the Skarvsnes ice-free area, Antarctica
Tsuji, Masaharu; Fujiu, Seiichi; Xiao, Nan; Hanada, Yuichi; Kudoh, Sakae; Kondo, Hidemasa; Tsuda, Sakae; Hoshino, Tamotsu
Fems Microbiology Letters, 346, 2, 121, 130, 2013, [Peer-reviewed]
English, Scientific journal

A simple medium enables bovine embryos to be held for seven days at 4 degrees C
Ideta, Atsushi; Aoyagi, Yoshito; Tsuchiya, Kanami; Kamijima, Tatsuro; Nishimiya, Yoshiyuki; Tsuda, Sakae
Scientific Reports, 3, 2013, [Peer-reviewed]
English, Scientific journal

Engineering a naturally inactive isoform of type III antifreeze protein into one that can stop the growth of ice
Garnham, Christopher P.; Nishimiya, Yoshiyuki; Tsuda, Sakae; Davies, Peter L.
Febs Letters, 586, 21, 3876, 3881, 2012, [Peer-reviewed]
English, Scientific journal

Ice-binding site of snow mold fungus antifreeze protein deviates from structural regularity and high conservation
Kondo, Hidemasa; Hanada, Yuichi; Sugimoto, Hiroshi; Hoshino, Tamotsu; Garnham, Christopher P.; Davies, Peter L.; Tsuda, Sakae
Proceedings of the National Academy of Sciences of the United States of America, 109, 24, 9360, 9365, 2012, [Peer-reviewed]
English, Scientific journal

Short term storage of Mouse epididymal spermatozoa by Antifreeze protein addition at cold temperature
Sakae Tsuda
Memoirs of Institute of Advanced Technology, Kinki University, 16, 51, 58, 近畿大学先端技術総合研究所, 2011, [Peer-reviewed]
Japanese, Scientific journal, [要約] 本研究では成熟B6D2F1マウスの精巣上体尾部由来精子を用いた冷蔵保存を行い、その後、冷蔵精子の評価および体外受精操作について検討した。精巣上体尾部精子を、各種保存液50μL（R18S3、R18、R18+AFP III 1mg/mL）中で4℃にて冷蔵保存した。加温後のそれらの運動性は、精子運動解析装置（IVOS：ニッコー・ハンセン（株））を用いて、保存期間１～ 5日を評価し、R18+AFP III の保存液では、5日目においても52％の運動性を保持していた。精子細胞膜損傷はLIVE/DEAD sperm viability kit を用いて評価し、R18S3 とR18+AFP IIIは5日目の細胞膜完全性の保持率は共に48％であった。受精能の確認では、常法により採卵し冷蔵精子と受精させた。また、一部の卵子卵丘複合体は透明帯穿孔処理を行い、冷蔵精子と受精させた。透明帯穿孔処理後の卵子との体外受精結果は冷蔵保存後3日目の冷蔵精子において37％（46/126）の受精率であった。冷蔵保存後5日目においても2％（4/162）の受精率を確認できた。 以上の結果より、マウス精子の冷蔵保存は低率ではあるが受精能を保持し、冷蔵保存が可能であることが示された。 [Abstract] The present study was performed to examine the storage of Slc : B6D2F1 mouse epididymal sperm at cold temperature. Afterwards, the evaluation and the in vitro fertilization operation of the refrigeration sperm were examined. The cauda epididymal spermatozoa from adult males were preserved with various solutions（ R18S3, R18, R18+AFP III 1mg/mL） at 4℃ . Sperm motility was assessed by Integrated Visual Optical System（ IVOS）. R18+AFP III maintained the movement of 52％ during 5 days. The integrity of the sperm membrane was determined using Live/Dead sperm viability kit. The percentage of membraneintact spermatozoa that stored in R18S3 and R18+AFP III was 48％ at day 5. Super ovulated eggs were fertilized in vitro with various refrigeration sperm. Moreover a part of eggs was treatment of zona drilled, and it was fertilized with the refrigeration sperm. In vitro fertilized rate of zona drilled oocyte withrefrigeration sperm of 3 days after preserved. The fertilization of the refrigeration sperm of the 5 day of preservation was 2％（ 4/162）. In conclusion were indicate that mouse epididymal spermatozoa stored at 4℃ maintain fertilization ability, although low rate, and can store at cold temperature.

Synthetic Study and Structural Analysis of the Antifreeze Agent Xylomannan from Upis ceramboides
Ishiwata, Akihiro; Sakurai, Ayaka; Nishimiya, Yoshiyuki; Tsuda, Sakae; Ito, Yukishige
Journal of the American Chemical Society, 133, 48, 19524, 19535, 2011, [Peer-reviewed]
English, Scientific journal

Antifreeze activities of fungi and sea ice diatoms from Antarcica.
Sakae Tsuda
2010, [Peer-reviewed]
Scientific journal

8. Crystal structure of Ca2+-dependent type II antifreeze protein from Japanese smelt
Kondo *Hidemasa; Nishimiya Yoshiyuki; Noro Natsuko; Takamichi Manabu; Yasui Masanori; Miura Ai; Tsuda Sakae
Cryobiology, 59, 3, 372, Elsevier BV, 2009, [Peer-reviewed]

New technologies developed for the practical use of antifreeze protein.
Sakae Tsuda
Cryobiology, 2009, [Peer-reviewed]
Scientific journal

Mutational and structural analysis of Ca2+-independent type II antifreeze protein from longsnout poacher.
Sakae Tsuda
Cryobiology, 2009, [Peer-reviewed]
Scientific journal

Cooperative ice growth inhibition of the isoforms of type III antifreeze protein
Sakae Tsuda
Cryobiology, 2009, [Peer-reviewed]
Scientific journal

Mass preparation and technological development of antifreeze protein
NISHIMIYA Yoshiyuki; MIE Yasuhiro; Hirano Yu; KONDO Hidemasa; MIURA Ai; TSUDA Sakae
Synthesiology, 1, 1, 7, 14, National Institute of Advanced Industrial Science and Technology, 2008
Japanese, Antifreeze protein has been known as an extraordinary biomolecule having the potential to both bind ice and preserve cell structure; the molecule was isolated from the blood serum of Arctic and Antarctic fishes. We have recently found that Japanese food fishes also contain an antifreeze protein, and have established a method of collecting the protein from fish muscle. The collected antifreeze protein is a mixture of many isoforms, which were found to be more active than any single isoform. Mass preparation of antifreeze protein is currently in progress to allow researchers in different fields to develop a variety of new technologies utilizing the functions of this protein.

COMP 207-Probing the origin of the hyperactivity of the two-domain type III antifreeze protein RD3
Holland, Nolan B.; Can, Oezge; Tsuda, Sakae; Soennichsen, Frank D.
Abstracts of Papers of the American Chemical Society, 234, 2007, [Peer-reviewed]
English, Scientific journal

Antifreeze protein from Japanese fish: An epoch in biotechnology
Tsuda, Sakae
Abstracts of Papers of the American Chemical Society, 234, 2007, [Peer-reviewed]
English, Scientific journal

Antifreeze Protein from Japanese Fish
TSUDA Sakae; MIURA Ai; NISHIMIYA Yoshiyuki
Kobunshi, 55, 7, 494, 495, The Society of Polymer Science, Japan, 01 Jul. 2006
Japanese

Antifreeze Glycoprotein: Elucidation of Antifreeze Mechanism
Masakazu Hachisu; Kohta Igarashi; Sakae Tsuda; Shuhei Koshida; Kenji Monde; Shin-Ichiro Nishimura
Polymer Preprints, Japan, 54, 1, 2168, 2005
Japanese, International conference proceedings

1P016 X-ray crystal structure analysis of type II antifreeze protein from Longsnout poacher
Nishimiya Y.; Sugimoto H.; Sato R.; Noro N.; Kondo H.; Takamichi M.; Miura A.; Tsuda S.
Seibutsu Butsuri, 45, S35, The Biophysical Society of Japan General Incorporated Association, 2005
Japanese

1P111 Characterization of a fish antifreeze protein from Sebastes steindachneri Hilgendorf
Yoshida T.; Tanaka S.; Nishimiya Y.; Miura K.; Miura A.; Tsuda S.
Seibutsu Butsuri, 45, S59, The Biophysical Society of Japan General Incorporated Association, 2005
Japanese

1P058 Functional and three-dimensional structure analysis of the Ca^<2+>-dependent type II antifreeze protein from Japanese smelt
Yasui M.; Kondo H.; Nishimiya Y.; Noro N.; Miura K.; Takamichi M.; Miura A.; Tsuda S.
Seibutsu Butsuri, 45, S46, The Biophysical Society of Japan General Incorporated Association, 2005
Japanese

2P019 X-ray crystal structure analysis of xyloglucan-specific exo- and endo-glucanases belonging to glycoside hydrolase family 74
Kondo H.; Yaoi K.; Noro N.; Hiyoshi A.; Sugimoto H.; Suzuki M.; Tsuda S.; Mitsuishi Y.
Seibutsu Butsuri, 45, S124, The Biophysical Society of Japan General Incorporated Association, 2005
Japanese

1P109 Screening research for antifreeze proteins using cold-finger device
Miura K.; Hoshino T.; Tsuda S.
Seibutsu Butsuri, 45, 0, S59, The Biophysical Society of Japan General Incorporated Association, 2005, [Peer-reviewed]
Japanese

1P110 Activity of recombinant antifreeze protein from basidiomycetes, Typhula ishikariensis
Suzuki K.; Fujiwara M.; Hoshino T.; Ohgiya S.; Tsuda S.
Seibutsu Butsuri, 45, 0, S59, The Biophysical Society of Japan General Incorporated Association, 2005, [Peer-reviewed]
Japanese

1P070 Detection of the ice-binding manner of antifreeze proteins by using Green Fluorescent Protein(GFP) labeling technique
Miura K.; Tsuda S.
Seibutsu Butsuri, 44, S47, The Biophysical Society of Japan General Incorporated Association, 2004
Japanese

1P071 Observation of the interaction manner between antifreeze proteins and ice crystal by using fluorescence labeling method
Yoshida T.; Miura K.; Tanaka S.; Tsuda S.
Seibutsu Butsuri, 44, S47, The Biophysical Society of Japan General Incorporated Association, 2004
Japanese

Antifreeze Protein
TANAKA Shotaro; KOBASHIGAWA Yoshihiro; MIURA Kazunori; NISHIMIYA Yoshiyuki; MIURA Ai; TSUDA Sakae
Seibutsu Butsuri, 43, 3, 130, 135, The Biophysical Society of Japan General Incorporated Association, 25 May 2003
Japanese, In 1969, the first antifreeze protein (AFP) was discovered from the blood plasma of Antarctic Nototheniids. In the past thirty years, different types of AFP have been found in many life forms that exhibit freezing tolerance, such as bacteria, fungi, plants, insects, and vertebrates. These discoveries have evoked us many questions regarding to the antifreeze mechanism and its biological significance for preventing their tissues from freezing damage. At present, ice physicist, biologist, chemist, biochemist, molecular biologist, physiologist, and NMR and X-ray structural biologists are subjecting AFP, which greatly improves our understandings about AFP and accelerates its applicability to various cryo-industries. In the present review we will describe an updated biophysical aspects of AFP to highlight the interests of this research field.

X-ray crystal structure analysis of Oligoxyloglucan reducing end-specific cellobiohydrolase
Kondo H.; Yaoi K.; Suzuki M.; Noro N.; Tsuda S.; Mitsuishi Y.
Seibutsu Butsuri, 43, S36, The Biophysical Society of Japan General Incorporated Association, 2003
Japanese

Antifreeze proteins from snow mold fungi
Hoshino, T; Kiriaki, M; Ohgiya, S; Fujiwara, M; Kondo, H; Nishimiya, Y; Yumoto, I; Tsuda, S
Canadian Journal of Botany-Revue Canadienne De Botanique, 81, 12, 1175, 1181, 2003, [Peer-reviewed]
English, Scientific journal

Letter to the Editor: Assignments of H-1, C-13, and N-15 resonances of human lysozyme at 4 degrees C
Kumeta, H; Kobashigawa, Y; Miura, K; Nishimiya, Y; Oka, C; Nemoto, N; Miura, A; Nitta, K; Tsuda, S
Journal of Biomolecular Nmr, 22, 2, 183, 184, 2002, [Peer-reviewed]
English, Scientific journal

NMR STRUCTURE OF INTRAMOLECULAR DIMER ANTIFREEZE PROTEIN RD3, 40 SA STRUCTURES
Sakae Tsuda
2001, [Peer-reviewed]

Letter to the Editor: Assignment of H-1, C-13, and 15N resonances of canine milk lysozyme
Kobashigawa, Y; Miura, K; Demura, M; Nemoto, N; Koshiba, T; Nitta, K; Tsuda, S
Journal of Biomolecular Nmr, 19, 4, 387, 388, 2001, [Peer-reviewed]
English, Scientific journal

p-COUMARIC ACID FROM WHEAT INACTIVATES ICE-NUCLEATING ACTIVITY BY ICE-NUCLEATING BACTERIUM
Sakae Tsuda
Plant and Cell Phisiology, 41, s50, Japanese Society of Plant Physiologists, 2000, [Peer-reviewed]
English, Scientific journal

Synthetic studies on specific mucins: Antifreeze glygoprotein.
Tachibana, Y; Tsuda, T; Matsubara, N; Tsuda, S; Nishimura, SI
Abstracts of Papers of the American Chemical Society, 219, U244, U244, 2000, [Peer-reviewed]
English, Scientific journal

Assignments of H-1, C-13, and N-15 resonances of intramolecular dimer antifreeze protein RD3
Miura, K; Ohgiya, S; Hoshino, T; Nemoto, N; Nitta, K; Tsuda, S
Journal of Biomolecular Nmr, 16, 3, 273, 274, 2000, [Peer-reviewed]
English, Scientific journal

Response of Plants to Environmental Stress.
Sakae Tsuda
Journal of plant research, 1999, [Peer-reviewed]
Scientific journal

Flora of the Russian Arctic Flora of the Russian Arctic.
Sakae Tsuda
Journal of Plant Research, 1999, [Peer-reviewed]
Scientific journal

Flexing Muscle With Just One Amino Acid.
Sakae Tsuda
Science, 271, 5245, 31, 31, 1996, [Peer-reviewed]
English, Scientific journal

