activity on a defective SP isoform of type IIIantifreeze protein Manabu Takamichi1,2, Yoshiyuki Nishimiya1, Ai Miura1and Sakae Tsuda1,2 1 Functional Protein Research Group, Research Inst
Trang 1activity on a defective SP isoform of type III
antifreeze protein
Manabu Takamichi1,2, Yoshiyuki Nishimiya1, Ai Miura1and Sakae Tsuda1,2
1 Functional Protein Research Group, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Japan
2 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan
Antifreeze proteins (AFPs) function to inhibit the
growth of naturally generated hexagonal ice crystals in
supercooled water by specific accumulation onto a set
of oxygen atoms constructing specific planes of the
crystals [1] The vacant narrow spaces on an ice plane
between the bound AFPs can undergo ice growth to
form a convex ice front, which is energetically
unfavor-able for the further incorporation of water molecules
(the ‘adsorption inhibition’ model [2–4]) The
tempera-ture at which ice growth is initiated is commonly
referred to as the hysteresis freezing point (Tf) A
tem-perature difference between the melting point (Tm)
and Tf observed for an ice crystal in the presence of AFPs has been defined as thermal hysteresis (TH) [5], which is now generally recognized as a measure of the potency of antifreeze activity
AFPs have been found in various organisms, such
as fish, insects, plants, fungi and bacteria, adapted to subzero temperature environments [1] Of these, fish express type I–IV AFPs and antifreeze glycoprotein (AFGP), which are structurally diverse and form the ice-binding surface in different ways Significantly, all types of AFP and AFGP are expressed as mixtures of several isoforms [1,4], and a cooperative effect between
Keywords
cooperative effect; ice growth inhibition;
notched-fin eelpout; thermal hysteresis;
type III antifreeze protein
Correspondence
S Tsuda, Functional Protein Research
Group, Research Institute of Genome-based
Biofactory, National Institute of Advanced
Industrial Science and Technology (AIST),
2-17-2-1 Tsukisamu-Higashi, Sapporo
062-8517, Japan
Fax: +81 11 857 8983
Tel: +81 11 857 8983
E-mail: s.tsuda@aist.go.jp
(Received 15 November 2008, revised 29
December 2008, accepted 5 January 2009)
doi:10.1111/j.1742-4658.2009.06887.x
Type III antifreeze protein is naturally expressed as a mixture of sulfopro-pyl-Sephadex (SP) and quaternary aminoethyl-Sephadex (QAE)-binding isoforms, whose sequence identity is approximately 55% We studied the ice-binding properties of a SP isoform (nfeAFP6) and the differences from those of a QAE isoform (nfeAFP8); both of these isoforms have been identified from the Japanese fish Zoarces elongatus Kner The two isoforms possessed ice-shaping ability, such as the creation of an ice bipyramid, but nfeAFP6 was unable to halt crystal growth and exhibited no thermal hysteresis activity For example, the ice growth rate for nfeAFP6 was 1000-fold higher than that for nfeAFP8 when measured for 0.1 mm protein solution at 0.25C below the melting point Nevertheless, nfeAFP6 exhib-ited full thermal hysteresis activity in the presence of only 1% nfeAFP8 (i.e [nfeAFP8]⁄ [nfeAFP6] = 0.01), the effectiveness of which was indistin-guishable from that of nfeAFP8 alone We also observed a burst of ice crystal growth from the tip of the ice bipyramid for both isoforms on low-ering the temperature These results suggest that the ice growth inhibitory activity of an antifreeze protein isoform lacking the active component is restored by the addition of a minute amount of the active isoform
Abbreviations
AFGP, antifreeze glycoprotein; AFP, antifreeze protein; IGM, ice growth modifier; nfeAFP6 and 8, SP and QAE isoforms of type III AFP from notched-fin eelpout; QAE, quaternary aminoethyl; SP, sulfopropyl; Tf,freezing point; TH, thermal hysteresis; Tm,melting point.
