The TH value is determined by observing the growth of an Keywords adsorption–inhibition; annealing time; notched-fin eelpout; thermal hysteresis; type III antifreeze protein Corresponden
Trang 1of 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), Toyohira, Sapporo, Japan
2 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita, Sapporo, Japan
Antifreeze proteins (AFPs) are a structurally diverse
class of macromolecules that interact with water
mole-cules located in the surface of an ice crystal at
temper-atures below the melting point of the solution [1]
Interaction of a substantial number of AFPs with the
ice surface modifies the shape of the ice crystal,
result-ing in unique morphologies, such as a hexagonal
bipyramid or hexagonal trapezohedron [2] AFPs
inhi-bit ice growth by adsorbing onto the ice surface
(adsorption–inhibition model) [3,4] as the temperature
is lowered, and such inhibition becomes insufficient
below a certain temperature that is favourable for the
initiation of crystal growth For AFP solutions, this
‘ice-growth initiation temperature’ (Tini) is different
from the melting point (Tm) of the ice crystal, and the
Tiniis referred to as the nonequilibrium freezing point (nonequilibrium Tf) The difference between Tm and the nonequilibrium Tf is defined as thermal hysteresis (TH), and is generally used as a measure of the growth inhibition ability of an AFP [5] Therefore, determina-tion of the Tiniand Tmof a seed ice crystal is an essen-tial procedure to evaluate the TH activity of AFP However, the mechanism of ice binding that alters the
TH value remains unclear [6]
The TH value of AFPs has been evaluated using
a nanolitre osmometer and the ‘capillary technique’ [5,7] In the former, submicrolitre volumes of an AFP solution are introduced into an oil droplet, the temper-ature of which is controlled by a Peltier device The
TH value is determined by observing the growth of an
Keywords
adsorption–inhibition; annealing time;
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 8912
E-mail: s.tsuda@aist.go.jp
Website: http://unit.aist.go.jp/rigb/fprg/
index_e.html
(Received 26 July 2007, revised 29
Septem-ber 2007, accepted 24 OctoSeptem-ber 2007)
doi:10.1111/j.1742-4658.2007.06164.x
Antifreeze proteins (AFPs) possess a unique ability to bind to a seed ice crystal to inhibit its growth The strength of this binding has been evalu-ated by thermal hysteresis (TH) In this study, we examined the dependence
of TH on experimental parameters, including cooling rate, annealing time, annealing temperature and the size of the seed ice crystal for an isoform of type III AFP from notched-fin eelpout (nfeAFP8) TH of nfeAFP8 dramat-ically decreased when using a fast cooling rate (0.20CÆmin)1) It also decreased with increasing seed crystal size under a slow cooling rate (0.01CÆmin)1), but such dependence was not detected under the fast cool-ing rate TH was enhanced 1.4- and 2.5-fold when ice crystals were annealed for 3 h at 0.05 and 0.25C below Tm, respectively After anneal-ing for 2 h at 0.25C below Tm, TH activity showed marked dependence
on the size of ice crystals These results suggest that annealing of an ice crystal for 2–3 h significantly increased the TH value of type III AFP Based on a proposed adsorption–inhibition model, we assume that type III AFP undergoes additional ice binding to the convex ice front over a 2–3 h time scale, which results in the TH dependence on the annealing time
Abbreviations
AFGP, antifreeze glycoprotein; AFP, antifreeze protein; nfeAFP8, an isoform of type III AFP from Notched-fin eelpout; TH, thermal
hysteresis.