H-1-NMR STUDY OF RABBIT SKELETAL-MUSCLE TROPONIN-C - CA2+-DEPENDENT INTERACTION WITH MASTOPARAN
TSUDA, S; HIKICHI, K
Biochimica Et Biophysica Acta, 1121, 1-2, 213, 220, 1992, [Peer-reviewed]
English, Scientific journal

H-1-NMR STUDY OF CA2+-DEPENDENT AND MG2+-DEPENDENT INTERACTION BETWEEN TROPONIN-C AND TROPONIN-I INHIBITORY PEPTIDE (96-116)
TSUDA, S; AIMOTO, S; HIKICHI, K
Journal of Biochemistry, 112, 5, 665, 670, 1992, [Peer-reviewed]
English, Scientific journal

1H-NMR Study on Amide Proton Exchange of Calmodulin-Mastoparan Complex.
Sakae Tsuda
J. Biochem., 1992, [Peer-reviewed]
Scientific journal

NUCLEOSIDES AND NUCLEOTIDES .46. CHEMICAL MODIFICATION OF GUANINE RESIDUES OF MOUSE 5S RIBOSOMAL-RNA WITH KETHOXAL
MIURA, K; TSUDA, S; UEDA, T; HARADA, F; KATO, N
Biochimica Et Biophysica Acta, 739, 3, 281, 285, 1983, [Peer-reviewed]
English, Scientific journal

NUCLEOSIDES AND NUCLEOTIDES .43. CHEMICAL MODIFICATION OF CYTOSINE RESIDUES OF MOUSE 5-S-RIBOSOMAL RNA WITH HYDROGEN-SULFIDE
MIURA, K; TSUDA, S; IWANO, T; UEDA, T; HARADA, F; KATO, N
Biochimica Et Biophysica Acta, 739, 2, 181, 189, 1983, [Peer-reviewed]
English, Scientific journal

CHEMICAL MODIFICATION OF CYTOSINE RESIDUES OF U-6 SNRNA WITH HYDROGEN-SULFIDE (NUCLEOSIDES AND NUCLEOTIDES .49.
MIURA, K; TSUDA, S; HARADA, F; UEDA, T
Nucleic Acids Research, 11, 17, 5893, 5901, 1983, [Peer-reviewed]
English, Scientific journal

Ice-binding proteins improve the survival rate of the nematode Caenorhabditis elegans at freezing and cold environments
倉持昌弘; 新井達也; 三尾和弘; 津田栄; 佐々木裕次, アグリバイオ, 6, 14, 2023

Ice-Binding Proteins in maintaining biological activity in frozen and sub-zero environments
倉持昌弘; 新井達也; 三尾和弘; 津田栄; 佐々木裕次, アグリバイオ, 7, 5, 2023

回折X線明滅法による不凍タンパク質分子表面の凍結水の動態観察
新井達也; 新井達也; 楊越; 津田栄; 三尾和弘; 佐々木裕次; 佐々木裕次, 日本蛋白質科学会年会プログラム・要旨集, 23rd (CD-ROM), 2023

Acquiring freezing and cold tolerance: The role of structure-specific functions in Ice-Binding Protein molecules
倉持昌弘; 新井達也; 三尾和弘; 津田栄; 佐々木裕次, アグリバイオ, 7, 10, 2023

Cryopreservation effects of C. elegans expressing ice-binding proteins
下谷祐平; 新井達也; 三尾和弘; 津田栄; 佐々木裕次; 倉持昌弘, 日本分子生物学会年会プログラム・要旨集(Web), 45th, 2022

化学合成により調製した不凍糖タンパク質を用いたO-GalNAc残基の機能解明
折井亮; 深見大地; 深見大地; 津田栄; 津田栄; 真木勇太; 和泉雅之; 梶原康宏; 岡本亮, 日本化学会春季年会講演予稿集(CD-ROM), 99th, 2019

カジカ科魚類の寒冷適応
山崎彩; 津田栄; 河田雅圭; 宗原弘幸, 日本魚類学会年会講演要旨, 50th, 148, 14 Sep. 2017
Japanese

氷の結晶成長を止める新しい不凍タンパク質の発見－食品の品質、細胞の生命力を維持できる凍結技術の実現に向けて
近藤 英昌; 津田 栄, 化学, 72, 8, 49, 52, Jul. 2017, [Invited]
化学同人, Japanese, Introduction commerce magazine

ギスカジカ属4種の不凍タンパク質アイソフォーム多型と不凍活性
山崎彩; 津田栄; 西宮佳志; 宗原弘幸, 日本進化学会大会プログラム・講演要旨集(Web), 17th, ROMBUNNO.P‐73 (WEB ONLY), 20 Aug. 2015
Japanese

北太平洋および北極海に生息するカジカ科魚類の不凍タンパク質活性
山崎彩; 花田祐一; 津田栄; 宗原弘幸, 日本魚類学会年会講演要旨, 47th, 74, 14 Nov. 2014
Japanese

Analysis of Cell-Preservation Function of Fish Antifreeze Protein
石原 和成; 三浦 愛; 津田 栄, Bio industry, 31, 6, 61, 66, Jun. 2014
シーエムシー出版, Japanese

コムギの新規強親水性天然変性タンパク質WCI16は耐凍性を向上させる
佐々木健太郎; CHRISTOV Nikolai Kirilov; 津田栄; 今井亮三, 農研機構北海道農業研究センター成果情報(Web), 2014, 2014

キノコの不凍タンパク質の分子構造と不凍機能のメカニズム
近藤 英昌; 津田 栄, 化学と生物, 52, 1, 10, 12, Jan. 2014, [Invited]
Japanese, Introduction other

Functional Analysis of Antifreeze Protein from Ascomycete
FUKAMI Daichi; HANADA Yuichi; CHENG Jing; TSUDA Sakae; KONDO Hidemasa, Cryobiology and cryotechnology, 59, 2, 157, 160, 2013
Antifreeze protein (AFP) specifically binds to ice crystal surface and inhibits its growth. In order to elucidate ice-binding mechanism of fungal AFPs, we performed measurements of thermal hysteresis (TH) activity and a recently developed method called "Fluorescence-based Ice Plane Affinity (FIPA)" analysis against AFP identified from Antarctic ascomycete, Antarctomyces psychrotrophicus (AnpAFP). AnpAFP showed a maximum TH of 0.8℃ and led to an ice growth along with the c-axis, which were typical for AFP with moderate antifreeze activity. In addition, FIPA analysis showed that AnpAFP bound to only prism planes of an ice crystal. Such an ice-binding manner of AnpAFP is different from that of the other known microbial AFPs, since they can bind to both prism and basal planes., Japanese Society for Cryobiology and Cryotechnology, Japanese

Fungal Antifreeze Protein Consists of a Unique β-Solenoid Structure
Kondo H; Hanada Y; Tsuda S, Photon Factory Activity Report 2012 Highlights, 30A, 58, 59, 2013
English, Introduction research institution

Antifreeze proteins from Japanese fish
津田 栄, 極限環境生物学会誌 = Journal of Japanese Society for Extremophiles, 11, 2, 64, 69, Dec. 2012
[極限環境生物学会]学会事務局, Japanese

キノコの不凍タンパク質 強力な不凍機能をもつタンパク質の立体構造を決定
津田栄; 近藤英昌; デイビス ピーター; ガーナム クリストファー; 杉本宏; 花田祐一; 星野保, 産総研TODAY(Web), 12, 12, 13 (WEB ONLY), Dec. 2012
Japanese

Variation of Antifreeze Proteins and their Antifreeze Effect
KONDO Hidemasa; TSUDA Sakae, 冷凍, 86, 1005, 557, 561, 15 Jul. 2011
日本冷凍空調学会, Japanese

Technological Developments utilizing Antifreeze Protein
NISHIMIYA Yoshiyuki; TSUDA Sakae, 冷凍, 86, 1005, 551, 556, 15 Jul. 2011
日本冷凍空調学会, Japanese

不凍タンパク質の種類と凍結抑制効果
近藤 英昌; 津田 栄, 冷凍, 86, 7, 15, 19, Jul. 2011, [Invited]
Japanese, Introduction other

ナガガジ由来変異型不凍タンパク質の構造安定性と氷成長抑制活性の相関
清水瑠美; 松本富美子; 新井栄揮; 大原高志; 安達基泰; 玉田太郎; 黒木良太; 西宮佳志; 近藤英昌; 津田栄, 日本蛋白質科学会年会プログラム・要旨集, 11th, 2011

INFULUENCE OF A TYPE III ANTIFREEZE PROTEIN ON HIGH-PRESSURE ICE VI
SAKASHITA Mami; NISHIMIYA Yoshiyuki; KONDO Hidemasa; TSUDA Sakae; YAMAWAKI Hiroshi; FUJIHISA Hiroshi; GOTOH Yoshito, 高圧討論会講演集, 51, 154, 154, 11 Oct. 2010
Japanese

不凍タンパク質 機能と応用
NISHIMIYA Yoshiyuki; KONDO Hidemasa; SAKASHITA Mami; MIURA Ai; TSUDA Sakae, KAGAKU TO SEIBUTSU, 48, 6, 381, 388, Jun. 2010, [Invited]
不凍タンパク質は，過冷却状態の水溶液中に生成する氷結晶の表面に特異的に結合し，その成長を抑制する機能を有する生体物質であり，様々な低温適応生物から様々な性状のものが発見されている．どのようにしてこれらのタンパク質が氷結晶に結合するのか?，不凍タンパク質間にはどのような共通性があるのか?，不凍タンパク質の機能発現機構を理解するための基礎研究に加え，医学や畜産分野をはじめとしてその成果を新しい技術開発に利用しようと実用化研究も進められている．ここでは，不凍タンパク質の氷結晶成長抑制機構に関する最近の研究状況について紹介する．, Japan Science and Technology Agency (JST), Japanese, Introduction other

変異導入とX線結晶構造解析によるナガガジ由来不凍タンパク質の抗凍結機構の解析
大原高志; 安達基泰; 清水瑠美; 玉田太郎; 黒木良太; 西宮佳志; 近藤英昌; 津田栄, 日本蛋白質科学会年会プログラム・要旨集, 10th, 2010

変異導入と結晶構造解析によるナガガジ由来不凍タンパク質の抗凍結機構の解析
大原高志; 安達基泰; 清水瑠美; 栗原和男; 玉田太郎; 黒木良太; 西宮佳志; 近藤英昌; 津田栄, 日本結晶学会年会講演要旨集, 2010, 2010

コムギWCI16は耐凍性に関与する新しいクラスのLEAタンパク質である
佐々木健太郎; KIRILOV CHRISTOV Nikolai; 津田栄; 今井亮三, 日本植物生理学会年会要旨集, 51st, 2010

Comparison of functional properties of two fungal antifreeze proteins from Antarctomyces psychrotrophicus and Typhula ishikariensis
Xiao, Nan; Suzuki, Keita; Nishimiya, Yoshiyuki; Kondo, Hidemasa; Miura, Ai; Tsuda, Sakae; Hoshino, Tamotsu, Febs Journal, 277, 2, 394, 403, 2010, [Peer-reviewed]
English

Comparison of functional properties of two fungal antifreeze proteins from Antarctomyces psychrotrophicus and Typhula ishikariensis
Nan Xiao; Keita Suzuki; Yoshiyuki Nishimiya; Hidemasa Kondo; Ai Miura; Sakae Tsuda; Tamotsu Hoshino, FEBS JOURNAL, 277, 2, 394, 403, Jan. 2010
English

Crystal structure and enhanced activity of a cutinase-like enzyme from Cryptococcus sp strain S-2
Yuya Kodama; Kazuo Masaki; Hidemasa Kondo; Mamoru Suzuki; Sakae Tsuda; Tomohiro Nagura; Nobuhisa Shimba; Ei-ichiro Suzuki; Haruyuki Iefuji, PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS, 77, 3, 710, 717, Nov. 2009
English

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
Katsuro Yaoi; Hidemasa Kondo; Ayako Hiyoshi; Natsuko Noro; Hiroshi Sugimoto; Sakae Tsuda; Kentaro Miyazaki, FEBS JOURNAL, 276, 18, 5094, 5100, Sep. 2009
English

NMRによるリニア不凍糖タンパク質(AFGP)のコンフォメーション解析
泉龍孝; 藤谷直樹; 松下隆彦; 比能洋; 高道学; 西宮佳志; 津田栄; 西村紳一郎, 日本糖質学会年会要旨集, 29th, 85, 10 Aug. 2009
Japanese

Fully active QAE isoform confers thermal hysteresis activity on a defective SP isoform of type III antifreeze protein
Manabu Takamichi; Yoshiyuki Nishimiya; Ai Miura; Sakae Tsuda, FEBS JOURNAL, 276, 5, 1471, 1479, Mar. 2009
English

環状不凍糖タンパク質の合成および不凍活性
八須匡和; 高道学; 比能洋; 清水弘樹; 津田栄; 西村紳一郎; 西村紳一郎, 高分子学会北海道支部研究発表会講演要旨集, 43rd, 2009

新規不凍糖タンパク質誘導体の合成と性質
八須匡和; 比能洋; 高道学; 三浦信明; 清水弘樹; 津田栄; 西村紳一郎; 西村紳一郎, 日本糖質学会年会要旨集, 29th, 2009

Crystal structure and enhanced activity of a cutinase-like enzyme from Cryptococcus sp strain S-2
Kodama, Yuya; Masaki, Kazuo; Kondo, Hidemasa; Suzuki, Mamoru; Tsuda, Sakae; Nagura, Tomohiro; Shimba, Nobuhisa; Suzuki, Ei-ichiro; Iefuji, Haruyuki, Proteins-Structure Function and Bioinformatics, 77, 3, 710, 717, 2009, [Peer-reviewed]
English

One-pot synthesis of cyclic antifreeze glycopeptides
Hachisu, Masakazu; Hinou, Hiroshi; Takamichi, Manabu; Tsuda, Sakae; Koshidaa, Shuhei; Nishimura, Shin-Ichiro, Chemical Communications, 13, 1641, 1643, 2009, [Peer-reviewed]
English

Fully active QAE isoform confers thermal hysteresis activity on a defective SP isoform of type III antifreeze protein
Takamichi, Manabu; Nishimiya, Yoshiyuki; Miura, Ai; Tsuda, Sakae, Febs Journal, 276, 5, 1471, 1479, 2009, [Peer-reviewed]
English