Trang 2isoforms with respect to the TH value has been
identi-fied [6–8] For example, an AFGP based on a
repeti-tive polypeptide consisting of Thr–Ala–Ala tripeptide
units changes the level of Tfdepression by the addition
of peptides of other lengths [6,7] Insect AFP from
Dendroides canadensis larva, an isoform mixture of
repetitive b-helical polypeptides, interacts among the
isoforms, which affects the observed TH value [8]
Thus, the characterization of such isoforms and their
cooperative effects on antifreeze activity will aid in our
understanding of the natural production of various
AFP isoforms
Type III AFP is a 7 kDa globular protein that
exhibits high sequence identity with the C-terminal
domain of human sialic acid synthase [9,10] Type III
AFP was first discovered in the ocean pout
Macrozoar-ces americanusas a mixture of 12 isoforms that can be
grouped into 11 sulfopropyl (SP)- and one quaternary
aminoethyl (QAE)-Sephadex-binding species [11] The
SP and QAE isoforms show approximately 55%
sequence identity [12] Immunological cross-reactivity
studies have shown a significant difference between the
two isoforms [9], and detailed structural
determina-tions by X-ray crystallography and NMR spectroscopy
have indicated that the SP and QAE isoforms
con-struct a very similar tertiary fold characterized by a
two-fold symmetric motif [13–20], which provides a
large, flat, amphipathic ice-binding surface [21–24]
Further, ice etching experiments [18,25] and computer
simulations [18,23,26] have shown that type III AFP
can undergo complementary binding to a set of oxygen
atoms located on the {10 1 0} prism plane
We have examined the cooperative effects between
several SP and QAE isoforms of type III AFP using a
commercial freezing point osmometer [27] This device
determines the Tfvalue by automatic measurement of
the ice–water equilibrium temperature of a sample
solution We found that the SP isoform has no
appre-ciable TH activity by itself However, we recently
reported that a recombinant SP isoform can modify
the shape of an ice crystal into a hexagonal bipyramid
below Tm, suggesting the possibility that the SP
iso-form itself possesses some ability to control the growth
of ice crystals, although the freezing point osmometer
was unable to detect this ability These considerations
led us to examine the details of ice growth inhibition
by the SP isoform and the difference between its
activ-ity and that of the QAE isoform
In this study, we observed the morphological change
in an ice crystal prepared in solutions of the SP
iso-form, the QAE isoform and their mixture employing
our custom-made photomicroscope system [28] to
evaluate the abilities of these isoforms to inhibit the
growth of ice (i.e TH activity) We used a recombi-nant nfeAFP6 as the SP isoform and nfeAFP8 as the QAE isoform, both of which were identified from Zoarces elongatus Kner [27] We discuss the ability of ice growth inhibition of the SP and QAE isoforms on the basis of a detailed analysis of the morphological change in the ice crystal
Results
Recombinant type III AFP isoforms from
notched-fin eelpout, nfeAFP6 and nfeAFP8, were used as the SP and QAE isoforms, respectively The primary sequences of the two isoforms are described in Materi-als and methods We first examined the ice growth inhibitory ability of each recombinant, as well as that
of a negative control (lysozyme), using a photomicro-scope system developed previously [28] In the nfeAFP8 solution, the morphology of a hexagonal ice nucleus was modified into a bipyramidal shape, as shown in Fig 1 (photograph and illustration a) When the crystal growth of this ice bipyramid was measured
at a cooling rate of 0.20 CÆmin)1, the growth was strongly inhibited at temperatures below Tm (Fig 1, photograph and illustrations a–c) On further cooling,
a bursting crystal elongation (i.e crystal growth) occurred suddenly and rapidly from the tip of the ice bipyramid (Fig 1, photograph and illustration d) Here, we define this bursting temperature from the bipyramidal tip as Tburst As no significant ice growth occurred for nfeAFP8 until Tburst, this temperature was identified as the ice growth initiation temperature,
