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7. Mutational and structural analysis of Ca2+-independent type II antifreeze protein from longsnout poacher
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Abstracts / Cryobiology 59 (2009) 370–418
the above two prominent functions, and to realize the practical use of AFP in a variety of industrial and medical fields, a ‘break through’ is anticipated that substantiates the preparation of unlimited amounts of the natural fish AFP (>1 kg). Our group has been trying to develop a method of isolating AFP from Japanese edible fish species for several years, and we have established a simple mass-preparation technique by utilizing fish muscle rather than blood serum as the source material. The best yield per week is currently obtained for type III AFP (AFPIII) in our laboratory out of the types I–III and antifreeze glycoprotein (AFGP). We have been examining the following with the AFPIII samples: (a) an inhibitory function of freeze-concentration phenomenon occurring in a gel (ex. agarose) below 0 °C, (b) ice-nucleation functioning by assembly of the AFP molecules on a metal surface (ex. aluminum) below 0 °C, and (c) preservation functions in various kinds of cell (HepG2, COS7, and IEC6) between 24 and 96 h above 0 °C. The presenter will show details and updates of these experiments in this meeting, and will discuss the possibility of ‘AFP technologies’ that possess environmentfriendly characteristics. (Conflicts of interest: None declared. Source of funding: Institutional sources of AIST.) doi:10.1016/j.cryobiol.2009.10.020
7. Mutational and structural analysis of Ca2+-independent type II antifreeze protein from longsnout poacher. *Yoshiyuki Nishimiya a, Hidemasa Kondo a, Manabu Takamichi a, Hiroshi Sugimoto b, Mamoru Suzuki c, Ai Miura a, Sakae Tsuda a, a Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517, Japan, b Biometal Science Laboratory, RIKEN SPring-8 Center, Harima Institute, 1-1-1, Kouto, Sayo, Hyogo 679-5148, Japan, c Institute for Protein Research, Osaka University, 3-2, Yamadaoka, Suita, Osaka 5650871, Japan Antifreeze proteins (AFPs) are able to bind specifically to the surface of ice that forms in an aqueous solution, and inhibits the growth of ice. Such ability of AFPs leads to a non-colligative freezing temperature depression of the aqueous solution; however, it does not significantly alter the melting point of ice. The difference between freezing temperature and the melting point is termed thermal hysteresis (TH), and is used as a measure of AFP activity. Fish type II AFPs are cysteine rich proteins ranging in molecular weight from 14 to 24 kDa, which have been identified from Atlantic herring, rainbow smelt, Japanese smelt and sea raven. They are further classified into Ca2+-dependent and -independent species based on the requirement of calcium ion for activity; only sea raven AFP (srAFP) has been reported as a Ca2+-independent species. The NMR structure of srAFP has been determined and mutational analyses have also been performed to identify its ice-binding site. However, the details of the ice-binding site and mechanism still remain unclear. We have isolated Ca2+-independent type II AFP (lpAFP) from longsnout poacher living in the northeast coast of Japan, and succeeded in expressing a recombinant lpAFP using Phichia pastoris. To obtain a higher resolution structure of Ca2+-independent species, we attempted X-ray crystallographic analysis of the recombinant lpAFP. The structure of lpAFP was determined at 1.34 Å resolution, and displayed a relatively planar protein surface, which appears to be suitable for binding to an ice crystal plane. Therefore, several mutants were prepared, focusing on the relatively planar surface, and its TH activities were examined. On the basis of the X-ray crystal structure and mutation analysis, we discuss the ice-binding site and further the ice-binding model of Ca2+-independent type II AFP. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2009.10.021
8. Crystal structure of Ca2+-dependent type II antifreeze protein from Japanese smelt. *Hidemasa Kondo a, Yoshiyuki Nishimiya a, Natsuko Noro a, Manabu Takamichi a, Masanori Yasui a, Ai Miura a, Sakae Tsuda a,b, a Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517, Japan, b Division of Biological Sciences, Graduate School of Science, Hokkaido University, N10 W8, Kita, Sapporo 060-0810, Japan Antifreeze proteins (AFPs) are preferentially adsorbed onto the surface of embryonic ice crystals to inhibit further growth of the crystal, leading to a reduction of the freezing point of the solution below the melting point. Various types of AFPs have been identified from cold-adapted organisms including fishes, insects and plants that can survive subzero temperature. Type II AFP from fish is a globular protein with a molecular weight of approximately 14 kDa and exhibits a sequence-similarity with the carbohydrate-recognition domain of C-type lectins. This type of AFP is further classified into two subgroups based on the dependence of Ca2+ ion on their antifreeze activity. Ca2+-dependent species have been isolated from herring and smelt, and require an equimolar amount of Ca2+ for the full activity. A wealth of structural and mutational analysis of the Ca2+-