Hypothermic preservation of cultured cells with using fish type III antifreeze protein from Noched-fin eelpout.
Warashina, A; Hirano, Y; Takamichi, M; Nishimiya, Y; Kondo, H; Tsuda, S, Cryobiology and Cryotechnology, 54, 2, 93-96., 2009

Enhancement of the activity of antifreeze protein by addition of a water soluble polymer.
Sakae Tsuda, Cryobiology, 54, 2, 89-92., 2009, [Peer-reviewed]

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
Yaoi, Katsuro; Kondo, Hidemasa; Hiyoshi, Ayako; Noro, Natsuko; Sugimoto, Hiroshi; Tsuda, Sakae; Miyazaki, Kentaro, Febs Journal, 276, 18, 5094, 5100, 2009, [Peer-reviewed]
English

One-pot synthesis of cyclic antifreeze glycopeptides
Masakazu Hachisu; Hiroshi Hinou; Manabu Takamichi; Sakae Tsuda; Shuhei Koshidaa; Shin-Ichiro Nishimura, CHEMICAL COMMUNICATIONS, 13, 1641, 1643, 2009
English

Construction of Time-Lapse Scanning Electrochemical Microscopy with Temperature, Control and Its Application To Evaluate the Preservation Effects of Antifreeze Proteins on Living Cells
Yu Hirano; Yoshiyuki Nishimiya; Keiko Kowata; Fumio Mizutani; Sakae Tsuda; Yasuo Komatsu, ANALYTICAL CHEMISTRY, 80, 23, 9349, 9354, Dec. 2008
English

Crystal structure and mutational analysis of Ca2+-independent type II antifreeze protein from longsnout poacher, Brachyopsis rostratus
Yoshiyuki Nishimiya; Hidemasa Kondo; Manabu Takamichi; Hiroshi Sugimoto; Mamoru Suzuki; Ai Miura; Sakae Tsuda, JOURNAL OF MOLECULAR BIOLOGY, 382, 3, 734, 746, Oct. 2008
English

Hypothermic preservation effect on mammalian cells of type III antifreeze proteins from notched-fin eelpout
Yu Hirano; Yoshiyuki Nishimiya; Shuichiro Matsumoto; Michiaki Matsushita; Satoru Todo; Ai Miura; Yasuo Komatsu; Sakae Tsuda, CRYOBIOLOGY, 57, 1, 46, 51, Aug. 2008
English

Two domains of RD3 antifreeze protein diffuse independently
Nolan B. Holland; Yoshiyuki Nishimiya; Sakae Tsuda; Frank D. Sonnichsen, BIOCHEMISTRY, 47, 22, 5935, 5941, Jun. 2008
English

Construction of Time-Lapse Scanning Electrochemical Microscopy with Temperature, Control and Its Application To Evaluate the Preservation Effects of Antifreeze Proteins on Living Cells
Hirano, Yu; Nishimiya, Yoshiyuki; Kowata, Keiko; Mizutani, Fumio; Tsuda, Sakae; Komatsu, Yasuo, Analytical Chemistry, 80, 23, 9349, 9354, 2008, [Peer-reviewed]
English

Mass preparation and technological development of an antifreeze protein - Toward a practical use of biomolecules –
Sakae Tsuda, Synthesiology, 1, 1, 7, 14, 2008, [Peer-reviewed]
Nat. Inst. of Adv. Industrial Science and Technology (AIST), English

Hypothermic preservation effect on mammalian cells of type III antifreeze proteins from notched-fin eelpout
Hirano, Yu; Nishimiya, Yoshiyuki; Matsumoto, Shuichiro; Matsushita, Michiaki; Todo, Satoru; Miura, Ai; Komatsu, Yasuo; Tsuda, Sakae, Cryobiology, 57, 1, 46, 51, 2008, [Peer-reviewed]
English

Hydroxyl groups of threonines contribute to the activity of Ca2+-dependent type II antifreeze protein.
Sakae Tsuda, Cryobiology and Cryotechnology, 54, 1, 1-8., 8, 2008, [Peer-reviewed]
Type II antifreeze protein from herring and smelt (AFPII) exhibits thermal hysteresis (TH) activity when the protein is saturated with Ca^<2+>. The ice-binding site of AFPII consists of Thr96, Thr98, and two Ca^<2+>-coordinating residues. Here we examined TH activity of AFPII from Japanese smelt (Hypomesus nipponensis) and its mutant proteins, T96S, T96V, T96A, T98S, T98V, and T98A. Note that T96S denotes a mutant whose Thr96 is replaced with Ser. It appeared that substitution of Thr96 with Ser and Val retained 90% and 45% of TH activity, respectively, while it becomes inactive when substituted with Ala. For Thr98, Ser substitution had no significant influence on TH, whereas it becomes less than 20% by Val and Ala substitutions. These results suggest that hydroxyl group rather than methyl group of Thrs are essential for TH activity of AFPII, which is quite distinct from the other types of AFP., Japanese Society for Cryobiology and Cryotechnology, English

Two domains of RD3 antifreeze protein diffuse independently
Holland, Nolan B.; Nishimiya, Yoshiyuki; Tsuda, Sakae; Sonnichsen, Frank D., Biochemistry, 47, 22, 5935, 5941, 2008, [Peer-reviewed]
English

Crystal structure and mutational analysis of Ca2+-independent type II antifreeze protein from longsnout poacher, Brachyopsis rostratus
Nishimiya, Yoshiyuki; Kondo, Hidemasa; Takamichi, Manabu; Sugimoto, Hiroshi; Suzuki, Mamoru; Miura, Ai; Tsuda, Sakae, Journal of Molecular Biology, 382, 3, 734, 746, 2008, [Peer-reviewed]
English

Hypothermic preservation of cultured cells with using fish type III antifreeze protein from Noched-fin eelpout.
Warashina, A; Hirano, Y; Takamichi, M; Nishimiya, Y; Kondo, H; Tsuda, S, Cryobiology and Cryotechnology, 54, 2, 93-96., 2008

Enhancement of the activity of antifreeze protein by addition of a water-soluble polymer.
Iwasaki, K; Kondo, H; Nishimiya, Y; Takamichi, M; Miura, A; Tsuda, S, Cryobiology & Cryotechnology, 54, 2, 89-92., 2008

Mass preparation and technological development of an antifreeze protein - Toward a practical use of biomolecules -.
Nishimiya, Y; Mie, Y; Hirano, Y; Kondo, H; Miura, A; Tsuda, S, Synthesiology, 1, 1, 7-14., 2008

Hydroxyl groups of threonines contribute to the activity of Ca2+-dependent type II antifreeze protein.
Yasui, M; Takamichi, M; Miura, A; Nishimiya, Y; Kondo, H; Tsuda, S, Cryobiology and Cryotechnology, 54, 1, 1-8., 8, 2008, [Peer-reviewed]
Type II antifreeze protein from herring and smelt (AFPII) exhibits thermal hysteresis (TH) activity when the protein is saturated with Ca^<2+>. The ice-binding site of AFPII consists of Thr96, Thr98, and two Ca^<2+>-coordinating residues. Here we examined TH activity of AFPII from Japanese smelt (Hypomesus nipponensis) and its mutant proteins, T96S, T96V, T96A, T98S, T98V, and T98A. Note that T96S denotes a mutant whose Thr96 is replaced with Ser. It appeared that substitution of Thr96 with Ser and Val retained 90% and 45% of TH activity, respectively, while it becomes inactive when substituted with Ala. For Thr98, Ser substitution had no significant influence on TH, whereas it becomes less than 20% by Val and Ala substitutions. These results suggest that hydroxyl group rather than methyl group of Thrs are essential for TH activity of AFPII, which is quite distinct from the other types of AFP., Japanese Society for Cryobiology and Cryotechnology, English

Effect of annealing time of an ice crystal on the activity of type III antifreeze protein
Manabu Takamichi; Yoshiyuki Nishimiya; Ai Miura; Sakae Tsuda, FEBS JOURNAL, 274, 24, 6469, 6476, Dec. 2007
English

The structural basis for the exo-mode of action in GH74 oligoxyloglucan reducing end-specific cellobiohydrolase
Katsuro Yaoi; Hidemasa Kondo; Ayako Hiyoshi; Natsuko Noro; Hiroshi Sugimoto; Sakae Tsuda; Yasushi Mitsuishi; Kentaro Miyazaki, JOURNAL OF MOLECULAR BIOLOGY, 370, 1, 53, 62, Jun. 2007
English

Type III antifreeze protein extremely enhances viability of cultured endothelial cells during hypothermic preservation.
Shuichiro Matsumoto; Michiaki Matsushita; Yoshiyuki Nishimiya; Yu Hirano; Sakae Tsuda; Satoru Todo, AMERICAN JOURNAL OF TRANSPLANTATION, 7, 509, 509, May 2007
English, Summary international conference

国産魚類からの不凍タンパク質の探索と機能解析
西宮佳志; 近藤英昌; 三重安弘; 平野悠; 宇梶愼子; 佐藤涼子; 野呂奈津子; 安井雅範; 高道学; 三浦愛; 津田栄; 津田栄, マリンバイオテクノロジー学会大会講演要旨集, 10th, 2007

Effect of annealing time of an ice crystal on the activity of type III antifreeze protein
Takamichi, Manabu; Nishimiya, Yoshiyuki; Miura, Ai; Tsuda, Sakae, Febs Journal, 274, 24, 6469, 6476, 2007, [Peer-reviewed]
English

Effects of additives on formation rates of CO2 hydrate films
T. Uchida; I. Y. Ikeda; R. Ohmura; S. Tsuda, PHYSICS AND CHEMISTRY OF ICE, 609, 618, 2007
English

Activity of a two-domain antifreeze protein is not dependent on linker sequence
Holland, Nolan B.; Nishimiya, Yoshiyuki; Tsuda, Sakae; Soennichsen, Frank D., Biophysical Journal, 92, 2, 541, 546, 2007, [Peer-reviewed]
English

Type III antifreeze protein extremely enhances viability of cultured endothelial cells during hypothermic preservation.
Matsumoto, Shuichiro; Matsushita, Michiaki; Nishimiya, Yoshiyuki; Hirano, Yu; Tsuda, Sakae; Todo, Satoru, American Journal of Transplantation, 7, 509, 509, 2007, [Peer-reviewed]
English

The structural basis for the exo-mode of action in GH74 oligoxyloglucan reducing end-specific cellobiohydrolase
Yaoi, Katsuro; Kondo, Hidemasa; Hiyoshi, Ayako; Noro, Natsuko; Sugimoto, Hiroshi; Tsuda, Sakae; Mitsuishi, Yasushi; Miyazaki, Kentaro, Journal of Molecular Biology, 370, 1, 53, 62, 2007, [Peer-reviewed]
English

Effects of additives on formation rates of CO2 hydrate films
Uchida, T.; Ikeda, I. Y.; Ohmura, R.; Tsuda, S., Physics and Chemistry of Ice, 609, 618, 2007, [Peer-reviewed]
English

Activity of a two-domain antifreeze protein is not dependent on linker sequence
Nolan B. Holland; Yoshiyuki Nishimiya; Sakae Tsuda; Frank D. Soennichsen, BIOPHYSICAL JOURNAL, 92, 2, 541, 546, Jan. 2007
English

Crystallization and preliminary X-ray crystallographic analysis of Ca2+-independent and Ca2+-dependent species of the type II antifreeze protein
Y Nishimiya; H Kondo; M Yasui; H Sugimoto; N Noro; R Sato; M Suzuki; A Miura; S Tsuda, ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 62, 538, 541, Jun. 2006
English

Thermal stabilization of Bacillus subtilis family-11 xylanase by directed evolution
K Miyazaki; M Takenouchi; H Kondo; N Noro; M Suzuki; S Tsuda, JOURNAL OF BIOLOGICAL CHEMISTRY, 281, 15, 10236, 10242, Apr. 2006
English

Crystallization and preliminary X-ray crystallographic analysis of Ca2+-independent and Ca2+-dependent species of the type II antifreeze protein
Nishimiya, Y; Kondo, H; Yasui, M; Sugimoto, H; Noro, N; Sato, R; Suzuki, M; Miura, A; Tsuda, S, Acta Crystallographica Section F-Structural Biology and Crystallization Communications, 62, 538, 541, 2006, [Peer-reviewed]
English

Thermal stabilization of Bacillus subtilis family-11 xylanase by directed evolution
Miyazaki, K; Takenouchi, M; Kondo, H; Noro, N; Suzuki, M; Tsuda, S, Journal of Biological Chemistry, 281, 15, 10236, 10242, 2006, [Peer-reviewed]
English

1P053 Functions of Characteristic C-term region of ASABF, Antibacterial Peptide Isolated from the Nematode Ascaris suum
Nakano M; Aizawa T; Miura K; Hoshino H; Miyazawa M; Kato Y; Kumaki Y; Demura M; Tsuda S; Kawano K; Nitta K, Biophysics, 45, 1, S45, 19 Oct. 2005
The Biophysical Society of Japan General Incorporated Association, Japanese

不凍糖タンパク質(AFGP)不凍活性メカニズムの解明
八須匡和; 五十嵐幸太; 津田栄; 越田周平; 門出健次; 西村紳一郎, 高分子学会予稿集(CD-ROM), 54, 1 Disk1, 2005

Crystal structure of PR-5 protein from winter wheat
Kuwabara Chikako; Kondo Hidemasa; Noro Natsuko; Takezawa Daisuke; Arakawa Keita; Tsuda Sakae, Plant and Cell Physiology Supplement, 2005, 0, 848, 848, 2005
WAS-3 protein, a member of PR-5 family, accumulates in an apoplastic space of winter wheat in response to cold acclimation. We previously revealed that recombinant WAS-3 protein (rWAS-3) produced using high efficiency wheat expression system inhibited hyphal growth of pink snow mold and Fusarium oxysporum. Furthermore, rWAS-3 bound to fungal cell wall itself and β-1,3-glucan that was main components of fungal cell walls. In order to clarify the biochemical interaction of WAS-3 and β-1,3-glucan, we determined the crystal structure of rWAS-3 using X-ray crystallographic analysis. The thin plate-shaped crystals of rWAS-3 were obtained by hanging-drop vapor diffusion method. The structure of rWAS-3 was determined at 1.8 angstrom resolution by molecular replacement method. The electrostatic potential analysis revealed that rWAS-3 had an acidic cleft region. Therefore, we concluded that this region might be β-1,3-glucan binding site of rWAS-3., The Japanese Society of Plant Physiologists