or simply the hysteresis freezing point (Tf) of this solu-tion [28] In the case of nfeAFP8, TH = |Tm) Tf| could hence be evaluated with Tmand Tburst
We also observed a bipyramidal ice crystal for nfeAFP6 at a cooling rate of 0.20CÆmin)1 (Fig 1, photograph and illustration e) This ice bipyramid, however, expanded rapidly and continuously with retention of the a- to c-axis ratio (1 : 3) (Fig 1, photo-graph and illustrations e–g), followed by a bursting elongation of the ice crystal suddenly from the tip of the ice bipyramid (Fig 1, photograph and illustra-tion h) Such a bursting elongaillustra-tion was similarly observed for nfeAFP8 (Fig 1, photograph d) The only difference was that the bursting elongation of nfeAFP6 accompanied rapid crystal expansion, whereas that of nfeAFP8 did not We can hence evalu-ate, for nfeAFP6, the burst initiation temperature of
an ice crystal as Tburst Rapid crystal expansion was observed even at a temperature only slightly below Tm (i.e Tm) 0.05 C), which was closely similar to the observation for the negative control (Fig 2C); the only
Trang 3difference was in the ice crystal morphology In other
words, the ice crystal created in nfeAFP6 and negative
control solution melted above Tm and grew below Tm
That is, each solution maintained an ice–water
equilib-rium state around Tm, for which no infinite value of
the non-equilibrium freezing point could be defined
We therefore conclude that Tm and Tf are equal for
nfeAFP6 solution, and that no TH activity could be
evaluated for nfeAFP6
Table 1 shows the growth rate of the ice bipyramid
estimated for each isoform; the a-axis length of the ice
bipyramid was used for evaluation The growth rates
were measured at two annealing temperatures between
Tm and Tburst (Tm) 0.05 C and Tm) 0.25 C) As
shown in Table 1, rapid and constant rates of ice
growth were evaluated for nfeAFP6, although the rates
were slower than that for the negative control
(lyso-zyme) It should be noted that, for nfeAFP6, the ice
growth (i.e expansion) rate was decelerated by
incre-asing the protein concentration and accelerated by
lowering the annealing temperature The crystal
expansion was observed even at a high concentration
of nfeAFP6 (5.0 mm) We also found very slow growth
(4· 10)2lmÆmin)1) of the ice bipyramid for diluted nfeAFP8 solution, which agrees with a previous report
by Deluca et al [29] When the nfeAFP8 concentration exceeded 0.1 mm, crystal growth was halted, making it difficult to measure the rate at Tm) 0.05 C We were able to compare the ice growth rates for the two isoforms for their 0.1 mm solutions at Tm) 0.25 C; the observed value for nfeAFP6 was approximately 1000-fold faster than that for nfeAFP8 In addition, the growth rate of nfeAFP8 decreased slightly with time, with only slight crystal growth along the c-axis (data not shown)
Figure 3A shows the ice bipyramid observed in a
1 : 1 mixture of nfeAFP6 (0.05 mm) and nfeAFP8 (0.05 mm) isoforms with a total concentration of 0.1 mm at Tm) 0.25 C Crystal growth was clearly inhibited between Tm and Tburst for 30 min, similar
to that in the case of nfeAFP8 only Figure 3B shows the growth rate of the ice bipyramid for some mix-tures of nfeAFP6 and nfeAFP8 at Tm) 0.05 C, the total concentration being adjusted to 0.1 mm The ice growth rate decreased dramatically from 6 to 0.1 lmÆmin)1 by the addition of only 1% nfeAFP8
a
A
B
e
f g h
Fig 1 Morphological change in an ice
crystal observed for 0.1 m M solutions of
nfeAFP8 (QAE isoform) and nfeAFP6
(SP isoform) (A) Photomicroscope images
for nfeAFP8 (a–d) and nfeAFP6 (e–h) were
obtained at a cooling rate of 0.20 CÆmin)1.
(a, e) T = Tm) 0.1 C; (b, f)
T = Tm) 0.2 C; (c, g) T = T burst ; (d, h)
T = T burst after 0.05 s (B) Illustrations of
crystal growth observed for solutions
of nfeAFP8 (a–d) and nfeAFP6 (e–h) at
different temperatures.