Trang 2ice crystal on the device through a microscope within a
cooling gradient Previous studies have used a similar
procedure for the capillary technique with a larger ice
crystal [5,6] Duman (2001) demonstrated that
consid-erably higher levels of TH are determined using the
nanolitre osmometer compared with the capillary
tech-nique for the same AFP sample [6] The sample used
was the AFP of the beetle Dendroides canadensis,
which displayed TH of 1.4C using the capillary
tech-nique, and 5.5C using the nanolitre osmometer [6]
In addition, several physicochemical factors influence
TH activity [8], and there is a linear and negative
cor-relation between TH activity and the logarithm of the
mass fraction of ice in a sample for cerambycid beetle
Rhagium inquisitor AFP [9,10] A similar observation
was also reported for the common mealworm Tenebrio
molitor, for which differential scanning calorimetry
was employed [11]
For fish AFPs, TH activity of approximately 1C is
close to the maximum value observed Fish AFPs can
be classified into four types (I–IV) or as antifreeze
gly-coproteins (AFGPs) Type I AFP, with relative
mole-cular weights ranging from 3.3 to 5 kDa, contains
alanine-rich amphipathic a-helices [12] Type II AFP is
a globular protein with an approximate relative
molecu-lar weight of 14 kDa that exhibits high structural
homology to a carbohydrate-recognition domain of
C-type lectin [13] Type III AFP is a 6.5 kDa compact
globular protein characterized by a unique internal
two-fold symmetry motif [14] Type IV is assumed to form a
four-helix bundle structure [15] AFGPs are
glycopro-teins whose relative molecular weight ranges from 3 to
34 kDa, and comprises a repetitive tripeptide
(Ala-Ala-Thr) whose Thr side chain is modified by a disaccharide
moiety [16] The structural characteristics of these fish
AFPs and their target ice crystal surface are different
from those of insect AFPs A faster cooling rate
decreases the apparent TH value of fish AFPs, and
lar-ger ice crystals tend to initiate crystal growth at higher
temperatures [17] The TH activity of a small species of
AFGP depends on the freezing rate (i.e cooling bath
temperature), while a large AFGP showed no such
dependence [18] Chapsky and Rubinsky (1997)
exam-ined the kinetic ice binding of fish type I AFP using a
technique called temperature-gradient thermometry
[19] For fish type III AFP, no time-dependence of TH
activity has been examined to date These data raise the
question about the key determinant that has a
signifi-cant influence on the TH activity of an AFP species
The present study examined the TH value of a
recombinant 65-residue type III AFP called nfeAFP8,
which was recently discovered in the Notched-fin
eelpout [20] nfeAFP8 exhibits 94% sequence identity
with HPLC12, a well-examined type III AFP isoform found in the ocean pout Macrozoarces americanus [21] For TH measurements, we utilized a custom-made photomicroscope system equipped with a temperature controller and a sample holder, for which data consis-tency with a commercial freezing-point osmometer had been verified We then examined the dependence of
TH value on the cooling rate, the seed ice crystal size, crystal annealing time, and annealing temperature The data obtained revealed that the TH value is signifi-cantly increased when the ice crystal is annealed for a long time at a lower annealing temperature Based on these results, we discuss the ice-binding mechanism of type III AFP that causes TH enhancement over a very slow time scale
Results
Before determining the TH activity of nfeAFP8, the performance of our photosystem was verified using solutions of NaCl and glucose The sample solution in the capillary tube (Fig 1) was initially cooled with the temperature controller until completely frozen The sample was then warmed until an ice crystal was apparent As expected, the ice crystal prepared in each sample exhibited a ‘disk-like’ morphology, which did not subsequently change [22] We carefully manipu-lated the temperature controller to fix the diameter of the seed crystal at approximately 20 lm, and examined whether there was a difference between Tini and Tm
It appeared that the temperatures were affected by