Functions and structures of xyloglucan hydrolases belonging to glycoside hydrolase family 74
YAOI K., J. Appl. Glycosci., 52, 2, 169, 176, 2005

A part of ice nucleation protein exhibits the ice-binding ability.
Y Kobashigawa; Y Nishimiya; K Miura; S Ohgiya; A Miura; S Tsuda, FEBS Letters, 579, 6, 1493, 1497, 2005
English

Co-operative effect of the isoforms of type III antifreeze protein expressed in Notched-fin eelpout, Zoarces elongatus Kner
Nishimiya, Y; Sato, R; Takamichi, M; Miura, A; Tsuda, S, Febs Journal, 272, 2, 482, 492, 2005, [Peer-reviewed]
English

A part of ice nucleation protein exhibits the ice-binding ability.
Sakae Tsuda, FEBS Letters, 579, 6, 1493, 1497, 2005, [Peer-reviewed]
English

Co-operative effect of the isoforms of type III antifreeze protein expressed in Notched-fin eelpout, Zoarces elongatus Kner
Y Nishimiya; R Sato; M Takamichi; A Miura; S Tsuda, FEBS JOURNAL, 272, 2, 482, 492, Jan. 2005
English

1P003 Structure and Activity of ASABFd18c, Antibacterial Peptide Isolated from the Nematode Ascaris suum
Nakano M; Aizawa T; Miura K; Hoshino H; Miyazawa M; Kato Y; Kumaki Y; Demura M; Tsuda S; Kawano K; Nitta K, Biophysics, 44, 1, S30, 10 Nov. 2004
The Biophysical Society of Japan General Incorporated Association, Japanese

Tandem repeat of a seven-bladed beta-propeller domain in oligoxyloglucan reducing-end-specific cellobiohydrolase
K Yaoi; H Kondo; N Noro; M Suzuki; S Tsuda; Y Mitsuishi, STRUCTURE, 12, 7, 1209, 1217, Jul. 2004
English

コムギの低温特異的に発現誘導される抗菌タンパク質遺伝子
今井亮三; 小池倫也; 津田栄, 研究成果情報 北海道農業, 2003, 66, 67, 30 Jun. 2004
Japanese

Tandem repeat of a seven-bladed beta-propeller domain in oligoxyloglucan reducing-end-specific cellobiohydrolase
Yaoi, K; Kondo, H; Noro, N; Suzuki, M; Tsuda, S; Mitsuishi, Y, Structure, 12, 7, 1209, 1217, 2004, [Peer-reviewed]
English

Antifreeze Glycoproteins: Elucidation of the Structural Motifs That Are Essential for Antifreeze Activity.
Sakae Tsuda, Angewandte Chemie International Edition, 43, 7, 856, 862, 2004, [Peer-reviewed]
English

A novel xyloglucan-specific glycosidase, olygoxyloglucan reducing end-specific cellobiohydrolase.
Yaoi, K; Kondo, H; Suzuki, M; Noro, N; Tsuda, S; Mitsuishi, Y, Biotech. Lignocel. Degrade Utilization (UNI Publishers, Co. Ltd.), 2004

Antifreeze glycoproteins: Elucidation of the structural motifs that are essential for antifreeze activity
Tachibana, Y; Fletcher, GL; Fujitani, N; Tsuda, S; Monde, K; Nishimura, SI, Angewandte Chemie-International Edition, 43, 7, 856, 862, 2004, [Peer-reviewed]
English

A novel xyloglucan-specific glycosidase, olygoxyloglucan reducing end-specific cellobiohydrolase.
Yaoi, K; Kondo, H; Suzuki, M; Noro, N; Tsuda, S; Mitsuishi, Y, Biotech. Lignocel. Degrade Utilization (UNI Publishers, Co. Ltd.), 2004

Antifreeze proteins from snow mold fungi
T Hoshino; M Kiriaki; S Ohgiya; M Fujiwara; H Kondo; Y Nishimiya; Yumoto, I; S Tsuda, CANADIAN JOURNAL OF BOTANY-REVUE CANADIENNE DE BOTANIQUE, 81, 12, 1175, 1181, Dec. 2003
English

Artificial multimers of the type III antifreeze protein - Effects on thermal hysteresis and ice crystal morphology
Y Nishimiya; S Ohgiya; S Tsuda, JOURNAL OF BIOLOGICAL CHEMISTRY, 278, 34, 32307, 32312, Aug. 2003
English

Type II antifreeze protein from a mid-latitude freshwater fish, Japanese smelt (Hypomesus nipponensis)
Y Yamashita; R Miura; Y Takemoto; S Tsuda; H Kawahara; H Obata, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 67, 3, 461, 466, Mar. 2003
English

Low-temperature-induced structural changes in human lysozyme elucidated by three-dimensional NMR spectroscopy
H Kumeta; A Miura; Y Kobashigawa; K Miura; C Oka; N Nemoto; K Nitta; S Tsuda, BIOCHEMISTRY, 42, 5, 1209, 1216, Feb. 2003
English

Crystallization and Preliminary X-Ray Crystallographic Study on Xyloglucan-Specific exo-beta-glycosidase, oligoxyloglucan reducing-end specific cellobiohydrolase.
Sakae Tsuda, Acta Crystallographica Section D, 59, 1838, 1839, 2003, [Peer-reviewed]
English

Low-temperature-induced structural changes in human lysozyme elucidated by three-dimensional NMR spectroscopy
Kumeta, H; Miura, A; Kobashigawa, Y; Miura, K; Oka, C; Nemoto, N; Nitta, K; Tsuda, S, Biochemistry, 42, 5, 1209, 1216, 2003, [Peer-reviewed]
English

Antifreeze proteins from snow mold fungi.
Hoshino, T; Kiriaki, M; Ohgiya, S; Fujiwara, M; Kondo, H; Nishimiya, Y; Yumoto, I; Tsuda, S, Can. J. Bot., 81, 1175-1181., 2003

Artificial multimers of the type III antifreeze protein - Effects on thermal hysteresis and ice crystal morphology
Nishimiya, Y; Ohgiya, S; Tsuda, S, Journal of Biological Chemistry, 278, 34, 32307, 32312, 2003, [Peer-reviewed]
English

Crystallization and preliminary X-ray crystallographic study on a xyloglucan-specific exo-beta-glycosidase, oligoxyloglucan reducing-end specific cellobiohydrolase
Yaoi, K; Kondo, H; Suzuki, M; Noro, N; Tsuda, S; Mitsuishi, Y, Acta Crystallographica Section D-Biological Crystallography, 59, 10, 1838, 1839, 2003
English

Type II antifreeze protein from a mid-latitude freshwater fish, Japanese smelt (Hypomesus nipponensis)
Yamashita, Y; Miura, R; Takemoto, Y; Tsuda, S; Kawahara, H; Obata, H, Bioscience Biotechnology and Biochemistry, 67, 3, 461, 466, 2003, [Peer-reviewed]
English

Efficient and versatile synthesis of mucin-like glycoprotein mimics
Y Tachibana; N Matsubara; F Nakajima; T Tsuda; S Tsuda; K Monde; SI Nishimura, TETRAHEDRON, 58, 51, 10213, 10224, Dec. 2002
English

A novel plant defensin-like gene of winter wheat is specifically induced during cold acclimation
M Koike; T Okamoto; S Tsuda; R Imai, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 298, 1, 46, 53, Oct. 2002
English

Production and characterization of recombinant tachycitin, the Cys-rich chitin-binding protein
T Suetake; T Aizawa; N Koganesawa; T Osaki; Y Kobashigawa; M Demura; S Kawabata; K Kawano; S Tsuda; K Nitta, PROTEIN ENGINEERING, 15, 9, 763, 769, Sep. 2002
English

21 Synthetic studies on glycopeptide polymers having ice crystal growth inhibition activity
Tachibana Yuki; Igarashi Kota; Monde Kenji; Nishimura Shin-Ichiro; Tsuda Sakae, Symposium on the Chemistry of Natural Products, symposium papers, 44, 0, 121, 126, 2002
Antifreeze glycoproteins [AFGPs: {Ala-Thr(Galβ1-3GalNAcα)-Ala}_n, n=4-50] in the serum of polar fishes have been known to depress the freezing point of their blood and help them to survive at the temperature below-1.9℃[1]. Though it is widely accepted that AFGPs bind to the ice surface and prevent its growth, the definitive molecular mechanism of the action has not been solved yet. In this study, structure-activity relationships in AFGPs were studied toward understanding of the mechanism, thorough chemical syntheses, activity evaluations, and conformational analyses of the AFGP and its analogues, in which sugar or peptide moieties were different from those of the wild-type AFGP. The AFGP (1) and its analogues (2-8) were successfully synthesized via simple polymerization of their repeating unit using a DPPA method without any protection of sugar -OH groups [2]. Additionally, sialyl T-bearing glycopolypeptide (9) was successfully prepared through enzymatic elongation of a sialic acid residue to C-3 position of the galactose residue in the AFGP (1), indicating the broad applicability of this methodology for the syntheses of various glycopeptide polymers. In the evaluation assay of the antifreeze activity, artificial AFGP (1) showed similar activity as that of the wild-type AFGP. On the contrary, neither an AFGP repeating unit glycopeptide (19) nor a sugar-lacked polypeptide (poly-ATA, 8) shows the activity. Interestingly, AFGP analogs having GalNAc (2) or LacNAc (3) as sugar moiety showed similar activities as AFGP, while that of Galactose (4), Lactose (5), β-O-linked (6), and ASA (7) did not. These results clearly indicate the importance of i) repeating structure of glycopeptide, ii) NHAc group at C-2 position of the sugar moiety directly attached to the peptide, iii) α-glycosidic linkage between the sugar and the threonyl residue, for the specific interaction with ice. Furthermore, secondary structural analyses of the synthetic compounds were carried out by means of a CD spectroscopic method, in order to clarify the relationships between their conformations and their activities. The CD spectra of active compounds (1-3) were obviously different from those of inactive analogues (4-8), suggesting the presence of an ordered structure, but not α-helix., Symposium on the Chemistry of Natural Products Steering Committee, Japanese

A novel plant defensin-like gene of winter wheat is specifically induced during cold acclimation
Koike, M; Okamoto, T; Tsuda, S; Imai, R, Biochemical and Biophysical Research Communications, 298, 1, 46, 53, 2002, [Peer-reviewed]
English

Alternate conformations observed in catalytic serine of Bacillus subtilis lipase determined at 1.3 angstrom resolution
Kawasaki, K; Kondo, H; Suzuki, M; Ohgiya, S; Tsuda, S, Acta Crystallographica Section D-Biological Crystallography, 58, 7, 1168, 1174, 2002
English

Efficient and versatile synthesis of mucin-like glycoprotein mimics
Tachibana, Y; Matsubara, N; Nakajima, F; Tsuda, T; Tsuda, S; Monde, K; Nishimura, SI, Tetrahedron, 58, 51, 10213, 10224, 2002, [Peer-reviewed]
English

Production and characterization of recombinant tachycitin, the Cys-rich chitin-binding protein
Suetake, T; Aizawa, T; Koganesawa, N; Osaki, T; Kobashigawa, Y; Demura, M; Kawabata, S; Kawano, K; Tsuda, S; Nitta, K, Protein Engineering, 15, 9, 763, 769, 2002, [Peer-reviewed]
English

Alternate conformations observed in catalytic serine of Bacillus subtilis lipase determined at 1.3 angstrome resolution.
Sakae Tsuda, Acta Crystallographica Section D, 58, 1168, 1174, 2002, [Peer-reviewed]
English

Assignments of 1H, 13C, and 15N resonances of human lysozyme at 4 °C [2]
Hiroyuki Kumeta; Yoshihiro Kobashigawa; Kazunori Miura; Yoshiyuki Nishimiya; Chitoshi Oka; Nobuaki Nemoto; Ai Miura; Katsutoshi Nitta; Sakae Tsuda, Journal of Biomolecular NMR, 22, 2, 183, 184, 2002
English, Report scientific journal

Letter to the Editor: Assignment of H-1, C-13, and 15N resonances of canine milk lysozyme
Y Kobashigawa; K Miura; M Demura; N Nemoto; T Koshiba; K Nitta; S Tsuda, JOURNAL OF BIOMOLECULAR NMR, 19, 4, 387, 388, Apr. 2001
English

Hydration of Glucose and Galactose Derivatives.
Sakae Tsuda, Bulletin of the Chemical Society of Japan, 74, 10, 1857, 1861, 2001, [Peer-reviewed]
English

NMR analysis of type III antifreeze protein intramolecular dimer - Structural basis for enhanced activity
Miura, K; Ohgiya, S; Hoshino, T; Nemoto, N; Suetake, T; Miura, A; Spyracopoulos, L; Kondo, H; Tsuda, S, Journal of Biological Chemistry, 276, 2, 1304, 1310, 2001, [Peer-reviewed]
English

NMR analysis of type III antifreeze protein intramolecular dimer - Structural basis for enhanced activity
K Miura; S Ohgiya; T Hoshino; N Nemoto; T Suetake; A Miura; L Spyracopoulos; H Kondo; S Tsuda, JOURNAL OF BIOLOGICAL CHEMISTRY, 276, 2, 1304, 1310, Jan. 2001
English

Hydration of Glucose and Galactose Derivatives.
H Uedaira; S Okouchi; S Tsuda; H Uedaira, Bulletin of the Chemical Society of Japan, 74, 10, 1857, 1861, 2001, [Peer-reviewed]
English

Assignment of 1H, 13C, and 15N resonances of canine milk lysozyme.
Sakae Tsuda, Journal of Biomolecular NMR, 19, 4, 387, 388, 2001, [Peer-reviewed]
English

Hydrogen exchange study of folding intermediate and molten globule state of canine milk lysozyme
Kobashigawa Y; Mizuguchi M; Demura M; Koshiba T; Tsuda S; Nitta K, Biophysics, 40, 1, S160, 05 Aug. 2000
The Biophysical Society of Japan General Incorporated Association, Japanese

Accelerated Publication - Chitin-binding proteins in invertebrates and plants comprise a common chitin-binding structural motif
T Suetake; S Tsuda; S Kawabata; K Miura; S Iwanaga; K Hikichi; K Nitta; K Kawano, JOURNAL OF BIOLOGICAL CHEMISTRY, 275, 24, 17929, 17932, Jun. 2000
English

