NMR Analysis of Type III Antifreeze Protein Intramolecular Dimer

Miura, Kazunori; Ohgiya, Satoru; Hashizume, Tamotsu; Nemoto, Nobuaki; Suetake, T.; Miura, Ai; Spyracopoulos, Leo; Kondo, Hidemasa; Tsuda, Sakae

doi:10.1074/jbc.m007902200

Cited by 49 publications

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References 31 publications

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“…One isoform (designated RD3) from the Antarctic eel pout (Lycodichthys dearborni) has two type III AFPs joined in tandem by a nine-amino acid linker peptide (23). This duplicated AFP with two similar AFP domains has been referred to in the literature as an intramolecular dimer (24) although there is no suggestion that the tandemly repeated AFPs contact each other. NMR analysis of the 14.7-kDa RD3 isoform indicates that the linker region is fairly flexible and may allow both ice-binding faces to engage the ice surface at the same time (24).…”

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“…This duplicated AFP with two similar AFP domains has been referred to in the literature as an intramolecular dimer (24) although there is no suggestion that the tandemly repeated AFPs contact each other. NMR analysis of the 14.7-kDa RD3 isoform indicates that the linker region is fairly flexible and may allow both ice-binding faces to engage the ice surface at the same time (24). RD3 was reported to have twice the molar activity of the monomeric isoforms RD1 and RD2 (23) and anywhere from 1.5 to six times the activity of the recombinant N-terminal domain alone depending on the concentration tested (24).…”

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Antifreeze Protein Dimer

Baardsnes

Kuiper

Davies

2003

Journal of Biological Chemistry

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A naturally occurring tandem duplication of the 7-kDa type III antifreeze protein from Antarctic eel pout (Lycodichthys dearborni) is twice as active as the monomer in depressing the freezing point of a solution. We have investigated the basis for this enhanced activity by producing recombinant analogues of the linked dimer that assess the effects of protein size and the number and area of the ice-binding site(s). The recombinant dimer connected by a peptide linker had twice the activity of the monomer. When one of the two ice-binding sites was inactivated by site-directed mutagenesis, the linked dimer was only 1.2 times more effective than the monomer. When the two monomers were linked through a C-terminal disulfide bond in such a way that their two ice-binding sites were opposite each other and unable to engage the same ice surface simultaneously, the dimer was again only 1.2 times as active as the monomer. We conclude from these analyses that the enhanced activity of the dimer stems from the two ice-binding sites being able to engage to ice at the same time, effectively doubling the area of the ice-binding site.Fish are protected from freezing by antifreeze proteins (AFPs), 1 which bind to the surface of nucleating ice crystals in their body fluids, thereby reducing their freezing point below that of the ocean (1-3). AFPs create a local curvature of the ice between adsorbed AFPs, which makes it energetically unfavorable for liquid water to join the ice surface (4). This process, known as the Kelvin effect, produces a non-equilibrium reduction of the freezing point of ice below the melting point (5). The difference between the melting and freezing points is the thermal hysteresis gap, and it is within this temperature range that ice growth is prevented and fish are protected from freezing.Type III AFPs belong to one of several structurally distinct antifreeze protein families found in fishes (6 -8). These 7-kDa proteins have a compact ␤-stranded structure (9 -13) that shows homology to the C-terminal domain of sialic acid synthase (14). Structure-function studies have localized the icebinding residues to a flat, amphipathic surface on the protein (11,15,16). This ice-binding face includes several conserved hydrophilic residues (Gln 9 , Asn 14 , Thr 15 , Thr 18 , Gln 44 ) that potentially form hydrogen bonds with water molecules on the ice surface (10, 17), flanked by hydrophobic residues (Leu 10 , Ile 13 , Leu 19 , Val 20 , Val 41 ) on the periphery. Altogether, these residues are thought to make favorable van der Waals contacts with the ice surface (10, 12, 18 -20). In addition, there may also be stabilizing entropic effects from bringing this somewhat hydrophobic surface into contact with ice (10, 21). Type III AFP was first reported to bind to the primary prism plane {10-10} of ice (22 Approximately 20 type III isoforms from five species of zoarcid fishes have now been sequenced, with an overall 50% sequence identity. One isoform (designated RD3) from the Antarctic eel pout (Lycodichthys dearborni) has two ...

