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org/JPCB Article

Revealing the Mechanism of Irreversible Binding of Antifreeze

Glycoproteins to Ice
Weijia Zhang,† Han Liu,† Haohao Fu, Xueguang Shao,* and Wensheng Cai*
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ABSTRACT: Antifreeze glycoproteins (AFGPs) are a special kind

of antifreeze proteins with strong flexibility. Whether their antifreeze
activity is achieved by reversibly or irreversibly binding to ice is
widely debated, and the molecular mechanism of irreversible binding
remains unclear. In this work, the antifreeze mechanism of the
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smallest AFGP isoform, AFGP8, is investigated at the atomic level.

The results indicate that AFGP8 can bind to ice both reversibly
through its hydrophobic methyl groups (peptide binding) and
irreversibly through its hydrophilic disaccharide moieties (saccharide
binding). Although peptide binding occurs faster than saccharide
binding, free-energy calculations indicate that the latter is energeti-
cally more favorable. In saccharide binding, at least one disaccharide
moiety is frozen in the grown ice, resulting in irreversible binding,
while the other moieties significantly perturb the water hydrogen-bonding network, thus inhibiting ice growth more effectively. The
present study reveals the coexistence of reversible and irreversible bindings of AFGP8, both contributing to the inhibition of ice
growth and further provides molecular mechanism of irreversible binding.

■ INTRODUCTION
Antifreeze proteins (AFPs) and antifreeze glycoproteins
(AFGPs) are a kind of special proteins with various antifreeze
properties, such as thermal hysteresis (TH),1 dynamic ice
shaping,2,3 and ice recrystallization inhibition,4,5 which can
ensure the survival of organisms in the subzero environment6,7
and have attracted extensive research.8−11 Among them,
AFGPs are the most effective ice recrystallization inhibitor,12
which can prevent the melting of small ice crystals and the
growth of large ice crystals (i.e., Ostwald ripening).13 Due to
this unique property, AFGPs have broad application prospects
in cryopreservation.14,15
AFGPs usually consist of tripeptide repeats of alanine-
alanine-threonine (Ala-Ala-Thr)n, in which the hydroxyl group
of the Thr is glycosylated with β-D-galactosyl-(1,3)-α-N-acetyl-
D-galactosamine. According to their size, AFGPs are divided
into two subtypes, AFGP1-6 and AFGP7-8. AFGP1 to AFGP8
corresponded to n = 50, 45, 35, 28, 17, 8, 5, and 4, respectively.
For the smaller AFGP7-8, Ala in some positions is substituted
by proline (Pro). Compared to native AFPs, AFGPs exhibit
high flexibility and do not have a well-defined structure.
Simulation and experimental studies show that although the
PPII helix is the dominant conformation, it is not the unique
one.16−19 This structure feature makes the antifreeze
mechanism of AFGPs widely debated.
Ahmed et al.20 found that the borate molecules would
combine with the cis-hydroxyl groups of β-D-galactosyl,
resulting in a significant decrease in the TH activity of
AFGP, indicating that the saccharide moieties play an
important role in inhibiting ice growth. In recent years,
perturbation of long-range water dynamics as the mechanism
of AFGPs has been proposed.21−23 The mechanism suggests
that the formation of water bridges on the surface of AFGPs
causes disorder in the H-bond network in the first solvation
shell, which propagates to the remaining solvation shells at low
temperatures, leading to a depressed freezing point.21−23 For
example, Ebbinghaus et al.23 used terahertz spectroscopy to
show that when the temperature is decreased to the biological
working temperature of AFGP, the terahertz absorption of
AFGP solution is stronger and the dynamics of water
molecules become faster. Recently, it was proposed that
AFGP inhibits ice growth by adsorbing on ice crystals, but its
ice-binding site and the reversibility of its adsorption are still
unclear. Mochizuki and Molinero17 suggested that AFGP is
reversibly bound to ice crystals through the methyl groups of
the peptide and disaccharides. They used molecular dynamics
(MD) simulations to prove that AFGP8 preferentially adopts
the PPII helix secondary structure in solution, which is
Received: August 29, 2022
Revised: November 5, 2022
Published: December 13, 2022