dependent type II AFP have been reported, showing that the Ca2+-binding site is involved in the ability of AFP to bind to the ice surface. Three-dimensional structure of this type of AFP in the Ca2+-free state, however, have not been reported, leaving the structural conversion between active and inactive states unclear. In this study we determined the crystal structure of both of the Ca2+-bound and free states of the Ca2+-dependent type II AFP from Japanese smelt (jsAFP). A recombinant jsAFP was crystallized in acetate buffer at pH 4.0 containing ammonium sulfate, and polyethylene glycol 3350. In order to prepare the crystal of a Ca2+-bound jsAFP the crystal was transferred into the crystallization solution containing 5 mM CaCl2, the pH of which was adjusted to 7.0. For preparation the Ca2+-free state of jsAFP, another crystal was soaked in the crystallization solution with the pH adjusted to 3.0. The diffraction data of the Ca2+-bound and -free crystals were collected 1.06 and 1.25 Å, respectively. The structures were determined by the molecular replacement method using the coordinate of the Ca2+-independent type II AFP from longsnout poacher as a search model, and refined against 20–1.06 Å for the Ca2+-bound state and 20–1.25 Å for the Ca2+-free state. Overall structures of both states of jsAFP were approximately identical and exhibited close similarity with other typeII AFPs previously reported. An apparent difference between two states was observed only around the Ca2+-binding site. The electron density map of the Ca2+-bound jsAFP showed an apparent density for Ca2+ ion which is coordinated by Asp and Glu residues located on the Ca2+-binding site. In the case of the Ca2+-free state the center of the Ca2+-binding site was occupied by a water molecule and side chains of Ca2+-binding residues oriented opposite to the water molecule. These observations suggest that conformational change of the side chains around the Ca2+-binding site affects the ice binding ability of Ca2+-dependent type II AFP. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2009.10.022
9. Cooperative ice growth inhibition of the isoforms of type III antifreeze protein. *Manabu Takamichi a, Yoshiyuki Nishimiya a, Ai Miura a, Sakae Tsuda a,b, a Functional Protein Research Group, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Toyohira, Sapporo, Hokkaido 062-8517, Japan, b Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita, Sapporo, Hokkaido 060-0810, Japan Antifreeze protein (AFP) is a biological polymer that inhibits the growth of a seed ice crystal (i.e., ice nuclei) generated in the chilled blood and/or body fluid, which confers protection of the host life from freezing [1]. AFP binds to the ice crystal surface as the initial step of ice growth inhibition, which prevents further addition of water molecules onto the ice surface, leading to depression of the freezing point Tf of the solution in a non-colligative manner. Type III AFP, a class of AFP originated from fish, is a globular protein with molecular weight of approximately 7 kDa. The AFP is naturally expressed as a mixture of several isoforms categorized into two sub-groups named QAE- and SP-isoforms, which shares 55% identity of the amino acid sequence [2]. In this study, we examined the ability of ice growth inhibition of QAE- and SPisoforms through the photomicroscope observations and considered the biological role of each isoform. Experiments were performed for a single ice crystal prepared in each isoform solution by employing our custom-made photo-system, which realizes both preparation and observation of a single ice crystal (20 lm) with manual manipulation of temperature settings using electronic thermal controlling system [3]. QAE-isoform firmly inhibited ice crystal growth and modified the crystal into a bipyramidal shape, and that exhibited Tf depression similarly to the all known active AFPs. Virtually no ice growth occurred between Tm and Tf, while rapid ice growth was initiated at Tf. SP-isoform also modified the crystal shape and depressed the growth rate of the ice bipyramid, but it could not halt the ice growth and allowed continuous growing without change of the morphology. The continuous growth did not stop even at a high concentration of SP-isoform (5.0 mM) and at a slight supercooling 0.05 °C), indicating that SP-isoform causes no Tf depression. Further crystal observation revealed that in SP solution, the bipyramid suddenly initiated ice growth from the tip of bipyramid at a certain minus temperature. For QAE, the tip of bipyramid was known to be the growth initiation point at Tf, suggesting that SP and QAE commonly possess the ice growth inhibitory function against the tip of ice bipyramid. Also, SP and QAE were nearly equivalent in the initiation temperature from tip (For QAE, the temperature is same as Tf. This functional equivalence will explain the comparable Tf depression of SP with QAE in the mixture, as involvement of SP-isoform in the ice growth inhibition of bipyramid tip. These results suggest that although the SP-isoform is a less active isoform, with insufficient ice growth inhibition, SP would assist the inhibition of the active QAE-isoform by participating in the inhibition of ice growth from the tip of bipyramid that is weakest inhibited point, and contribute to further depression of Tf of the solution. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2009.10.023