小麦由来のパラクマル酸による氷核活性細菌の不活性化
大平万里; 星野保; 吉田みどり; 神繁樹; 津田栄; 扇谷悟; 石崎紘三, 日本植物生理学会年会要旨集, 40th, 100, 20 Mar. 2000
Japanese

Letter to the editor: Assignments of 1H, 13C and 15N resonances of intramolecular dimer antifreeze protein RD3 [3]
Kazunori Miura; Satoru Ohgiya; Tamotsu Hoshino; Nobuaki Nemoto; Katsutoshi Nitta; Sakae Tsuda, Journal of Biomolecular NMR, 16, 3, 273, 274, 2000
English, Report scientific journal

Accelerated Publication - Chitin-binding proteins in invertebrates and plants comprise a common chitin-binding structural motif
Suetake, T; Tsuda, S; Kawabata, S; Miura, K; Iwanaga, S; Hikichi, K; Nitta, K; Kawano, K, Journal of Biological Chemistry, 275, 24, 17929, 17932, 2000, [Peer-reviewed]
English

Determination of the solution structure of the N-domain plus linker of antarctic eel pout antifreeze protein RD3
K Miura; S Ohgiya; T Hoshino; N Nemoto; M Odaira; K Nitta; S Tsuda, JOURNAL OF BIOCHEMISTRY, 126, 2, 387, 394, Aug. 1999
English

Physiological and biochemical significance of antifreeze substances in plants
T Hoshino; M Odaira; M Yoshida; S Tsuda, JOURNAL OF PLANT RESEARCH, 112, 1106, 255, 261, Jun. 1999
English

水の凍結温度を制御するバイオ物質の分子構造解析
近藤 英昌; 津田 栄, 北海道工業技術研究所報告, 73, 73, 7, 13, 1999
経済産業省産業技術総合研究所北海道工業技術研究所, Japanese

Low-Temperature-Induced Structural Changes in The Apo Regulatory Domain of Skeletal Muscle Troponin C.
S Tsuda; A Miura; SM Gagne; L Spyracopoulos; BD Sykes, Biochemistry, 38, 18, 5693, 5700, 1999, [Peer-reviewed]
English

Physiological and biochemical significance of antifreeze substances in plants
Hoshino, T; Odaira, M; Yoshida, M; Tsuda, S, Journal of Plant Research, 112, 1106, 255, 261, 1999, [Peer-reviewed]
English

Determination of the solution structure of the N-domain plus linker of antarctic eel pout antifreeze protein RD3
Miura, K; Ohgiya, S; Hoshino, T; Nemoto, N; Odaira, M; Nitta, K; Tsuda, S, Journal of Biochemistry, 126, 2, 387, 394, 1999, [Peer-reviewed]
English

Low-Temperature-Induced Structural Changes in The Apo Regulatory Domain of Skeletal Muscle Troponin C.
Sakae Tsuda, Biochemistry, 38, 18, 5693, 5700, 1999, [Peer-reviewed]
English

小麦の新規不凍活性物質の検索と評価
大平 万里; 星野 保; 吉田 みどり; 津田 栄; 扇谷 悟; 石崎 紘三, 日本植物学会大会研究発表記録 = Proceedings of the annual meeting of the Botanical Society of Japan, 62, 190, 190, 01 Sep. 1998
Japanese

SEARCHING FOR NEW ANTIFREEZE SUBSTANCES FROM WHEAT
ODAIRA Masato; HOSHINO Tamotsu; YOSHIDA Midori; TSUDA Sakae; OHGIYA Satoru; ISHIZAKI Kozo, Plant Cell Physiol., 39, S140, S140, May 1998
English

Three-Dimensional Structure of Tachycitin in Solution Determined by 1H NMR.
Suetake, T; Tsuda, S; Kawabata, S; Kawano, K; Miura, K; Hikichi, K; Nitta, K, Rept. Progr. Polym. Phys. Jpn., 41, 67-69., 1998

Backbone and Methyl Dynamics of the Regulatory Domain of Troponin C: Anisotropic Rotational Diffusion and Contribution of Conformational Entropy to Calcium Affinity.
Sakae Tsuda, J. Mol. Biol., 278, 3, 667, 686, 1998, [Peer-reviewed]
English

Three-Dimensional Structure of Tachycitin in Solution Determined by 1H NMR.
Suetake, T; Tsuda, S; Kawabata, S; Kawano, K; Miura, K; Hikichi, K; Nitta, K, Rept. Progr. Polym. Phys. Jpn., 41, 67-69., 1998

Backbone and Methyl Dynamics of the Regulatory Domain of Troponin C: Anisotropic Rotational Diffusion and Contribution of Conformational Entropy to Calcium Affinity.
SM Gagne; S Tsuda; L Spyracopoulos; LE Kay; BD Sykes, J. Mol. Biol., 278, 3, 667, 686, 1998
English

Solution Structure of the N-domain with A Linker Portion of Antarctic Eel Pout Antifreeze Protein RD3.
Miura, K; Ohgiya, S; Hoshino, T; Nemoto, N; Hikichi, K; Tsuda, S, Rept. Progr. Polym. Phys. Jpn., 41, 71-74., 1998

氷結晶と相互作用するタンパク質の研究-NMR構造解析から-
津田 栄, 資源・素材, 1997, 3, 19, 22, 23 Sep. 1997
Japanese

Synthesis and modification of new biodegradable copolymers: Serine/glycolic acid based copolymers
George John; Sakae Tsuda; Mikio Morita, Journal of Polymer Science, Part A: Polymer Chemistry, 35, 10, 1901, 1907, 30 Jul. 1997, [Peer-reviewed]
John Wiley and Sons Inc., English

A hairpin-loop conformation in tandem repeat sequence of the ice nucleation protein revealed by NMR
S Tsuda; A Ito; N Matsushima, FASEB JOURNAL, 11, 9, A910, A910, Jul. 1997
English, Summary international conference

A hairpin-loop conformation in tandem repeat sequence of the ice nucleation protein revealed by NMR spectroscopy.
Sakae Tsuda, FEBS Lett, 409, 2, 227, 231, 1997, [Peer-reviewed]
English

A hairpin-loop conformation in tandem repeat sequence of the ice nucleation protein revealed by NMR spectroscopy.
Sakae Tsuda, FEBS Lett, 409, 2, 227, 231, 1997
English

Synthesis and Modification of New Biodegradable Co-polymers: Serine/Glycolic acid Alternating Polymer abd Copolymers of L-Lactide or e-Caprolactone.
Sakae Tsuda, J. Polym. Sci., 35, 10, 1901, 1907, 1997, [Peer-reviewed]
John Wiley and Sons Inc., English

Secondary structure and Ca2+-binding property of the N-terminal half domain of calmodulin from yeast Saccharomyces cerevisiae as studied by NMR
SY Ohki; K Miura; M Saito; K Nakashima; H Maekawa; M Yazawa; S Tsuda; K Hikichi, JOURNAL OF BIOCHEMISTRY, 119, 6, 1045, 1055, Jun. 1996
English

Secondary Structure and Ca2+-Binding Property of the N-Terminal Half Domain of Calmodulin from Yeast Saccaromyces cerevisiae as Studied by NMR.
Sakae Tsuda, J Biochem, 119, 6, 1045, 1055, 1996, [Peer-reviewed]
English

Modern NMR Spectroscopy and X-ray Crystallography: a Different approach to Study the Structure and its Function of a Protein.
Tsuda, S, J. Crystallographic Soc. Jpn., 38, 84-88., 1996

Modern NMR Spectroscopy and X-ray Crystallography: a Different approach to Study the Structure and its Function of a Protein.
Tsuda, S, J. Crystallographic Soc. Jpn., 38, 1, 84-88., 88, 1996
The NMR spectroscopy has been utilized widely for a elucidation of the structural change of a protein caused by the change of pH, ionic strength, temperature, and ligand concentration in solution. The X-ray was less utilized for these study excutable easily in solution, but is utilized much for the structural determination of a protein. Such difference has ever lead to the situation that the NMR relied on the structure solved by X-ray and the X-ray argued its struture in reference to the conformational change elucidated by NMR. However, recent developments of NMR spectroscopy made it possible to determine the three-dimensional structure, and the X-ray techniques has also been developped to clarify the structural change of a protein. This review compares the recent development of these two techniques, and will discuss about the future collaborating interaction between NMR and X-ray., The Crystallographic Society of Japan, Japanese

STRUCTURES OF THE TROPONIN-C REGULATORY DOMAINS IN THE APO AND CALCIUM-SATURATED STATES
SM GAGNE; S TSUDA; MX LI; LB SMILLIE; BD SYKES, NATURE STRUCTURAL BIOLOGY, 2, 9, 784, 789, Sep. 1995
English

Structural Features of Bovine Brain S100b as Studied by 1H-NMR Spectroscopy.
Tsuda, S; Matsuda, S; Izumi, Y; Hikichi, K; Matsushima, N, Rept. Progr. Polym. Phys. Jpn., 38, 509-510., 1995

Secondary Structure of N-Terminal-Half Domain of Yeast Calmodulin in the Presence of Ca2+ as Studied by 1H-NMR.
Miura, K; Saito, M; Ohki, S; Tsuda, S; Nakashima, K; Yazawa, M; Hikichi, K, Rept. Progr. Polym. Phys. Jpn., 38, 501-502., 1995

Interaction between Troponin-C and Inhibitory Region Peptide of Troponin-I as Studied by 1H-NMR.
Tsuda, S; Aimoto, S; Hikichi, K, Rept. Progr. Polym. Phys. Jpn., 35, 583-584., 1995

NMR Studies of the Calcium-Induced Structural lChanges that Triggers Muscle Contraction.
Sykes, B.D; Audette, G; Gagne, S.M; Li, M.X; Slupsky, C.M; Tsuda, S, 34th. Hanford Symposium on Health and the Environment, "Biomolecules: From 3-D Structure to Applications"(ed. Ornstein, R.L.), 35, 11-19., S92, 1995
The Biophysical Society of Japan General Incorporated Association, Japanese

Calcium Binding to the Regulatory N-Domain of Skeletal Muscle Troponin C Occurs in a Stepwise manner.
Sakae Tsuda, Biochemistry, 34, 26, 8330, 8340, 1995, [Peer-reviewed]
English

Structures of the troponinC regulatory domains in the apo and calcium-saturated states.
Sakae Tsuda, Nat Struct Biol, 2, 9, 784, 789, 1995, [Peer-reviewed]
English

NMR Studies of the Calcium-Induced Structural lChanges that Triggers Muscle Contraction.
Sykes, B.D; Audette, G; Gagne, S.M; Li, M.X; Slupsky, C.M; Tsuda, S, 34th. Hanford Symposium on Health and the Environment, "Biomolecules: From 3-D Structure to Applications"(ed. Ornstein, R.L.), 35, 11-19., S92, 1995
The Biophysical Society of Japan General Incorporated Association, Japanese

Structural Features of Bovine Brain S100b as Studied by 1H-NMR Spectroscopy.
Tsuda, S; Matsuda, S; Izumi, Y; Hikichi, K; Matsushima, N, Rept. Progr. Polym. Phys. Jpn., 38, 509-510., 1995

Secondary Structure of N-Terminal-Half Domain of Yeast Calmodulin in the Presence of Ca2+ as Studied by 1H-NMR.
Miura, K; Saito, M; Ohki, S; Tsuda, S; Nakashima, K; Yazawa, M; Hikichi, K, Rept. Progr. Polym. Phys. Jpn., 38, 501-502., 1995

Calcium Binding to the Regulatory N-Domain of Skeletal Muscle Troponin C Occurs in a Stepwise manner.
MX LI; SM GAGNE; S TSUDA; CM KAY; LB SMILLIE; BD SYKES, Biochemistry, 34, 26, 8330, 8340, 1995, [Peer-reviewed]
English

Interaction between Troponin-C and Inhibitory Region Peptide of Troponin-I as Studied by 1H-NMR.
Tsuda, S; Aimoto, S; Hikichi, K, Rept. Progr. Polym. Phys. Jpn., 35, 583-584., 1995

QUANTIFICATION OF THE CALCIUM-INDUCED SECONDARY STRUCTURAL-CHANGES IN THE REGULATORY DOMAIN OF TROPONIN-C
SM GAGNE; S TSUDA; MX LI; M CHANDRA; LB SMILLIE; BD SYKES, PROTEIN SCIENCE, 3, 11, 1961, 1974, Nov. 1994
English

NMR-STUDIES OF CALCIUM-BINDING TO N-DOMAIN OF CHICKEN TROPONIN-C
MX LI; SM GAGNE; S TSUDA; LB SMILLIE; BD SYKES, BIOPHYSICAL JOURNAL, 66, 2, A310, A310, Feb. 1994
English, Summary international conference

Quantification of the calcium-induced secondary structural changes in the regulatory domain of troponin-C.
Sakae Tsuda, Protein Sci, 3, 11, 1961, 1974, 1994, [Peer-reviewed]
English

DEVELOPMENT OF A LINKER WITH AN ENHANCED STABILITY FOR THE PREPARATION OF PEPTIDE THIOESTERS AND ITS APPLICATION TO THE SYNTHESIS OF A STABLE-ISOTOPE-LABELED HU-TYPE DNA-BINDING PROTEIN
H HOJO; Y KWON; Y KAKUTA; S TSUDA; TANAKA, I; K HIKICHI; S AIMOTO, BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN, 66, 9, 2700, 2706, Sep. 1993
English

IDENTIFICATION OF THE HYDROPHOBIC LIGAND-BINDING REGION IN RECOMBINANT GLUTATHIONE S-TRANSFERASE-P AND ITS BINDING EFFECT ON THE CONFORMATIONAL STATE OF THE ENZYME
J NISHIHIRA; T ISHIBASHI; M SAKAI; S TSUDA; K HIKICHI, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 302, 1, 128, 133, Apr. 1993
English

IDENTIFICATION OF THE HYDROPHOBIC LIGAND-BINDING REGION IN RECOMBINANT GLUTATHIONE S-TRANSFERASE-P AND ITS BINDING EFFECT ON THE CONFORMATIONAL STATE OF THE ENZYME
NISHIHIRA, J; ISHIBASHI, T; SAKAI, M; TSUDA, S; HIKICHI, K, Archives of Biochemistry and Biophysics, 302, 1, 128, 133, 1993, [Peer-reviewed]
English