a change in the temperature controller of only
± 0.01C, consistent with equality between Tini and
Tm for the solutions of NaCl and glucose, affirming that they possess no specific ice-binding ability (i.e
Tini¼ equilibrium Tf) In Fig 2A, the concentration dependence of the equilibrium Tf(¼ Tm) for NaCl and glucose are shown by open circles and open squares, respectively The corresponding data measured using a freezing-point osmometer are also plotted in Fig 2A (closed circles and closed squares) The linear profiles obtained with our photosystem and the osmometer perfectly overlapped for the NaCl and glucose solu-tions These results indicated that our system precisely evaluates the Tiniand Tmvalues of a seed ice crystal in
a solution
An example of TH determination for nfeAFP8 using our photosystem is shown in Fig 2B, for which nfeAFP8 was dissolved to a final concentration of 0.1 mm The figure depicts a series of images of an ice crystal formed in solution at)0.46 C; the crystals had
a bipyramidal structure due to adsorption of nfeAFP8 onto their surfaces Adsorption of the AFP stopped ice
Trang 3crystal growth during the 0.01CÆmin)1 temperature decrease (images a and b), but failed to stop growth at )0.81 C (image c) and allowed subsequent crystal growth (images d–h) The nonequilibrium Tf of nfeAFP8 was hence determined to be )0.81 C As a separate experiment determined the Tmto be)0.30 C for an ice crystal in the same sample, 0.51C is the
TH value of 0.1 mm nfeAFP8 under these experimen-tal conditions
Figure 3A shows the concentration dependence of the TH activity of nfeAFP8 measured at cooling rates
of 0.01CÆmin)1 (closed circles) and 0.20CÆmin)1 (open circles) The TH activity obtained with a slow cooling rate (0.01CÆmin)1) was significantly (1.7-fold) higher than that obtained with the fast cooling rate (0.20CÆmin)1) Figure 3B is a plot of the TH depen-dence on the cooling rate of 0.1 mm nfeAFP8 The TH value decayed exponentially with increasing cooling rate, and reached a plateau at a rate of approximately 0.10CÆmin)1
When the relationship between TH and crystal size was assessed, a marked difference was noted between the cooling rates of 0.01 and 0.20CÆmin)1 (Fig 3C) The TH activity for a slow rate of cooling (0.01CÆmin)1) markedly decreased with increasing ice crystal size, while that measured using a fast cooling rate (0.20CÆmin)1) exhibited only a slight decrease A high TH value (approximately 0.7C) was obtained only when a slow cooling rate (0.01C min)1) was used for a small ice crystal
Figure 4A is a plot of TH activity measured for 0.1 mm nfeAFP8 after the annealing of an ice bipyra-mid for 0–3 h The annealing was performed at a Tm
of )0.05 C or )0.25 C Each experiment used the same cooling rate of 0.20CÆmin)1 The TH value
Photomicroscope
CCD Camera
Object glas s
Display
A
B
Crystal & Temp monitor)
Stage
Capillary holder
Viewing hole
Capillary (sample) Capillary
Sample solution Oil
Air
Capillary holde r
Freezing plate
Fig 1 Experimental set-up (photosystem)
for viewing the growth and melting of ice
crystal to determine thermal hysteresis
value of an AFP solution (A) The
photosys-tem composed of a photomicroscope (Leica
DMLB100), a temperature controller
(Lin-kam THMS 600), a Colorvideo 3CCD camera
(Sony), and an image-processing computer.
(B) The capillary cell containing a 0.75 lL
sample solution, and setting of the sample
into the holder.
4.0
3.5
A
B
0.5
1.0
1.5
2.0
2.5
3.0
0
NaCl
Glucose
photo-system & osmometer
o C)
Concentration ( M )
20 μm
h g
f e
d c
b a
Fig 2 (A) Concentration dependence of the equilibrium freezing
point of NaCl and glucose solutions Open symbols represent the
data determined using the photosystem, and closed symbols are
those obtained using a commercial osmometer (Vogel, model
OM802) The error bars represent standard deviations of the data
obtained from at least three experiments (B) Snapshots showing
the change in an ice bipyramid created in a 0.1 m M solution of
type III AFP from Notched-fin eelpout (denoted nfeAFP8, cooling
rate 0.01 CÆmin)1) (a) T ¼ –0.46 C; (b) T ¼ –0.61 C; (c)–(h)
T ¼ –0.81 C The nonequilibrium freezing point of )0.81 C was
evaluated from this experiment.