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Antifreeze Protein Dimer

Baardsnes

Kuiper

Davies

2003

Journal of Biological Chemistry

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“…The currently available structural database for AFPIII includes NMR structures (Sönnichsen et al 1996;Miura et al 2001), X-ray structures (Graether et al 1999;Antson et al 2001), and a neutron structure (Howard et al 2011). On the basis of these data, Howard et al identified an ice-like geometry for four of the water molecules bound to a pocket of the IBS formed by Gln 9 , Thr 18 , Val 20 , and Met 21 , for which the anchoring role that leads to the AFP-ice interaction was assumed.…”

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NMR structure note: a defective isoform and its activity-improved variant of a type III antifreeze protein from Zoarces elongates Kner

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Biological contextAntifreeze proteins (AFPs) produced in various cold-adapted animals and plants can specifically bind to ice crystals and inhibit their growth (Fletcher et al. 2001;Graether and Sykes, 2004). The ice-binding ability of AFPs depresses the freezing temperature (T f ) of water non-colligatively, which leads to the protection of cells and tissues from freezing. The level of T f depression has been evaluated by measuring the T f and melting temperature (T m ) for an ice crystal created in an aqueous solution of an AFP. The difference between these two temperatures is defined as the thermal hysteresis (TH), which is a measure of the AFP's ice-growth inhibition. AFPs also modify the shape of an ice crystal uniquely, hexagonal bipyramid for example, in the temperature range of TH. These two activities have been assumed to be common for all AFPs; however, it has recently been found that they are not always the rule for every species, such as the AFP type III (denoted AFPIII) found in fish (Takamichi et al. 2008).Fish AFPIII is a globular protein made up of many short β-strands and one helical turn. AFPIII is generally produced in vivo as a mixture of quaternary-amino-ethyl (QAE)-Sephadex-and sulfopropyl (SP)-Sephadex-binding isoforms. These two isoforms possess different pIs and share approximately 50% sequence identity. The Japanese notched-fin eelpout, Zoarces elongates Kner, produces 13 different AFPIII isoforms (denoted nfeAFP), which have been divided into six SP (nfeAFP1-6) and seven QAE (nfeAFP7-13) isoforms, and the latter was further divided into QAE1 (nfeAFP7-10) and QAE2 (nfeAFP11-13) isoforms (Nishimiya et al. 2005). Among them, only the QAE1 isoforms are fully active variants exhibiting both TH and ice-shaping activities, while the others only shape the ice morphology. The reason for such defective activity for the SP-and QAE2-isoforms is not well understood.Recently, we found that alterations of the 9 th , 19 th , and 20 th residues of a QAE2 isoform make them the same as their counterparts in the QAE1 isoform, leading to the successful conversion of the former into a QAE1-like fully active isoform . That is, the QAE-2 isoform nfeAFP11 can only shape ice crystals, but has no TH activity, while its triple mutant nfeAFP11-V9Q/V19L/G20V (denoted nfeAFP11-tri) perfectly exhibits both activities. These residues were thought to construct a "compound" ice-binding site (IBS) that consists of two adjacent flat surfaces inclined at an angle of approximately 150° to each other (Garnham et al. 2010). To determine if the triple mutations in nfeAFP11 led to changes in the structure of the IBS that could affect its icebinding activity, the multidimensional NMR spectra of both nfeAFP11 and nfeAFP11-tri were examined. Special attention was paid to the surface-bound water molecules, for which the unique role of anchoring the IBS to ice lattice has been postulated (Garnham et 3 al. 2011;Kondo et al. 2012).The tertiary structures of AFPIII have been determined mostly for the QAE1-isoform HPLC12, which was ...