Trang 4(i.e [nfeAFP8]⁄ [nfeAFP6] = 0.01), and reached a
pla-teau (0.06 lmÆmin)1) at 12.5% nfeAFP8 At 25%
nfeAFP8, the growth rate was indistinguishable from
that at 100% nfeAFP8
Figure 4A shows time-course snapshots of a busting
growth of an ice bipyramid observed in a 0.1 mm
solu-tion of the 1 : 1 mixture of the two isoforms, detected
at the temperature Tburst Bursting growth occurred
from the tip of the ice bipyramid, as similarly observed
for the two isoforms (Fig 1Ad and h) We were able
to estimate the TH value for this mixture, as the ice
crystal did not expand between Tm and Tburst
(Fig 3A), but underwent bursting crystal growth at
Tburst (i.e Tburst = Tf) TH activities of nfeAFP6,
nfeAFP8 and their 1 : 1 mixture plotted against their
A
B
C
Fig 2 Photomicroscope images of an ice crystal observed for
0.1 m M solutions of nfeAFP8 and nfeAFP6 between Tmand Tburst.
(A) An ice bipyramid observed for nfeAFP8 at T m ) 0.25 C; the
crystal growth was strongly inhibited for longer than 30 min (B)
Expansion of an ice bipyramid observed for nfeAFP6 at
T m ) 0.25 C The snapshots were taken before and after 2 min of
annealing time (C) Snapshots of 0.1 m M solution of negative
con-trol (lysozyme) annealed slightly below its Tmvalue (Tm) 0.05 C).
The horizontal bars and arrows indicated in the photographs
repre-sent a scale of 20 lm and the direction of the c-axis, respectively.
The c-axis is vertical to the paper for the negative control.
nfeAFP8 and the negative control (lysozyme) between T m and
Tburst.
Sample
Concentration (m M )
Annealing temperature (C)
Ice growth rate (lmÆmin)1)
T m ) 0.25 13.7
T m ) 0.25 1 · 10)2 Lysozyme
(negative control)
A
B
Fig 3 (A) Photomicroscope images of an ice crystal observed for
a 0.1 m M solution of a 1 : 1 mixture of nfeAFP6 and nfeAFP8 annealed at T m ) 0.25 C The crystal showed no significant change for 30 min The horizontal scale bar represents 20 lm; the arrow indicates the direction of the c-axis, which is perpendicular to the a-axis (B) Crystal growth rates of an ice bipyramid determined
at Tm) 0.05 C for nfeAFP6 in the presence of various amounts of nfeAFP8, with the total protein concentration adjusted to 0.1 m M The x-axis indicates the percentage of nfeAFP8 For example, 25% indicates 0.075 m M nfeAFP6 plus 0.025 m M nfeAFP8 To evaluate the growth rate, we measured the length of the middle of the bipyramid along the a-axis.
Trang 5concentrations are shown in Fig 4B Interestingly, the
TH activity of the 1 : 1 mixture was closely equivalent
to that of nfeAFP8 when plotted against the total
AFP concentration Figure 4C plots the TH activity of
a 0.1 mm solution of the mixture against the
percent-age content of nfeAFP8 Full activity was observed in
the presence of only 1% of the QAE isoform, as
sug-gested by Fig 3B These results indicate that nfeAFP6
exhibited full TH activity in the presence of only a
minute amount of nfeAFP8
Figure 5 shows the value of |Tm) Tburst| plotted
against the protein concentration (mm) for each
nfeAFP6 and nfeAFP8 isoform Interestingly, the two
profiles show closely similar hyperbolic curves, which
largely overlap the whole concentration range This
suggests that the SP and QAE isoforms possess similar
levels of growth inhibitory ability for the tip of the ice
bipyramid
Discussion
This study reveals that the SP isoform (nfeAFP6) lacks
the ability to inhibit ice growth (Figs 2 and 3), similar
A
a b c d
B
Fig 4 (A) Burst elongation (crystal growth) arising from the tip of an ice bipyramid in a 0.1 m M solution of a 1 : 1 mixture of nfeAFP6 and nfeAFP8 (a) An ice bipyramid just before initiation of burst growth (b) Initiation of the crystal burst from the tip of the ice bipyramid (c, d) The crystal burst rapidly proceeds and leads to complete freezing of the solution Insets show expanded views of the growth initiation point Horizontal bar represents 20 lm Arrow shows the direction of the c-axis (B) TH activities of nfeAFP6 (filled triangles), nfeAFP8 (filled circles) and a 1 : 1 mixture of the two isoforms (open circles) as a function of each total concentration TH was evaluated as the difference between T f and T m (i.e TH = |T m ) T f |) For nfeAFP6, T f = T m For nfeAFP8 and the 1 : 1 mixture, T burst = T f (C) TH activity of nfeAFP6 in the presence of various proportions of nfeAFP8, with the total protein concentration adjusted to 0.1 m M
0
0 0.1 0.2 0.3 0.4 0.1
0.2 0.3 0.4 0.5 0.6
Concentration (mM)
Tburst
nfeAFP6 (SP) nfeAFP8 (QAE)
Fig 5 Concentration dependence of |Tm) T burst | for nfeAFP6 (open circles) and nfeAFP8 (filled circles) Tburstrepresents the tem-perature at which a burst of ice crystal growth occurs from the tip
of the ice bipyramid We were unable to define TH activity, but obtained a |Tm) T burst | value for nfeAFP6 For nfeAFP8, this value was identical to the TH activity (Fig 4B).