Trang 4tended to increase in proportion to the crystal anneal-ing time When the ice bipyramid was annealed for 3 h
at a Tm of )0.05 C, TH activity increased 1.4-fold compared with time zero This TH increase reached 2.5-fold when the 3 h annealing was performed at a
Tmof)0.25 C
Dependence of TH on the size of the seed ice crystal was also examined for 0.1 mm nfeAFP8 at a cooling rate of 0.20 CÆmin)1 The closed circles in Fig 4B rep-resent TH values obtained after 2 h annealing of the ice bipyramid at a Tmof )0.25 C, and the open cir-cles, which are reproduced from Fig 3C, depict com-parable data obtained without ice crystal annealing
TH decreased exponentially with increasing crystal size only after 2 h annealing at a Tmof )0.25 C, and no
TH dependence on crystal size was evident when TH was measured soon after creation of the ice bipyramid
Discussion
In the present study, we first examined the TH depen-dence of nfeAFP8 on the rate of cooling (Fig 3), uti-lizing our in-house-built photomicroscope system Data consistency between the system and a freezing-point osmometer (Fig 2) validated the performance of the system for evaluating TH value Using this system,
we demonstrated that a slower rate of cooling ampli-fies TH, while a faster rate of cooling progressively diminishes TH until it reaches a plateau A similar, but not identical, observation has been made for a small species of AFGP using a freezing-point osmome-ter; the AFGP exhibited significant antifreeze activity upon slow cooling, but lacked such activity if cooled quickly [18] In addition, a time-dependent change in
Tf of a type I AFP solution has been demonstrated
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.1 0.2 0.3 0.4 0.5 0.6
Concentration (m M )
0.01 o C/min
0.20 o C/min
0 0.05 0.10 0.15 0.20 Cooling rate ( o C/min)
o C)
o C) (slow)
(fast)
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
0.01 o C/min
0.20 o C/min
Ice crystal size ( μm)
o C)
(slow)
(fast)
C
Fig 3 (A) Concentration dependence of TH for a 20 lm long ice crystal measured using 0.01 CÆmin)1(closed circles) and 0.20 CÆmin)1 (open circles) rates of cooling (B) Dependence of TH on the rate of cooling (CÆmin)1) (C) Dependence of TH on the size of the ice crystal Each point represents the mean of three experiments and the error bar represents the standard deviation All experiments were performed immediately after preparation of an ice crystal in a 0.1 m M solution of nfeAFP8.
Annealing time (h) 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Annealing Temp.
= Tm - 0.25 °C
1.4 2.5
1.0
Ice crystal size (μm) 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Annealing Time
= 2 h
Annealing Time = 0 h
A
B
Annealing Temp.
= Tm - 0.05 °C
Fig 4 Influence of annealing time and ice crystal size on TH (A)
Thermal hysteresis of nfeAFP8 measured after annealing of an ice
bipyramid for a certain period of time (0–3 h) The measurement
was repeated for three times at the annealing temperatures of
Tm)0.25 C (closed circles) and T m )0.05 C (open circles) using a
cooling rate of 0.20 CÆmin)1 (B) Dependence of thermal hysteresis
of nfeAFP8 on the size of the seed ice crystal Closed circles are
the data obtained after 2 h of annealing, and open circles are those
without annealing All other experimental parameters are the same
as Fig 3.
Trang 5using temperature-gradient thermometry [19] No such
time-dependent change in TH was reported for insect
AFPs, while a linear and negative correlation was
detected between TH and the logarithm of the mass
fraction of ice crystal versus the total water mass for
AFP from cerambycid beetle R inquisitor [9,10]
We examined the dependence of TH on the crystal
size under fast (0.20CÆmin)1) and slow
(0.01CÆmin)1) cooling rates to clarify the correlation
between the cooling rate and seed ice crystal size
These results show that TH decreases with increasing
crystal size at the slow cooling rate, and does not
depend on the crystal size at the fast cooling rate
These results imply that the cooling rate dominates the
TH dependence on the seed crystal size As actual
tem-perature control of the freezing plate in our system is
stepwise, a different cooling rate will produce a
differ-ent time lapse of cooling on a sample In other words,
there is a longer annealing period before starting the
TH measurement at a slower cooling rate We
there-fore examined whether an annealing period of 2–3 h
influenced the TH value of nfeAFP8 An annealing
experiment was performed within the hysteresis gap
(0.25 and 0.05C below Tm), and TH was measured at
a fixed cooling rate (0.20CÆmin)1) These parameters
revealed that the annealing time amplifies TH, the
amplification level is enhanced by lowering of the
annealing temperature, and TH shows dependence on
crystal size only after 2–3 h of annealing As the