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“…Experimental accounts report that the binding of fish AFP to hexagonal pyramids is indexed frequently as (201) (14,16,17,19,21,23); but more elongated pyramids with higher indices such as (301) or (401) (24,25) and even extremely elongated pyramids with indices (501) or higher (15) have been reported. Less elongated pyramids with indices in the range (102)-(201), although not excluding the occurrence of (101), have also been observed (26,27) as well as combinations of such pyramids with the basal face (001) and various prisms (hk0) (17).…”

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“…The appearance of pyramidal forms on ice crystals grown under the influence of the AFP has thus far been explained (2,14,35) as the occurrence of an intermediate orientation between the basal face and the primary prism. For example, it is held in Refs.…”

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Antifreeze Protein-induced Morphological Modification Mechanisms Linked to Ice Binding Surface

Strom

Liu

Jia

2004

Journal of Biological Chemistry

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The mechanisms by which the antifreeze protein (AFP) modifies the ice morphology are identified precisely as surface poisoning by the ice binding surface (IBS) of insect AFPs and as bridge-induced surface reconstruction by the IBS of fish AFPs and antifreeze glycoproteins. The primary surfaces of hexagonal ice have predetermined face indices. The "two-dimensional" insect type IBS has regularly spaced binding intervals in two directions. It causes surface poisoning by matching and reinforcing simultaneously intersecting strong bonding directions on the primary ice surfaces. The secondary ice surfaces have variable face indices. The "onedimensional" and "irregular" IBS variants of fish AFPs and antifreeze glycoproteins are either linearly extended with regular ice binding intervals or have ice binding sites lacking spacing regularity. These variants can bridge transversely lattice periods or shorter oxygen-oxygen distances between parallel adjacent strong bonding directions that do not intersect. Thus, one-dimensional and irregular IBS variants induce supplementary bridges cross-wise on selected secondary surfaces by mimicking strong bonding directions that are not present in the ice structure. These proteins cause surfaces with variable face indices, which in the absence of the AFPs would not grow flat, to appear in the morphology. Whereas for the primary ice surfaces it is only the morphological importance that is determined by the experimental conditions, for the secondary ice surfaces it is the face indices themselves that become adjusted in the process of maximizing the AFP-substrate interaction through attainment of the best structural match. The growth morphology of the AFP-ice system is derived from various factors, including the face indices, surface molecular compositions, relative growth rates, and the mechanisms responsible for that morphology. The theoretical formulation agrees with experiments over a wide range and resolves these, to date, unexplained phenomena.Freezing is a process of ice crystallization from supercooled water. Ice should first experience ice nucleation, followed by growth (1). Whether or not freezing takes place is determined to a large extent by ice nucleation (2, 35). There is evidence that fish antifreeze proteins bind to and reduce the efficiency of heterogeneous nucleation sites rather than bind to embryonic ice nuclei (2, 35). Similar phenomena were obtained and explained in Refs. 3-5. The inhibition of ice nucleation is important in any antifreeze process. The antifreeze action of the AFP 1 is actually first to inhibit nucleation by terminating the relevant kinetics (3-5). When the inhibition of ice nucleation fails, the AFP proceeds to inhibit the growth of ice.In this work the growth morphology is derived to first-order by taking into account the AFP-ice system. For the first time, the face indices, the surface molecular compositions, and the assessment of relative growth rates of the ice crystals as well as the AFP-induced morphological modification mechanisms are predict...

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NMR Analysis of Type III Antifreeze Protein Intramolecular Dimer

Cited by 49 publications

References 31 publications

Antifreeze Protein Dimer

Antifreeze Protein Dimer

NMR structure note: a defective isoform and its activity-improved variant of a type III antifreeze protein from Zoarces elongates Kner

Antifreeze Protein-induced Morphological Modification Mechanisms Linked to Ice Binding Surface

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