Trang 6to the negative control, although nfeAFP6 can
specifi-cally interact with an ice nucleus to form an ice
bipyra-mid (i.e it has ice-shaping ability) Consequently,
nfeAFP6 exhibits no TH activity and shows an
ordin-ary ice–water equilibrium phenomenon (i.e Tm= Tf)
(Fig 2) It is interesting that, although this SP isoform
was found to be a dominant component of a purified
fish type III AFP [27], it cannot function as an AFP,
but should rather be termed an ‘ice growth modifier’
(IGM), according to the nomenclature proposed by
Harding et al [30] Similar observations have been
reported in studies of artificial AFP mutants [31–34]
For example, a flounder type I AFP lost 90% of its
inherent TH activity and allowed continuous growth
of ice bipyramids when Ala21 was replaced with Leu
[31] Recombinant type I AFP from shorthorn sculpin
that lacks N-terminal blocking (denoted rSS3) as well
as that of the lysine mutant of Ala25 also failed to
inhibit the growth of ice bipyramids [32,33] For
type II AFP from longsnout poacher, continuous
growth of an ice bipyramid was observed by mutation
of Ile58, a residue located within a planar ice-binding
surface [34] HPLC12, a QAE isoform of type III AFP
with 94% sequence identity to nfeAFP8, permits
con-tinuous growth of an ice bipyramid that expands
rap-idly on amino acid replacement of Ala16, a residue
located at the center of the ice-binding surface [29]
It has been observed that the bipyramidal ice crystal
grows continuously with retention of the c- : a-axis
ratio at approximately 3.3 for flounder type I AFP
mutants that accumulate onto a {20 21} pyramidal
plane Significantly, a similar continuous growth of the
ice bipyramid with retention of the c- : a-axis ratio of
approximately 3 was observed for nfeAFP6 (Fig 2),
suggesting that nfeAFP6 binds to the pyramidal plane
In contrast, significant ice growth along the c-axis was
identified for ordinary type III AFP and sculpin type I
AFP, which bind to the prism plane nfeAFP8 also
permitted the growth of an ice bipyramid along the
c-axis at a low concentration (0.005 mm), suggesting
that nfeAFP8 binds to the prism plane It should be
noted that a recent study on ice etching revealed that
type III AFP can bind to several ice planes, including
the {10 10} prism and the {2021} pyramidal plane [18]
The preliminary X-ray structure of nfeAFP6 (data not
shown) obtained was indistinguishable from that of
ordinary type III AFP (i.e HPLC12) [14,18] There
is a 41% sequence difference between nfeAFP6 and
AFP8, which includes Leu19 and Val20 locating at the
edge of the putative ice-binding region Hence, we
assume that amino acid replacements of Leu19 and
Val20 differentiate the manner of ice binding between
nfeAFP6 and nfeAFP8
The fast and slow ice growth rates evaluated for nfeAFP6 and nfeAFP8 reveal a difference in their growth inhibitory function An extremely slow growth rate was found for 0.01 mm nfeAFP8 (4· 10)2lmÆmin)1, Table 1) and for the mixture of approximately 0.01 mm nfeAFP8 plus 0.09 mm nfeAFP6 (10% nfeAFP8) (Fig 3B) As nfeAFP6 could not halt ice growth by itself, it may be assumed that the two iso-forms act cooperatively for ice growth inhibition Indeed, TH activity of the 1 : 99 mixture containing approximately 0.001 mm nfeAFP8 (TH = 0.33C) (Fig 4C) was higher than that of 0.05 mm nfeAFP8 alone (TH = 0.26C) (Fig 4B) We examined the
1H-15N heteronuclear single quantum coherence spec-trum of 15N-labeled nfeAFP6 in the absence and pres-ence of 20% of non-labeled nfeAFP8, and found that the two spectra were virtually identical (Fig S1) Hence, we can assume no significant protein–protein interaction between the two isoforms, which is in good agreement with the proposed independent ice-binding model of AFP [35,36] Kristiansen and Zachariassen [4] proposed a two-step irreversible adsorption of AFPs to the ice surface; i.e following the initial adsorption controlled by hydrophobic forces, perma-nent adsorption occurs on this plane by hydrophilic forces This proposition may account for the weak ice growth inhibition of nfeAFP6 That is, nfeAFP6 can undergo initial adsorption to a pyramidal ice plane, but fails to undergo the secondary permanent adsorp-tion on this plane As nfeAFP8 presumably possesses