Abstracts / Cryobiology 59 (2009) 370–418
the above two prominent functions, and to realize the practical use of AFP in a variety of industrial and medical fields, a ‘break through’ is anticipated that substantiates the preparation of unlimited amounts of the natural fish AFP (>1 kg). Our group has been trying to develop a method of isolating AFP from Japanese edible fish species for several years, and we have established a simple mass-preparation technique by utilizing fish muscle rather than blood serum as the source material. The best yield per week is currently obtained for type III AFP (AFPIII) in our laboratory out of the types I–III and antifreeze glycoprotein (AFGP). We have been examining the following with the AFPIII samples: (a) an inhibitory function of freeze-concentration phenomenon occurring in a gel (ex. agarose) below 0 °C, (b) ice-nucleation functioning by assembly of the AFP molecules on a metal surface (ex. aluminum) below 0 °C, and (c) preservation functions in various kinds of cell (HepG2, COS7, and IEC6) between 24 and 96 h above 0 °C. The presenter will show details and updates of these experiments in this meeting, and will discuss the possibility of ‘AFP technologies’ that possess environmentfriendly characteristics. (Conflicts of interest: None declared. Source of funding: Institutional sources of AIST.) doi:10.1016/j.cryobiol.2009.10.020
7. Mutational and structural analysis of Ca2+-independent type II antifreeze protein from longsnout poacher. *Yoshiyuki Nishimiya a, Hidemasa Kondo a, Manabu Takamichi a, Hiroshi Sugimoto b, Mamoru Suzuki c, Ai Miura a, Sakae Tsuda a, a Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517, Japan, b Biometal Science Laboratory, RIKEN SPring-8 Center, Harima Institute, 1-1-1, Kouto, Sayo, Hyogo 679-5148, Japan, c Institute for Protein Research, Osaka University, 3-2, Yamadaoka, Suita, Osaka 5650871, Japan Antifreeze proteins (AFPs) are able to bind specifically to the surface of ice that forms in an aqueous solution, and inhibits the growth of ice. Such ability of AFPs leads to a non-colligative freezing temperature depression of the aqueous solution; however, it does not significantly alter the melting point of ice. The difference between freezing temperature and the melting point is termed thermal hysteresis (TH), and is used as a measure of AFP activity. Fish type II AFPs are cysteine rich proteins ranging in molecular weight from 14 to 24 kDa, which have been identified from Atlantic herring, rainbow smelt, Japanese smelt and sea raven. They are further classified into Ca2+-dependent and -independent species based on the requirement of calcium ion for activity; only sea raven AFP (srAFP) has been reported as a Ca2+-independent species. The NMR structure of srAFP has been determined and mutational analyses have also been performed to identify its ice-binding site. However, the details of the ice-binding site and mechanism still remain unclear. We have isolated Ca2+-independent type II AFP (lpAFP) from longsnout poacher living in the northeast coast of Japan, and succeeded in expressing a recombinant lpAFP using Phichia pastoris. To obtain a higher resolution structure of Ca2+-independent species, we attempted X-ray crystallographic analysis of the recombinant lpAFP. The structure of lpAFP was determined at 1.34 Å resolution, and displayed a relatively planar protein surface, which appears to be suitable for binding to an ice crystal plane. Therefore, several mutants were prepared, focusing on the relatively planar surface, and its TH activities were examined. On the basis of the X-ray crystal structure and mutation analysis, we discuss the ice-binding site and further the ice-binding model of Ca2+-independent type II AFP. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2009.10.021
8. Crystal structure of Ca2+-dependent type II antifreeze protein from Japanese smelt. *Hidemasa Kondo a, Yoshiyuki Nishimiya a, Natsuko Noro a, Manabu Takamichi a, Masanori Yasui a, Ai Miura a, Sakae Tsuda a,b, a Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517, Japan, b Division of Biological Sciences, Graduate School of Science, Hokkaido University, N10 W8, Kita, Sapporo 060-0810, Japan Antifreeze proteins (AFPs) are preferentially adsorbed onto the surface of embryonic ice crystals to inhibit further growth of the crystal, leading to a reduction of the freezing point of the solution below the melting point. Various types of AFPs have been identified from cold-adapted organisms including fishes, insects and plants that can survive subzero temperature. Type II AFP from fish is a globular protein with a molecular weight of approximately 14 kDa and exhibits a sequence-similarity with the carbohydrate-recognition domain of C-type lectins. This type of AFP is further classified into two subgroups based on the dependence of Ca2+ ion on their antifreeze activity. Ca2+-dependent species have been isolated from herring and smelt, and require an equimolar amount of Ca2+ for the full activity. A wealth of structural and mutational analysis of the Ca2+-