Development of a Linker with an Enhanced Stability for the Preparation of a Peptide Thioesters and Its Application to the Systhesis of a Stable-Isotope-Labeled HU-Type DNA-Binding Protein.
Sakae Tsuda, Bull. Chem. Soc. Jpn., 66, 9, 2700, 2706, 1993, [Peer-reviewed]
English

H-1-NMR STUDY OF CA2+-DEPENDENT AND MG2+-DEPENDENT INTERACTION BETWEEN TROPONIN-C AND TROPONIN-I INHIBITORY PEPTIDE (96-116)
S TSUDA; S AIMOTO; K HIKICHI, JOURNAL OF BIOCHEMISTRY, 112, 5, 665, 670, Nov. 1992
English

CHARACTERIZATION OF CYSTEINE RESIDUES OF GLUTATHIONE S-TRANSFERASE-P - EVIDENCE FOR STERIC HINDRANCE OF SUBSTRATE BINDING BY A BULKY ADDUCT TO CYSTEINE-47
J NISHIHIRA; T ISHIBASHI; M SAKAI; S NISHI; T KUMAZAKI; Y HATANAKA; S TSUDA; K HIKICHI, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 188, 1, 424, 432, Oct. 1992
English

1H-NMR study of rabbit skeletal muscle troponin C: Ca2+-dependent interaction with mastoparan
Sakae Tsuda; Kunio Hikichi, Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular, 1121, 1-2, 213, 220, 22 May 1992
English

H-1-NMR STUDY OF RABBIT SKELETAL-MUSCLE TROPONIN-C - CA2+-DEPENDENT INTERACTION WITH MASTOPARAN
S TSUDA; K HIKICHI, BIOCHIMICA ET BIOPHYSICA ACTA, 1121, 1-2, 213, 220, May 1992
English

The Ca2+-Binding Property of Carp Parvalbumin 5a as Studied by 1H-NMR Spectroscopy.
Moriyasu, M; Ohki, S; Tsuda, S; Hikichi, K, Rept. Progr. Polym. Phys. Jpn., 35, 579, 580, 1992

CHARACTERIZATION OF CYSTEINE RESIDUES OF GLUTATHIONE S-TRANSFERASE-P - EVIDENCE FOR STERIC HINDRANCE OF SUBSTRATE BINDING BY A BULKY ADDUCT TO CYSTEINE-47
NISHIHIRA, J; ISHIBASHI, T; SAKAI, M; NISHI, S; KUMAZAKI, T; HATANAKA, Y; TSUDA, S; HIKICHI, K, Biochemical and Biophysical Research Communications, 188, 1, 424, 432, 1992, [Peer-reviewed]
English

The Ca2+-Binding Property of Carp Parvalbumin 5a as Studied by 1H-NMR Spectroscopy.
Moriyasu, M; Ohki, S; Tsuda, S; Hikichi, K, Rept. Progr. Polym. Phys. Jpn., 35, 579-580., 1992

1H-NMR study of Ca2+- and Mg2+-dependent interaction between troponin C and troponin I inhibitory peptide (96-116)1
Sakae Tsuda; Saburo Aimoto; Kunio Hikichi, Journal of Biochemistry, 112, 5, 665, 670, 1992
Oxford University Press, English

H-1-NMR STUDY OF CASEIN PHOSPHOPEPTIDE (1-25) - ASSIGNMENT AND CONFORMATION
S TSUDA; R NIKI; T KUWATA; TANAKA, I; K HIKICHI, MAGNETIC RESONANCE IN CHEMISTRY, 29, 11, 1097, 1102, Nov. 1991
English

H NMR-STUDY ON AMIDE PROTON-EXCHANGE OF CALMODULIN-MASTOPARAN COMPLEX
S OHKI; S TSUDA; S JOKO; M YAZAWA; K YAGI; K HIKICHI, JOURNAL OF BIOCHEMISTRY, 109, 2, 234, 237, Feb. 1991
English

Two-Dimensional NMR Studies of the synthetic repeat pentapeptide.
Kumaki, Y; Tsuda, S; Hikichi, K; Hojo, H; Aimoto, S; Matsushima, N, Rept. Progr. Polym. Phys. Jpn., 34, 475-478., 1991

Analysis of Ca2+-binding to Troponin-C as Studied by 1H-NMR.
Tsuda, S; Kakita, S; Hikichi, K, Rept. Progr. Polym. Phys. Jpn., 34, 461-462., 1991

Two-Dimensional NMR Studies of the synthetic repeat pentapeptide.
Kumaki, Y; Tsuda, S; Hikichi, K; Hojo, H; Aimoto, S; Matsushima, N, Rept. Progr. Polym. Phys. Jpn., 34, 475-478., 1991

Analysis of Ca2+-binding to Troponin-C as Studied by 1H-NMR.
Tsuda, S; Kakita, S; Hikichi, K, Rept. Progr. Polym. Phys. Jpn., 34, 461-462., 1991

1H NMR study of casein phosphopeptide (1–25): Assignment and conformation
Sakae Tsuda, Magn. Reson. Chem, 29, 11, 1097, 1102, 1991, [Peer-reviewed]
English

HYDRATION OF OLIGOSACCHARIDES
H UEDAIRA; M ISHIMURA; S TSUDA; H UEDAIRA, BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN, 63, 12, 3376, 3379, Dec. 1990
English

PRACTICAL IMPLEMENTATION OF THE SELF-REFOCUSED 1331 SOLVENT-SUPPRESSION SEQUENCE
K TAKEGOSHI; S TSUDA; K HIKICHI, JOURNAL OF MAGNETIC RESONANCE, 89, 2, 399, 405, Sep. 1990
English, Introduction scientific journal

H-1-NMR STUDY OF RABBIT SKELETAL-MUSCLE TROPONIN-C - MG-2+-INDUCED CONFORMATIONAL CHANGE
S TSUDA; K OGURA; Y HASEGAWA; K YAGI; K HIKICHI, BIOCHEMISTRY, 29, 20, 4951, 4958, May 1990
English

1H-NMR Spectral Assignment of RNA HexamerGGICCC.
Tsuda, S; Sasaki, T; Ohtsuka, E; Hikichi, K, Rept. Progr. Polym. Phys. Jpn., 33, 583-584., 1990

Two-Dimensional 1H-NMR Study of Troponin C in The Mg2+-Saturated State.
Tsuda, S; Yazawa, M; Morita, F; Hikichi, K, Rept. Progr. Polym. Phys. Jpn., 33, 587-588., 1990

The Interaction between Trifluoperazine and Troponin C as Studied by 1H-NMR.
Kakuta, Y; Tsuda, S; Yazawa, M; Hikichi, K, Rept. Progr. Polym. Phys. Jpn., 33, 585-586., 1990

Interaction between mastoparan and Troponin-C as Studied by Nuclear Magnetic Resonance.
Tsuda, S; Yazawa, M; Yagi, K; Hikichi, K, Rept. Progr. Polym. Phys. Jpn., 32, 531-532., 1990

Chemical synthesis of oligoribonucletides for structural studies.
Sakae Tsuda, Nucleic Acids Research, 22, 43, 44, 1990, [Peer-reviewed]
English

1H NMR Study of Rabbit Skeletal Muscle Troponin C: Mg2+-Induced Conformational Change.
Sakae Tsuda, Biochemistry, 29, 20, 4951, 4958, 1990, [Peer-reviewed]
English

Practical Implementation of the Self-Refocused 1331 Solvent Suppression.
Sakae Tsuda, J. Magn. Reson., 89, 2, 399, 405, 1990, [Peer-reviewed]
English

Two-Dimensional 1H-NMR Study of Troponin C in The Mg2+-Saturated State.
Tsuda, S; Yazawa, M; Morita, F; Hikichi, K, Rept. Progr. Polym. Phys. Jpn., 33, 587-588., 1990

CHEMICAL SYNTHESIS OF OLIGORIBONUCLEOTIDES FOR STRUCTURAL STUDIES
T SASAKI; S IWAI; S TSUDA; K HIKICHI; E OHTSUKA, SYMPOSIUM ON NUCLEIC ACIDS TECHNOLOGY, 22, 43, 44, 1990
English

1H-NMR Spectral Assignment of RNA HexamerGGICCC.
Tsuda, S; Sasaki, T; Ohtsuka, E; Hikichi, K, Rept. Progr. Polym. Phys. Jpn., 33, 583-584., 1990

Hydration of Oligosaccharides
Sakae Tsuda, Bull. Chem. Soc. Jpn, 63, 12, 3376, 3379, 1990, [Peer-reviewed]
English

The Interaction between Trifluoperazine and Troponin-C as Studied by 1H-NMR.
Kakuta, Y; Tsuda, S; Yazawa, M; Hikichi, K, Rept. Progr. Polym. Phys. Jpn., 33, 585-586., 1990

Interaction between mastoparan and Troponin-C as Studied by Nuclear Magnetic Resonance.
Tsuda, S; Yazawa, M; Yagi, K; Hikichi, K, Rept. Progr. Polym. Phys. Jpn., 32, 531-532., 1990

SELF-REFOCUSED BINOMINAL PULSE SEQUENCE FOR SOLVENT SUPPRESSION
K TAKEGOSHI; S TSUDA; K HIKICHI, JOURNAL OF MAGNETIC RESONANCE, 85, 1, 198, 202, Oct. 1989
English

A 1H-NMR Study on Hydrogen Exchange of Rabbit Skeletal Muscle Troponin-C.
Yamazaki, Y; Tsuda, S; Yagi, K; Hikichi, K, Rept. Progr. Polym. Phys. Jpn., 32, 533-534., 1989

Self-Refocused Binominal Pulse Sequence for Solvent Suppression
Sakae Tsuda, J. Magn. Reson., 85, 1, 198, 202, 1989, [Peer-reviewed]
English

A 1H-NMR Study on Hydrogen Exchange of Rabbit Skeletal Muscle Troponin-C.
Yamazaki, Y; Tsuda, S; Yagi, K; Hikichi, K, Rept. Progr. Polym. Phys. Jpn., 32, 533-534., 1989

Magnesium Binding to the Low Affinity Calcium Sites of Troponin C as determined by Nuclear Magnetic Resonance.
Tsuda, S; Ogura, K; Hasegawa, Y; Yagi, K; Hikichi, K, Rept. Progr. Polym. Phys. Jpn., 31, 565-566., 1988

Nuclear magnetic resonance study on rabbit skeletal troponin C: calcium-induced conformational change
S TSUDA; Y HASEGAWA; M YOSHIDA; K YAGI; K HIKICHI, Biochemistry, 27, 11, 4120, 4126, 1988, [Peer-reviewed]
English

Magnesium Binding to the Low Affinity Calcium Sites of Troponin C as determined by Nuclear Magnetic Resonance.
Tsuda, S; Ogura, K; Hasegawa, Y; Yagi, K; Hikichi, K, Rept. Progr. Polym. Phys. Jpn., 31, 565-566., 1988

Nuclear magnetic resonance study on rabbit skeletal troponin C: calcium-induced conformational change
Sakae Tsuda, Biochemistry, 27, 11, 4120, 4126, 1988, [Peer-reviewed], [International Magazine]
English

Functions and Applications of Antifreeze Preotein
TSUDA Sakae
シーエムシー出版, 2018, 9784781313399, viii, 289p, Japanese, [Supervisor]

タンパク質の辞典
朝倉書店, 2008, 9784254171280

TANPAKUSITSU-NO-JITEN (Japanese)
ASAKURA SHUPPAN, 2008, 9784254171280

産総研のすごい仕事
日系BP出版センター, 2006, 9784861530067

SANSOUKEN-NO-SUGOI-SHIGOTO
NIKKEI BP SHUPPAN Center, 2006, 9784861530067

不凍タンパク質とは何か？
津田 栄
第74回日本生物工学会大会, 18 Oct. 2022
[Invited]

Fish ice-binding proteins differently function at higher concentrations
Sakae Tsuda
International meeting of ice binding protein (IBP2022), 01 Aug. 2022, English
[Invited]

不凍タンパク質／凍ってもおいしいのはどうして？冷凍物の品質を向上させる技術
津田 栄
STEAMライブラリー・未来の教室, 04 Feb. 2022
[Invited]

Ice and Antifreeze Protein - How does water freezes? -
Sakae Tsuda
Cryopreservation Conference 2021, 11 Nov. 2021, Japanese, Invited oral presentation
[Invited]

What is Antifreeze Protein? - from basic to application -
Sakae Tsuda
61st Summer Seminar of Life Science, 27 Aug. 2021, Japanese, Public discourse
[Invited]

Discovery of the hyperactive antifreeze protein from a Japanese stag beetle
Sakae Tsuda
IBP International Seminar Series 2021-2022, 28 Jul. 2021, English, Invited oral presentation
28 Jul. 2021 - 28 Jul. 2021, [Invited]

Practical use of new quality products of antifreeze protein
Sakae Tsuda
7th annual conference of AnalytiX-2019, 14 Nov. 2019, English, Invited oral presentation
[Invited]

ICE RECRYSTALLIZATION IS EFFICIENTLY TERMINATED BY ANTIFREEZE PROTEIN-BINDING TO MULTIPLE ICE PLANES
津田 栄
国際低温生物学会 Cryobiology 2019, 23 Jul. 2019
[Invited]

Application of new quality products of type I & III antifreeze proteins and antifreeze glycoprotein
津田 栄
11th European Biosimilars Congress, 26 Apr. 2018
[Invited]

不凍タンパク質の分子機能と技術応用
津田 栄
石川県立大学食品科学科公開セミナー, 04 Dec. 2017
[Invited]

不凍タンパク質とは何か？ー分子機能解明から産業医学応用までー
津田 栄
防衛大学校応用化学科課外講演, 28 Nov. 2017
[Invited]

不凍タンパク質の分子生物学
津田 栄
2017年度日本魚類学会年会, 18 Sep. 2017
[Invited]

不凍タンパク質の分子機能と技術応用
津田 栄
第15回Spring-8先端研究ワークショップ, 24 Aug. 2017
[Invited]

A new type I antifreeze protein BpAFP undergoes oligomerization to bind to whole surface of an ice crystal
津田 栄
2nd International Conference on Bioscience, 19 Jun. 2017
[Invited]

不凍タンパク質の生産と産業医学応用
津田 栄
TIAかけはし第3回ナノバイオ・コンソーシアム討論会, 27 Feb. 2017
[Invited]

不凍タンパク質の機能解明に基づく産業応用展開
Sakae Tsuda
H24中国地域産総研技術セミナーin米子. 米子, 07 Dec. 2012
[Invited]