an ability to execute the two-step irreversible adsorp-tion to the prism plane, nfeAFP6 may irreversibly bind
to the prism plane in the presence of nfeAFP8
In the case of nfeAFP8, we observed that crystal bursting was initiated from the tapered tip of the ice bipyramid at Tf (= Tburst) (Fig 1Ad), implying that the tip is the weakest point Significantly, even nfeAFP6 can inhibit crystal bursting from the tip between Tmand Tburst(Fig 1Ae,f), similar to nfeAFP8 (Fig 1Aa–c) The TH activities obtained for nfeAFP8 and the 1 : 1 mixture of nfeAFP6 and nfeAFP8 were indistinguishable (Fig 4B) The level of depression of
Tburst for nfeAFP6 was also indistinguishable from that for nfeAFP8 (Fig 5) These results suggest that the functions of nfeAFP6 and nfeAFP8 are equivalent with regard to the inhibition of growth from the tips
of the ice bipyramid To our knowledge, there is little documentation about such growth inhibition of the bipyramidal tip by type III AFP We offer a plausible explanation below
The origin of an ice bipyramid is a hexagonal ice unit (i.e ice nucleus) generated in supercooled water under a pressure of 1013 hPa [37] A scheme of
Trang 7transi-tion from an ice nucleus to an ice bipyramid in the
presence of AFP (Fig 6), which was proposed many
years ago [38], is still widely accepted with no
signifi-cant revisions Inherently, the ice growth rate on the
six prism planes is much faster than that on the basal
plane [37,39] When AFPs are present, they accumulate
on the six prism planes and inhibit their growth along
the a1-, a2- and a3-axes (first layer in Fig 6), but
can-not inhibit the generation of a new ice nucleus on the
first layer, namely ice growth along the c-axis [38]
AFPs further accumulate on the prism planes grown
from the new ice nucleus and a hinge region between
the second prism and the first basal plane [14], thereby
creating a hexagonal ice plate that is smaller than the
first layer Repeated binding of AFP to the prism
plane and the generation of a smaller ice nucleus cause
successive stacking of hexagonal ice plates on the basal
plane, leading to the formation of an ice bipyramid, as
illustrated in Fig 6 When pyramidal planes are
cre-ated by the adsorption of AFPs, the 12 equivalent
planes construct the bipyramidal ice crystal [40]
Hence, one can imagine that the tip of the ice
bipyr-amid is the basal plane or a part of the basal plane,
which is of ultimately small size Therefore, such an
extremely narrow space of the top plane scarcely
allows elongation of the ice crystal along the c-axis
between Tm and Tburst, thereby maintaining
bipyrami-dal morphology (Fig 1) Explosive ice growth along
the c-axis may be induced by slight expansion of the
narrow top plane through ice growth towards the
other axes on lowering the temperature to Tburst (Fig 1) That is, an increase in isoform concentration (Fig 5) may contribute to the stability of the top plane
of the ice bipyramid by stabilizing its prism or pyra-midal planes, and this increases the value of
|Tm) Tburst| These suppositions do not contradict the recent observations of Scotter et al [41] These authors showed that the crystal burst always occurred from the tip (basal plane) of the ice bipyramid for most fish AFPs, and occurred from the prism plane for insect hyperactive AFPs They ascribed the former observa-tion to no binding ability of fish AFPs to the basal plane and the latter to the binding of hyperactive AFPs not only to the prism but also to the basal plane We assume that the fully active isoform of type III AFP strongly binds to the prism plane, whereas a defective isoform weakly interacts with the pyramidal plane, but they possess similar abilities to inhibit ice growth from the tip of the ice bipyramid