time-dependent amplification of TH explains the detection
of a higher TH value at the slower cooling rate, it may
be concluded that the time of annealing is an essential
influence on the TH value of nfeAFP8
For type III AFP, the 3D structure and the
ice-bind-ing mechanism have been extensively examined
High-resolution X-ray and NMR structures have revealed
that a remarkably flat and amphipathic surface is
constructed on type III AFP, enabling complementary binding to the flat ice prism plane [14,21] When the type III AFP solution contains a seed ice crystal, the AFP molecules come closer to the ice through the dif-fusion process, for which an association constant of
107)108s)1Æm)1 can be assumed from the Smulochow-ski model [23] The AFP molecules then undergo prompt binding onto the flat ice plane, which has been thought to progress irreversibly according to the adsorption–inhibition mechanism at the ice–water interface [3,4] Subsequently, convex ice fronts are formed between the ice-bound AFPs on the flat plane,
as depicted in Fig 5A At this stage, the ice crystal adopts a bipyramidal shape During subsequent
TH measurements, the convex ice front overgrows (Fig 5B, dashed line), allowing uncontrolled growth at the nonequilibrium Tf This leads to the detection of a nonzero TH value at time zero (0 h of annealing) The height and curvature of the convex ice front are defined by spacing between adsorbed AFP molecules (i.e the Kelvin effect [24]), such that the TH value reflects the fraction of ice-bound AFPs on the flat ice plane Indeed, TH increases in proportion with the total concentration of nfeAFP8, as can be seen in Fig 3A The fraction of AFPs on the flat ice plane is not, however, changed during the present time-depen-dent experiment on TH (Fig 4) as we fixed the con-centration of AFP at 0.1 mm A plausible explanation for the time-dependence of TH is that nfeAFP8 under-goes ‘secondary’ binding onto the convex ice front over a very slow time scale (2–3 h; Fig 5C) Indeed, a recent study on ice etching revealed that type III AFP can bind to several ice planes in addition to the (10– 10) prism plane [14] TH is the level of supercooling required to nucleate ice growth from the ‘weak’ convex ice fronts, the growth of which is not strongly inhib-ited by AFP If AFP undergoes secondary binding
C
AFP
low TH
TH measurement
A
annealing time (2–3 h)
TH measurement
B
D
solution
ice
ice
ice
high TH
ice
Fig 5 Schematic drawing of ice growth
inhibition of type III AFP based on the
‘adsorption–inhibition’ model [3,4] (A) A
convex ice front is created after primary
binding of type III AFP onto the flat ice
plane (B) Overgrowth of the convex ice
front, giving a low TH value (C) Possible
secondary ice binding of type III AFP onto
the convex ice front during the annealing
period (2–3 h) (D) AFPs bound onto the
convex ice front narrow the growth area
(dashed line), leading to an enhancement of
TH activity.
Trang 6over a very slow time scale after the primary ice
bind-ing, it stabilizes the convex ice front (Fig 5D), leading
to the time-dependent change in TH activity This
con-cept further explains the increased level of
time-depen-dent amplification of TH at the lower annealing
temperature (Fig 4A) The convex ice front will
become more accentuated by lowering the annealing
temperature, which will raise the opportunity for
bind-ing of AFPs to the growbind-ing convex ice front, thereby
enhancing TH activity We recently monitored the
flu-orescence of seed ice crystals at 30 min intervals in a
solution of a fusion protein comprising nfeAFP8 and
green fluorescence protein (nfeAFP8–GFP) (see
supple-mentary Fig S1) We observed a slight increase of
fluorescence intensity in a region proximal to the
seed ice surface, accompanying a slight growth of ice
crystals in proportion with the annealing time (0–3 h)
It is noteworthy that such an intensity change was
observed only at the lower annealing temperature
(Tm)0.25 C) These preliminary data are also
consis-tent with the idea of progressive binding of AFP over
a time scale of 2–3 h
A larger number of convex ice fronts are presumably
located on a larger-sized ice crystal If secondary
binding of AFP has the ability to stabilize convex ice
surfaces, such stabilization becomes imperfect with an
increasing number of weak points on the ice crystal
This supposition is consistent with the data obtained
after 2 h of annealing (Fig 4B, closed circles), for
which a reduction in TH was apparent with increasing
size of the ice crystal The lack of significant size
dependence of TH activity (Fig 4B, open circles)
might be ascribed to nonstabilization of the weak
point of any size of the crystals, because of the lack of
secondary binding of AFP at time zero (0 h of
anneal-ing) It should be noted that no apparent