不凍蛋白質の機能と産業応用に関する研究
Sakae Tsuda
第9回メタンハイドレート産業創出イノベーション講演会．東京, 05 Nov. 2012
[Invited]

食品分野における不凍蛋白質（AFP）の活用技術について
津田 栄
日本食品工業倶楽部 東京部会（品質保証懇話会）, 24 Apr. 2012
[Invited]

不凍タンパク質の大量精製と新たな応用開拓
津田 栄
H24産総研新規採用職員初期研修ロールモデル懇談会, 11 Apr. 2012
[Invited]

Recent technological developments utilizing antifreeze protein
津田 栄
1st International Ice-Binding Protein (IBP) Conference and Workshop, 03 Aug. 2011
[Invited]

不凍タンパク質の分子機能解明に基づく新技術の創成
津田 栄
極限環境生物学会第12回シンポジウ, 13 Jun. 2011
[Invited]

不凍タンパク質の分子機能解析と応用技術研究
津田 栄
酵素工学研究会第65回講演会, 22 Apr. 2011, Japanese
[Invited]

New technologies utilizing the functions of antifreeze protein
12th Annual Meeting of Japanese Society of Extremophiles, 2011

Functional analyses of antifreeze protein and its practical applications
65th Annual Meeting of Japanese Society of Enzyme Engineering, 2011

NEW TECHNOLOGIES UTILIING ANTIFREEZE PROTEIN
12th INTERNATIONAL CONFERENCE ON THE PHYSICS AND CHEMISTRY OF ICE (PCI-2010), 2010

INTERACTION BETWEEN ICE AND A TYPE III ANTIFREEZE PROTEIN UNDER PRESSURE
12th INTERNATIONAL CONFERENCE ON THE PHYSICS AND CHEMISTRY OF ICE (PCI-2010), 2010

Molecular analyses of antifreeze protein for its industrial applications
5th alliance seminar for methane-hydrate research laboratory of AIST, 2010

Functional analyses of antifreeze protein for a new technology
10th annual meeting of protein science society of Japan, 2010

Structure and function analyses of antifreeze protein for a novel technology
Annual Seminar for the national newtron diffraction center of Ibaraki prefecture, 2010

不凍タンパク質の分子機能解明と産業応用に関する研究（依頼セミナー）
室蘭工業大学・創製機能工学専攻セミナー, 2009

不凍タンパク質の分子機能解明と食品分野への応用（依頼講演）
第56回日本食品科学工学会, 2009

New technologies developed for practical use of antifreeze protein (基調講演)
46th Annual Meeting of the Society for Cryobiology, 2009

Analysis of antifreeze function for technological application
Annual seminar held at Division of Engineering for Composite Functions in Muroran Institute of Technology, 2009

Functional analysis of antifreeze protein and its application to food technologies
56th Annual Meeting of the Japanese Society for Food Science and Technology, 2009

New technologies developed for practical use of antifreeze protein
46th Annual Meeting of the Society for Cryobiology (CRYO2009), 2009

Antifreeze Protein - from functional analysis to technological applications- (基調講演)
2nd International Symposium on the Environmental Physiology of Ectotherms and Plants (ISEPEP2, Duniden, NZ)., 2007

Antifreeze Protein from Japanese Fish: An epoch in biotechnology.
The 234th. American Chemical Society Meeting - Antifreeze Proteins: A Memorial Synposium for Robert Feeney. (Boston, USA), 2007

魚類由来不凍タンパク質を用いた冷却エネルギー削減技術の創生（依頼講演）
九州大学WSフォーラム「タンパク質・ペプチド研究の現状と展望」(福岡), 2007

魚肉すり身由来不凍タンパク質による新しい凍結保存技術の開発（依頼講演）
産総研研究講演会in中部（名古屋）, 2007

不凍タンパク質（ＡＦＰ）の謎と産業利用の可能性（招待講演）
極限環境下の生物に学ぶ食品技術フォーラム（東京）, 2007

Development of practical freezing techniques by using antifreeze protein
AIST Forum in Nagoya, 2007

Saving of the freezing energy by utilizing antifreeze protein
Kyusyu University WS-Forum "Current situation and future aspects of protein and peptides, 2007

Antifreeze Protein from Japanese Fish: An epoch in biotechnology
The 234th American Chemical Society Meeting - Antifreeze Proteins: A Memorial Synposium for Robert Feeney (USA), 2007

Antifreeze Protein - from functional analysis to technological applications-
2nd International Symposium on the Environmental Physiology of Ectotherms and Plants (ISEPEP2, NZ), 2007

Functions of antifreeze protein (AFP) and its practical use in industry
Food Technology Forum, 2006

北の自然からの贈り物ー不凍タンパク質ー（依頼講演）
市民講演会 ー世界物理年にあたり、生命を新しい視点から解き明かすー（札幌）, 2005

純国産不凍蛋白質のバイオテクノロジー
Ｈ１６産業技術連携推進会議生命工学部会（福岡）, 2005

A special gift from nothern nature - antifreeze protein -
A forum for the citizen of Sapporo, 2005

Biotechnology utilizing Japan-original antifreeze protein
2005 AIST Life Science Meeting, 2005

不凍タンパク質ー３次元分子構造解明から産業応用までー（依頼セミナー）
北大創成科学研究機構セミナー（札幌）, 2004

Antifreeze Protein - from structural analysis to industrial application -
産総研-韓国工業科学技術会ジョイントワークショップ（札幌）, 2004

Antifreeze Protein - from structural basis to industrial application -
Hokkaido University Sousei-kagaku seminar, 2004

Antifreeze Protein - from structural basis to industrial application -
AIST-Korea Joint Workshop, 2004

純国産不凍蛋白質の研究（依頼講演）
第30回日本低温医学会総会（札幌）, 2003

純国産不凍蛋白質の研究－臓器保存等への応用の可能性－
第２回北海道海洋生物科学シンポジウム（札幌）, 2003

不凍蛋白質の３次元構造解析および産業応用研究
Ｈ１５産業技術連携推進会議生命工学部会（札幌）, 2003

不凍蛋白質－分子構造解明から産業応用まで－（招待講演）
Ｈ１５低温生物工学会年会（札幌）, 2003

純国産不凍蛋白質研究の最新状況（依頼講演）
第３回蛋白質科学会（札幌）, 2003

Japanese Antifreeze Proteins
Symposium on Stress Proteins: From Antifreeze to Heat Shock（California, USA）, 2003

極地魚類の低温適応 -凍結から身を守る氷晶形成阻害蛋白質-
大阪大学蛋白質研究所セミナー（大阪）, 2003

Antifreeze Protein from Japanese Fish
30th. Annual Meeting of the Japan Society for Low Temperature Medicine, 2003

Antifreze Protein from Japanese Fish - application for tissue preservation -
2nd Hokkaido Marine Bioscience Symposium, 2003

Three-Dimensional Structure of Fish Antifreeze Protein and its Industrial Applications
2003 AIST Life Science Meeting, 2003

Antifreeze Protein - from structural basis to industrial application -
2003 Annual Meeting of Japanese Society for Cryobiology and Cryotechnology, 2003

Antifreeze Protein from Japanese Organisms
3rd Annual meeting of Japanese Society for Protein Science, 2003

Cold Adaptation of Arctic Fish - Expression of Ice binding Protein -
Special Seminar at the Institute for Protein Research in Osaka University, 2003

キノコの不凍タンパク質の分子構造と不凍機能のメカニズムを解明
Sakae Tsuda
第21回産総研･新技術セミナーin弘前．弘前

不凍タンパク質とは何か？ー凍結技術分野での応用可能性ー
Sakae Tsuda
株式会社サイエンスフォーラム主催講演会．東京

Antifreeze Protein from Japanese Organisms: Functional Analysis for the General Use
Sakae Tsuda
50th Annual meeting of the society for cryobiology (CRYO2013). Washington DC, USA

新技術の創生をもたらす不凍蛋白質の分子機能解明
Sakae Tsuda
H25北海道地区国立大学法人技術職員研修セミナー

不凍蛋白質の機能解明に基づく技術創生
Sakae Tsuda
第1回オープンファシリティシンポジウム．札幌

The membrane-binding ability of fish IBP prolongs the life-time of a cell dramatically
Sakae Tsuda
2nd International Ice-Binding Protein Conference (IBP2014)

不凍蛋白質の分子機能解明に基づく新規細胞保存技術の開発
Sakae Tsuda
基礎生物学研究所IBBPセンター主催 Cryopreservation conference 2014. 岡崎

不凍タンパク質とは何か？ーその構造多様性と分子機能の関係ー
Sakae Tsuda
大阪大学大学院理学研究科講演会. 大阪

不凍タンパク質の構造ー氷結晶結合機能の解明とその応用ー
Sakae Tsuda
2016日本真空学会年会．札幌

Mass preparation of fish antifreeze protein.
Sakae Tsuda
4th International conference on Bioprocess and Bio Thrapeutics (Bioprocess-2016). Houston, USA.

高純度魚類不凍タンパク質を用いた細胞保存技術の創生
Sakae Tsuda
基礎生物学研究所IBBPセンター主催 Cryopreservation conference 2016. 岡崎

不凍物質の科学
Sakae Tsuda
第3回低温科学技術交流調査会（ちよだプラットフォーム）. 東京

Editorial Board Member of Scientific Reports (nature.com)
01 Apr. 2019 - 30 Sep. 2024, [Others]

水の凍結を制御する水分子凝集層の開発
科学研究費助成事業
01 Apr. 2024 - 31 Mar. 2027
津田 栄
日本学術振興会, 基盤研究(C), 東京大学, 24K08487

Application of hyperactive antifreeze from a cold-tolerant insect
Grants-in-Aid for Scientific Research Challenging Research (Exploratory)
28 Jun. 2019 - 31 Mar. 2022
TSUDA SAKAE
This research clarified that a stag beetle (Dorcus hopei binodulosus) synthesizes a hyperactive antifreeze protein (DhbAFP), which unexpectedly shares a significant identity with an AFP identified from a phylogenetically distant beetle discovered in 1990s. It also appeared that Dhb larvae are not frozen during minus 5 deg C storage for 24 hrs. The native DhbAFP sample appeared to be a mixture of 6 isoforms, each of which consists of tandem repeats of a 12-residue consensus sequence. A special instrument to purify this beetle AFP by employing ice-probe was also prepared, which simplified the AFP preparation procedure. A supercooled cell storage solution containing DhbAFP appeared to improve the survival rate (%) of rat insulinoma cells, which suggests that AFP binds to embryo ice crystals generated in supercooled water to make them "nano" ice crystals, thereby minimizing physical damages onto the preserved cells. These results were published in world-wide.
Japan Society for the Promotion of Science, Challenging Research (Exploratory), National Institute of Advanced Industrial Science and Technology, 19K22989

Development of new cell preservation technologies utilizing antifreeze protein
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
01 Apr. 2019 - 31 Mar. 2022
Tsuda Sakae
The present study revealed that (1) a phenomenon called "freeze-induced concentration" is strongly inhibited by using liquid nitrogen or AFP, among which the latter consumes quite a few energy cost, (2) the survival rate (%) is not so improved when rat insulinoma cells (RIN-5F) were cooled to be filled with relatively small ice crystals, and (3) the survival rate (%) is dramatically improved when the RIN-5F cells were cooled to make it supercooling state with AFP, or make it to be filled with ultimately small "nano" ice crystals. In this study supercooling state was achieved by using insect-derived AFP, which realized significantly high (~53%) survival rate (%) of RIN-5F cells even after 20 days of preservation at -5 deg C. These new findings were opened to public though many journals, seminars, and invited lectures. Dr. Tsuda received "2019 Society Award" from Japanese society for cryobiology and cryotechnology.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), National Institute of Advanced Industrial Science and Technology, 19H02529

Development of a cooling preservation method that secures the function of retinal tissue for regenerative medicine
Grants-in-Aid for Scientific Research
Apr. 2016 - Mar. 2019
Koide Naoshi; SUNAGAWA Genshiro; TSUDA Sakae
In this research, we worked on development of preservation technology that applied fluorescence anisotropy and antifreeze protein. With regard to fluorescence anisotropy, it was not possible to minimize the light scattering by water due to the thickness problem unique to the three-dimensional structure and the floating state, and it could not be completed. Antifreeze protein was confirmed to improve cell viability by the addition of type III AFP in a refrigerated environment. On the other hand, with regard to cell transport in clinical research conducted in our laboratory, we have reached the determination of transport conditions in clinical research by making use of the know-how acquired in this task. As evidence from scientific evidence, cytotoxicity due to overcooling was also clearly confirmed, and vibration verification was found to be sufficient to provide limited consideration in preparing transport materials at a certain level.
Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B), Institute of Physical and Chemical Research, 16K21633

Quantitative analysis of the ability of ice recrystallization inhibition of antifreeze protein
Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
01 Apr. 2015 - 31 Mar. 2017
Tsuda Sakae
Numerous embryo single ice crystals are generated in water when it is frozen. They are progressing crystal growth and combined together to form a multi crystalline state, whose process is called ice recrystallization. Antifreeze protein (AFP) has a strong ability of ice recrystallization inhibition (IRI) , while it has not been established an efficient method to analyze the IRI activity quantitatively. Here we developed a new method, which is different from the known splat cooling assay, to precisely evaluate IRI rate of AFP based on the equation (r3 = r3(0) + Kt) of the Ostwald Ripening. We also identified a new species of AFP (BpAFP) from righteye flounder that showed a strong IRI and thermal hysteresis activity in concentration-dependent manner.
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research, National Institute of Advanced Industrial Science and Technology, 15K13760

不凍ペプチドを用いた牛生殖細胞と初期胚の超高性能保存液の開発
イノベーション創出基礎的研究推進事業
Apr. 2011 - Mar. 2014
青柳敬人; 津田 栄
生物系特定産業技術研究支援センター（生研セ）, Principal investigator, Competitive research funding