In summary, we have found that a minute amount
of the active QAE isoform of type III AFP confers
TH activity on the SP isoform, which possesses no TH activity by itself This may imply that the large amount
of the SP isoform contained in the body fluid makes a significant contribution to ice growth inhibition with the help of the active isoform, thereby enabling the host fish to survive in seawater at subzero tempera-tures The present findings may further suggest that any defective antifreeze analog (e.g IGM) could be used as an effective TH substance by the addition of a minute amount of the fully active antifreeze substance
Materials and methods
Sample preparation Recombinant proteins of the type III AFP isoforms nfeAFP6 and nfeAFP8 were prepared as described previously [27] with some modifications After sonication of genetically trans-formed Escherichia coli BL21 (DE3), a soluble fraction con-taining a recombinant isoform was dialyzed against 50 mm citric acid buffer (pH 2.9) Cation exchange chromatography was then performed using an Econo-Pac High S cartridge (Bio-Rad, Hercules, CA, USA) with a linear NaCl gradient (0–0.5 m) with 50 mm citric acid buffer (pH 2.9) The amino acid sequences of nfeAFP6 and nfeAFP8 are as follows: nfeAFP6, G1ESVVATQLIPINTALTPAMMEGKVTNPS GIPFAEMSQIVGKQVNTPVAKGQTLMPGMVKTYVP AK66; nfeAFP8, N1QASVVANQLIPINTALTLVMMRA
VKGYTPA65 The concentration of each purified sample was measured by UV absorption (280 nm) using a DU 530 spectrophotometer (Beckman Coulter, Fullerton, CA, USA)
c-axis
Basal plane
Prism plane
a1 a2
a3
1 st layer
2 nd layer
3 rd layer
Fig 6 Left: illustration of the proposed transition scheme from ice
nucleus to ice bipyramid based on previous ideas [38] Right:
illus-tration of an ice bipyramid observed in a solution of type III AFP.
Detailed explanations are given in the text.
Trang 8Measurement of ice growth rate and TH activity
Ice crystal morphology was observed and the crystal growth
rate was measured for solutions of nfeAFP6, nfeAFP8 and
their mixtures using a custom-made photomicroscope
system described in [28] Detailed procedures for the
evalua-tion of TH activity are also described in [28] using a cooling
rate of 0.20CÆmin)1 The sample solution was placed in a
capillary tube and frozen at approximately )30 C; it was
then warmed by manipulation of the temperature control
device until a single ice crystal was observed ( 10–30 lm)
The observed ice crystal was annealed slightly below its Tm,
and recorded using a Color-video 3CCD camera (Sony,
Tokyo, Japan) The annealing period was 0.5–1 h for
nfeAFP6 and 3 h for nfeAFP8 The ice growth rate was
examined in five to ten still images captured at regular
inter-vals; the length of the middle of the bipyramid along the
a-axis was used for evaluations In all solutions tested here,
the value of Tmwas)0.3 C This was equivalent to that of
the buffer solution (0.1 m ammonium bicarbonate, pH 7.9)
Acknowledgements
The authors thank Dr Hidemasa Kondo for providing
them with a preliminary X-ray structure of nfeAFP6
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Supporting information
The following supplementary material is available: Fig S1 500 MHz 1H-15N heteronuclear single quan-tum coherence spectra of a recombinant 15N-labeled protein of the defective isoform nfeAFP6 dissolved in water in the absence and presence of non-labeled nfeAFP8 (temperature, 4C; pH 6.7; [nfeAFP6] : [nfeAFP8] = 1 : 4; total concentration, 1 mm)
This supplementary material can be found in the online version of this article
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