time-depen-dent TH increase was detected for different types of
AFP (type I AFP from great sculpin and type II AFP
from Japanese smelt; data not shown) Therefore, it is
not clear whether such a slow accumulation process is
a general mechanism for any type of AFP Several past
studies have shown that the bipyramidal ice crystal
can be maintained for a long time [25], but no TH
data have been reported for such an ice bipyramid We
believe that a simple TH measurement before and after
2 h will enable us to learn more about the secondary
binding of AFP over a very slow time scale, which will
be detected as enhancement of the observed TH value
To summarize, we have successfully prepared a
recombinant type III AFP isoform and carefully
mea-sured its TH activity using an in-house-built
photomi-croscope system Measurement of TH dependence on
various experimental parameters revealed, for the first
time, that annealing time significantly causes an enhancement of TH activity of fish type III AFP The adsorption–inhibition model may explain this time-dependent change in TH if type III AFP undergoes sec-ondary binding to the convex ice front following bind-ing to the primary ice plane
Experimental procedures
Sample preparation of AFPIII
A sample of the type III AFP isoform nfeAFP8 was pre-pared for TH measurements as described previously [20] with the following modifications A soluble fraction con-taining a recombinant nfeAFP8 after sonication of
acid buffer (pH 2.9) After dialysis, cation-exchange chro-matography was performed using an Econo-Pac High S cartridge (Bio-Rad, Hercules, CA, USA) with a linear NaCl gradient (0–0.5 m) in 50 mm sodium citrate buffer (pH 2.9) The purified nfeAFP8 was concentrated (approximately
For all the TH experiments, the sample was dissolved in 0.1 m ammonium bicarbonate (pH 7.9)
TH measurement system The experimental set-up using a commercial photomicro-scope system (denoted as the ‘photosystem’) is illustrated in Fig 1 The two main instruments in this system were a Leica DMLB100 photomicroscope (Leica Microsystems, Wetzlar, Germany) and a Linkam THMS 600 temperature controller (Linkam Scientific Instruments Ltd, Tadworth, Surrey, UK) equipped with a liquid nitrogen Dewar flask The latter con-trolled the temperature of the freezing plate with an accuracy
liquid nitrogen The left portion of Fig 1(B) illustrates the position of the sample solution in a quarter of a Hirschmann capillary tube (Hirschmann Labogera¨te, Heilbronn, Ger-many) (length 30 mm, diameter 0.92 mm); each end of the tube was sealed using mineral oil (approximately 1 lL each)
to prevent vaporization of the sample solution (approxi-mately 0.75 lL) The sample-containing capillary was then loaded into a custom-made copper capillary holder (diameter
17 mm, thickness 2.5 mm) (Fig 1B, right), which was placed into the freezing plate on the cooling stage (Fig 1A) After positioning of the capillary tube, 15 lL of ethylene glycol was poured through the viewing hole (Fig 1B, right) to achieve thermal conductivity between the holder and capil-lary tube The ice crystal image was captured using a Color-video 3CCD camera (Sony, Tokyo, Japan) (Fig 1A), and the temperature status was simultaneously viewed on a dis-play and saved as a video file on a personal computer
Trang 7playback of the video The size and length of the ice
bipyra-mid was evaluated from the crystal image captured by the
video For accurate determination of the crystal length, we
initially captured the image of a 30 lm fiber inserted into the
sample solution, and found that the captured length needed
to be multiplied by 1.2 to determine the actual length of the
at least three times, and the values were averaged More
details of the TH measurement procedure are described in
Results
A model OM802 commercial freezing-point osmometer
(Vogel, Giessen, Germany) was used to check the
perfor-mance of the photosystem We initially placed a 50 lL
automatically inserted a frosty probe into the sample
solu-tion to initiate the growth of ice crystals Accordingly,
latent heat was emitted owing to the phase transition from
liquid to solid state, which raised the temperature of the
sample After completion of the rise in temperature, an ice–
water equilibrium state was achieved at a certain negative
Before performing measurements, the osmometer was
nor-malized with a standard fluid of 300 mOsmol
Acknowledgements
We are grateful to Brian Sykes at the University of
Alberta (Edmonton, Canada) for fruitful discussions
on the ice-binding mechanism of nfeAFP8
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Supplementary material
The following supplementary material is available
online:
Fig S1 Time-dependent changes in photomicroscope image of an ice bipyramid in the solution of nfeAFP– GFP
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