dependent type II AFP have been reported, showing that the Ca2+-binding site is involved in the ability of AFP to bind to the ice surface. Three-dimensional structure of this type of AFP in the Ca2+-free state, however, have not been reported, leaving the structural conversion between active and inactive states unclear. In this study we determined the crystal structure of both of the Ca2+-bound and free states of the Ca2+-dependent type II AFP from Japanese smelt (jsAFP). A recombinant jsAFP was crystallized in acetate buffer at pH 4.0 containing ammonium sulfate, and polyethylene glycol 3350. In order to prepare the crystal of a Ca2+-bound jsAFP the crystal was transferred into the crystallization solution containing 5 mM CaCl2, the pH of which was adjusted to 7.0. For preparation the Ca2+-free state of jsAFP, another crystal was soaked in the crystallization solution with the pH adjusted to 3.0. The diffraction data of the Ca2+-bound and -free crystals were collected 1.06 and 1.25 Å, respectively. The structures were determined by the molecular replacement method using the coordinate of the Ca2+-independent type II AFP from longsnout poacher as a search model, and refined against 20–1.06 Å for the Ca2+-bound state and 20–1.25 Å for the Ca2+-free state. Overall structures of both states of jsAFP were approximately identical and exhibited close similarity with other typeII AFPs previously reported. An apparent difference between two states was observed only around the Ca2+-binding site. The electron density map of the Ca2+-bound jsAFP showed an apparent density for Ca2+ ion which is coordinated by Asp and Glu residues located on the Ca2+-binding site. In the case of the Ca2+-free state the center of the Ca2+-binding site was occupied by a water molecule and side chains of Ca2+-binding residues oriented opposite to the water molecule. These observations suggest that conformational change of the side chains around the Ca2+-binding site affects the ice binding ability of Ca2+-dependent type II AFP. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2009.10.022
9. Cooperative ice growth inhibition of the isoforms of type III antifreeze protein. *Manabu Takamichi a, Yoshiyuki Nishimiya a, Ai Miura a, Sakae Tsuda a,b, a Functional Protein Research Group, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Toyohira, Sapporo, Hokkaido 062-8517, Japan, b Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita, Sapporo, Hokkaido 060-0810, Japan Antifreeze protein (AFP) is a biological polymer that inhibits the growth of a seed ice crystal (i.e., ice nuclei) generated in the chilled blood and/or body fluid, which confers protection of the host life from freezing [1]. AFP binds to the ice crystal surface as the initial step of ice growth inhibition, which prevents further addition of water molecules onto the ice surface, leading to depression of the freezing point Tf of the solution in a non-colligative manner. Type III AFP, a class of AFP originated from fish, is a globular protein with molecular weight of approximately 7 kDa. The AFP is naturally expressed as a mixture of several isoforms categorized into two sub-groups named QAE- and SP-isoforms, which shares 55% identity of the amino acid sequence [2]. In this study, we examined the ability of ice growth inhibition of QAE- and SPisoforms through the photomicroscope observations and considered the biological role of each isoform. Experiments were performed for a single ice crystal prepared in each isoform solution by employing our custom-made photo-system, which realizes both preparation and observation of a single ice crystal (20 lm) with manual manipulation of temperature settings using electronic thermal controlling system [3]. QAE-isoform firmly inhibited ice crystal growth and modified the crystal into a bipyramidal shape, and that exhibited Tf depression similarly to the all known active AFPs. Virtually no ice growth occurred between Tm and Tf, while rapid ice growth was initiated at Tf. SP-isoform also modified the crystal shape and depressed the growth rate of the ice bipyramid, but it could not halt the ice growth and allowed continuous growing without change of the morphology. The continuous growth did not stop even at a high concentration of SP-isoform (5.0 mM) and at a slight supercooling 0.05 °C), indicating that SP-isoform causes no Tf depression. Further crystal observation revealed that in SP solution, the bipyramid suddenly initiated ice growth from the tip of bipyramid at a certain minus temperature. For QAE, the tip of bipyramid was known to be the growth initiation point at Tf, suggesting that SP and QAE commonly possess the ice growth inhibitory function against the tip of ice bipyramid. Also, SP and QAE were nearly equivalent in the initiation temperature from tip (For QAE, the temperature is same as Tf. This functional equivalence will explain the comparable Tf depression of SP with QAE in the mixture, as involvement of SP-isoform in the ice growth inhibition of bipyramid tip. These results suggest that although the SP-isoform is a less active isoform, with insufficient ice growth inhibition, SP would assist the inhibition of the active QAE-isoform by participating in the inhibition of ice growth from the tip of bipyramid that is weakest inhibited point, and contribute to further depression of Tf of the solution. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2009.10.023