Functional analysis and application of an effective cell-preservation peptide CPP
Grants-in-Aid for Scientific Research
2011 - 2014
TSUDA SAKAE; KONDO Hidemasa; NISHIMIYA Yoshiyuki; SAKASHITA Mami
Antifreeze proteins (AFPs) found from many organisms can inhibit ice crystal growth for cold-survival. We preliminarily found that some AFPs also show cell-preservation ability and named them CPP (Cell-Preservation Peptide). During the 3-year research period, we have been tested structure-function relationship of various AFPs to obtain the most promising CPP. In 2013, we finally could develop a method to alive rat insulinoma cells for more than 5 days at +4 degree C by using a flat-fish-derived CPP (PLoS ONE, 2013). In addition, we published the 1st crystal structure of fungal AFP (PNAS, 2012) and structure-function of CPP3 based on the multi-dimensional NMR experiments (JBNMR, 2013). Biochechemical characterizations and many applicational results about AFP and CPP were also published: 23 publications (incl.19 peer-reviewed articles), 40 presentations (incl.14 invited speeches), and 1 patent claim.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), 独立行政法人産業技術総合研究所, Principal investigator, Competitive research funding, 23310171

Elucidation of protein architecture and molecular evolution of microbial antifreeze proteins
Grants-in-Aid for Scientific Research
2008 - 2010
KONDO Hidemasa; NISHIMIYA Yoshiyuki; TSUDA Sakae; HOSHINO Tamotsu
Antifreeze proteins bind preferentially to the surfaces of embryonic ice crystals and inhibit further growth of ice. In the present study we analyzed three-dimensional structures of microbial antifreeze proteins and revealed their possible ice-binding site by structure-based site-directed mutagenesis. Antifreeze activities measured for various kinds of microbial antifreeze proteins exhibited wide diversity both in thermal hysteresis value and ice crystal shape, in spite of their high sequence homologies. From these observations it is suggested that microbial antifreeze proteins share a common structural scaffold for their interaction with ice and adopt their own antifreeze activities according to their growing environment.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), National Institute of Advanced Industrial Science and Technology, Coinvestigator not use grants, Competitive research funding, 20570118

不凍蛋白質・氷核蛋白質の探索、分子機構解明および食品・医学・工学分野への応用
ライフサイエンス基礎科学研究
2000
Competitive research funding

Antifreeze protein - from structural basis to technological applications -
2000
Antifreeze protein (AFP) initially isolated from the blood serum of polar fish in 1960's is an extraordinary bio-molecule that specifically binds to both ice and cell surfaces. Tsuda discovered for the first time that approximately 50 kinds of Japanese edible fish also contain AFP, and developed an easy method to obtain ~kg order of natural AFPs from the fish muscle, but not from the blood serum. He also found that an AFP isoform having no thermal hysteresis exhibits an extreme cell-preservation ability. Additionally, he determined the first NMR structure of a natural intra-molecular AFPIII dimmer RD3 and the first X-ray structure of Ca2+-independent AFPII LpAFP.
Competitive research funding

NMRを用いたトロポニンCとトロポニンエペプチドの相互作用の研究
科学研究費助成事業
1992 - 1992
津田 栄
日本学術振興会, 奨励研究(A), 北海道大学, 04780272

カルシウム結合タンパク質の構造のNMRによる研究
科学研究費助成事業
1992 - 1992
引地 邦男; 津田 栄; 竹腰 清乃理; 田中 勲
真核生物のカルモデュリン(CaM)は、EF-ハンド構造を持つ4つのカルシウムイオン(Ca)結合部位を持つ。CaMの1次構造は種の違いを問わず90%以上の高い相同性がある。Ca結合部位はN,C両末端側に2つづつ対になって存在しており、C末端側の2つが高親和性部位である。酵母菌のカルモデュリン(YCMO)は他のCaMと1次構造の相同性が60%程度しかない。YCMOでは最もC末端側に位置するCa結合部位が潰れており1分子当たり3個しかCaを結合しないことが知られているが、どのCa結合部位が高親和性であるのかが明らかでなかった。我々は、YCMOのCa結合特性についてNMRを用いて調べた。YCMOにはN末端側とC末端側にそれぞれ1個のHis残基(61,107)がある。His107は他のCaMでもよく保存されているが、61番はCaMではGlyである。遺伝子操作によってHis61をGlyに置換した変異体(YCM61G)を発現させた。YCMOとYCM61Gの重水溶液の1H-NMRスペクトルを比較することによりHis61,His107のC2プロトンのシグナルを帰属した。YCMOの重水および軽水溶液にCaを滴定し、スペクトルを測定した。Caを加えるにつれてスペクトルは大きく変化し、この変化は[Ca]/[YCMO]=3で終了した。スペクトル変化を解析した結果、[Ca]/[YCMO]=0-2で大きく変化するシグナルと[Ca]/[YCMO]=2-3で大きく変化するシグナルの2種類に大別できることがわかった。先に帰属したHis61のシグナルは[Ca]/[YCMO]=0-2で変化するタイプに属することがわかった。また、[Ca]/[YCMO]=0-2で現われるアミドプロトンのシグナルにはCaMのNMRスペクトルとの比較からGly25と11e27と考えられるものが含まれていた。この結果は、YCMOはN端側の2つのCa結合部位が高親和性部位であることを示している。YCMOのCa結合特性はCaMとは異なることが明らかになった。この結果は、2つのCa結合部位が対になって何らかの安定な高次構造を形成することが高いCa親和性を持つための重要な鍵であることを強く示唆している。
日本学術振興会, 重点領域研究, 北海道大学, 04225202

Development of New Methods for NMR of Spin*1 in Solids
Grants-in-Aid for Scientific Research
1990 - 1992
HIKICHI Kunio; HIGUCHI Keiichiro; TSUDA Sakae; TAKEGOSHI Kiyonori
We examined several NMR approaches to observe NMR of nuclei with spin*1. Firstly, we modified our commercial NMR apparatus to incorporate a new versatile pulse programmer and a fast A/D converter. The modifications makes it possible to observe NMR signal with wide line-shape of spin*1 nuclei. Secondly, we observed the overtone NMR of ^<14>N in which the "overtone" transition (1 <1> -1) is observed.
In the overtone approach, the large first-order quadrupole interaction is eliminated for ^<14>N. The remaining line-broadening mainly comes from the second-order quadrupole splitting. Effects of sample spinning on the "overtone" NMR of quadrupole nuclei with spin=1 in the solid are investigated to exploit a possibility for removing the second-order quadrupole broadening from the "overtone" spectra. A good averaging of the second-order quadrupole broadening and a high sensitivity are achieved at spinning angles of 60-70ﾟ It is also shown that the magic angle (54.7ﾟ) produces a rather narrow single-peak for the second-order quadrupole broadening. Since the magic-angle spinning averages other anisotropies such as dipole-dipole and chemical shift interactions, it is concluded that the magic angle spinning can be used for samples with a large chemical shift anisotropy and/or dipole-dipole interactions.
Furthermore, the "overtone" NMR approach was used to determine a quadrupole coupling tensor of the ^<14>N nucleus in a single crystal. The theoretical basis of analysis of overtone NMR for a single crystal is also developed. It was found that the present overtone approach has some advantages, although it is not very promising for the accurate determination of quadrupole coupling constant. It demands less hardware requirement than the conventional ^<14>N single quantum approach ; one can observe all of ^<14>N signals in one spectrum. This allows us to conduct the two-dimensional exchange NMR among ^<14>N resonances. It is easy to observe the temperature dependence of quadrupole coupling constant of all ^<14>N nuclei.
Japan Society for the Promotion of Science, Grant-in-Aid for Developmental Scientific Research (B), Hokkaido University, 02554014

マストパランを結合したトロポニンCの2次元NMRによる高次構造解析
科学研究費助成事業
1991 - 1991
津田 栄
日本学術振興会, 奨励研究(A), 北海道大学, 03780241

Study of Sol-gel Transition using Various NMR Techniques
Grants-in-Aid for Scientific Research
1989 - 1990
HIKICHI Kunio; TSUDA Sakae; TAKEGOSHI Kiyonori
Kappa -Carrageenan gel which is formed by the addition of Rb^+ ion to its aqueous solution undergoes a transition into sol in the vicinity of 55^ﾟC with increasing temperature. In order to examine the behavior of water molecules in the gel, we measured the self-diffusion constant of water molecules by the nuclear magnetic resonance method.
The kappa -carrageenan gel was prepared by adding 50mM RbCl to 5wt% kappa -carrageenan aqueous solution. The self-diffusion constant was measured by the combined use of the Carr-Purcell-Meiboom-Gill method and the field gradient method using JEOL JNM-FX60 equipped with a PL502 field gradient unit. The field gradient unit was calibrated using the known self-diffusion constant of pure water at room temperature.
We measured the temperature dependence of the self-diffusion constant of pure water molecules in the temperature range from 25 to 75^ﾟC. The observed value was found to agree well with the value in the literature. We also measured the temperature dependence of the self-diffusion constant of water molecules of the 5wt% kappa -carrageenan aqueous solution. The values are almost the same as those of pure water.
The temperature dependence of the self-diffusion constant of the kappa -carrageenan gel was measured. It was found that the self-diffusion constant of water molecules in the gel state is smaller than that of pure water, indicating the restricted translational motion of water molecules inside the gel network. In the vicinity of 55^ﾟC where the gel-to-sol transition occurs, the self diffusion constant abruptly increases and approaches the value of pure water. The results indicate that the transition releases the translational freedom of motion of water. When the temperature is lowered, the transition region shifts to lower temperature, showing the hysteresis. The data were found to scatter in the transition region, supposedly implying the critical relaxation.
Japan Society for the Promotion of Science, Grant-in-Aid for General Scientific Research (C), Hokkaido University, 01550684

常磁性金属イオンをプローブとする高分子金属錯体の構造とゆらぎの研究
科学研究費助成事業
1988 - 1988
引地 邦男; 津田 栄; 鴇田 昌之; 平沖 敏文; 戸倉 清一
グルタミン酸がCu(II)あるいはMn(II)と錯体を形成する際にロイシンがどのように影響するかを調べるため、グルタミン酸とロイシンとのコポリマーについてNMRの研究を行った。グルタミン酸とリシンのコポリマーについて、グルタミン酸残基とリシン残基の近距離相互作用が錯体形成にどのように影響するかを調べた。 中性pH、室温においてpoly(glu、leu)にCu(II)あるいはMn(II)を加えると、グルタミン残基のシグナルの線幅が広がるが、ロイシンのシグナルにはほとんど影響が現れない。この結果は、Cu(II)あるいはMn(II)はグルタミン酸残基とのみ相互作用することを示しており、疎水性残基は大きな影響を与えないことを示唆している。グルタミン酸とリシンのコポリマーにCu(II)を加えると、グルタミン酸のCrとC_8シグナルが著しく影響を受けるが、リシン残基のシグナルはほとんど影響されない。pH10で、Cu(II)を加えた場合、リシン残基のC_ε、C_8のシグナルの線幅が広がり、更に、グルタミン酸のC_8、Crのシグナルの線幅も広がることがわかった。これらのことは、酸性pHでCu(II)はグルタミン酸のカルボキシラートに配位しアルカリpHでリシンのεアミノ基に配位するというグルタミン酸及びリシンのホモポリマーでこれまでに得られていた結果と一致する。しかし、アルカリpHでグルタミン酸の側鎖炭素も影響を受けることはホモポリマーでは見られなかったことで、リシンのεアミノ基とグルタミン酸のカルボキシラートの両者がCu(II)に配位していることを示唆している。酸性pHでは、錯体の平均寿命はT_<1M>(〜1_)より短い。アルカリpHでは、平均寿命は1_より長い。いずれの温度においてもT_<2M>が10^<-8>_s程度と小さいためで、錯体の平均寿命は10^<-6>_sよりは長いことがわかる。pH5.6でCu(II)-グルタミン酸C_8の距離は2.3A、Crまでの距離は2.4Aと求められた。
日本学術振興会, 重点領域研究, 北海道大学, 63612501

Characterization of Polymers by Use of New NMR Techniques
Grants-in-Aid for Scientific Research
1986 - 1988
HIKICHI Kunio; TSUDA Sakae; TOKITA Masayuki
The putpose of this research is to establish a new method for characterizing polymers by use of the new NMR techniques. For the past three years we have been studying methodology which does not depend on empirical knowledge and uses twodimensional NMR methods. We propose a new method for characterization of vinyl type synthetic polymers. The method is as follows; (1) measure 1H J-resolved 2D spectra, obtain different chemical shifts for methins and methylenes, and disclose equivalent methylenes, (2) measure broad-band decoupled 1H COSY and correlate methin and methylene peaks with reference of the connectivity, (3) measure CHCOSY and assign 13C resonances through the results of 1H assignments, (4) measure INADEQUATE 2D spectra to assign 13C resonances in detail, and (5) characterize configurations from 13C resonances with help of determined assignments. The conventional method is baed on the assumption of a statistical distribution of configurations. The present method can be applied to resonances which can not be determined by the conventional method. So far, we have successfully studied poly(vinyl alcohol), poly(acryl amide), and poly(vinyl chloride). INADEQUATE 2D experiments are useful for determination of the distribution of comonomers in ethylene propylene copolymers. A newly developed HMQC method is quite helpful for correlating 1H and 13C resonances.
Japan Society for the Promotion of Science, Grant-in-Aid for General Scientific Research (A), Hokkaido University, 61430021

層及び構造体
Patent right
特許7708403

生体材料保護用ペプチド
Patent right
特許6664738

南極産子嚢菌の産生する新規不凍タンパク質
Patent right
特許5397848

不凍タンパク質を用いる多孔体の製造方法
Patent right
特許5536874

水又は含水物の凍結を促進するための材料
Patent right
特許4748480
特許 第7442769号（米国）

不凍タンパク質混合物
Patent right
特許4446058
特許 第4332646号（日本）

魚類由来の不凍タンパク質
Patent right
特許4332646

担子菌類の生産する不凍タンパク質
Patent right
特許4257970
特許 第4257970号（日本）、第7442769号（米国）、第1344827号（欧州）

氷核蛋白質の配列を含む不凍蛋白質
Patent right
特許4231928
特許 第4231928号（日本）

魚類由来の不凍タンパク質
Patent right
特許4228068
特許 第4228068号および 第4332646号（日本）、第1452539号（欧州）

不凍蛋白質を用いた包摂化合物の生成制御法
Patent right
特許4106440
特許 第4106440号（日本）

マルチマー化した高機能不凍タンパク質
Patent right
特許3906357
特許 第3906357号（日本）、第7041788号（米国）、第1334980号（欧州）

含水物中における物質の凍結濃縮を抑制する方法、生理活性物質の失活を抑制する方法、および成分が均質に拡散した凍結物又は凍結乾燥物を製造する方法
Patent right
WO 2004/082378 A1, 30 Sep. 2004