© 2022 American Chemical Society https://doi.org/10.1021/acs.jpcb.2c06183

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Figure 1. (A) Saccharide moiety toward ice. Structure of AFGP8 is shown in the inset. (B) Peptide moiety toward ice. Center of mass of AFGP8 is
about 1 nm from the ice surface.
consistent with the experimental results.24−26 This structure
makes the hydrophilic and hydrophobic groups separated on
both sides, which is vital for ice binding, and the decrease of
antifreeze activity after adding borate buffer may be due to the
destruction of the PPII helix structure after the saccharide
moieties are combined with borate. However, Meister et al.27
confirmed that both AFGP isoforms bind irreversibly to ice by
microfluidic solution exchange experiments. The results based
on two-dimensional infrared spectroscopy showed that the
addition of borate buffer did not lead to significant changes in
the conformation of AFGP and had no effect on the dynamics
of water. Therefore, they suggested that AFGPs bind to ice
with their saccharide moieties.27 Groot et al.28 used polar-
ization-resolved femtosecond infrared spectroscopy to analyze
the picosecond reorientation dynamics of water molecules
around AFGP and obtained the same conclusion. Recently, by
measuring the antifreeze activities of AFGP variants, Sun et
al.19 found that both hydrogen-bonding through the hydroxyl
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groups of the disaccharide and hydrophobic interactions
through the polypeptide backbone are important for the
antifreeze activities of AFGPs. However, the details of the
AFGP-ice interaction at the atomic level are unclear.
The abovementioned results indicate that the antifreeze
mechanism of AFGPs is widely debated. For example, whether
the antifreeze effect is exerted by adsorption is still
inconclusive, and the role of its unique disaccharide moieties
in it is not clear. In this work, through simulations with a time
amounted to 31 μs, the atomic details of the antifreeze
mechanism of the smallest AFGP isoform�AFGP8 were
revealed. We found that AFGP8 can bind to the prism plane of
ice crystals through both its peptide and the saccharide
moieties and explain the mechanisms of these two modes of
inhibiting the growth of ice crystals at the atomic level. We
further studied the binding ability of these two binding ways by
free-energy calculations and the perturbation of water
molecules by their nonice binding sides to reveal their
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Table 1. Molecular Assemblies Investigated

no. molecular assemblies atoms box (nm3) time (ns) runsa
1 AFGP8−water 32,627 6.3 × 6.3 × 6.3 150 1 (269 K)
1500 5 (269 K)
2 AFGP8−ice-water (sugar down) 36,995 11.8 × 6.7 × 6.4 1500 3 (268 K)
300 1 (263 K)
1500 5 (269 K)
3 AFGP8−ice-water (sugar up) 36,995 11.8 × 6.7 × 6.4 1500 3 (268 K)
300 1 (263 K)
1 (269 K)
4 ice-water 37,104 11.8 × 6.7 × 6.4 300 1 (268 K)
1 (263 K)
5 AFGP8−ice-water(saccharide binding, SMD) 51,859 6.4 × 6.6 × 15.8 75 30 (270.8 K)
6 AFGP8−ice-water(peptide binding, SMD) 52,039 6.4 × 6.6 × 15.8 75 30 (270.8 K)
a
Independent MD runs.

Figure 2. Snapshots of AFGP8 bound to the prism plane of ice (cyan lines). (A) Saccharide binding. (B) Peptide binding, front view (top), and
enlarged view of the bound methyl groups of AFGP8 (bottom). CH3 of the N-acetyl group is shown as yellow bold sphere, CH3 of 2-, 5-, 8-, 11-Ala
as orange one, CH3 of 4-, 10-Ala as green one, CH2 of 7-, 13-Pro as green one, and CH3 of Thr as purple one. Free water molecules are not shown.
Saccharide moieties and peptide backbone are shown in PaperChain and NewRibbons representations, respectively. These representations are used
throughout the study unless otherwise stated.

differences in the degree of inhibiting the growth of ice crystals. water model is 270 K,34 in good agreement with the
In the present contribution, we reveal the multiple antifreeze experimental value (273.15 K).35 AFGP8 is represented by
mechanism of AFGP8, which provides a theoretical basis for Amber ff14SB36 and the GLYCAM_06j-137 force field. Free-
the design of efficient ice crystal recrystallization inhibitors. energy calculations characterizing the binding ability were

■ METHODS
To explore the mechanism of AFGP8 inhibiting ice growth,
two AFGP8 ice-water assemblies with saccharide side facing
ice (sugar down) and peptide side facing ice (sugar up) were
set up. Because the binding of AFGPs to the prism plane of ice
crystals is more stable than that of other ice planes,17,29 the
saccharide/peptide side of AFGP8 was placed toward the
prism plane of ice crystals, as depicted in Figure 1.
CHARMMGUI30 was employed to prepare the structure of
the AFGP8 with the sequence of Ala-Ala-T*-Ala-Ala-T*-Pro-
Ala-T*-Ala-Ala-T*-Pro-Ala, in which T* denotes the glycosy-
lated Thr. All the MD simulations in this study were carried
out using GROMACS 2021.2,31,32 with the TIP4P/ICE33
water model. The melting point of hexagonal ice with this
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performed using the utilizing steered MD (SMD).38 For SMD
simulations, an external force was applied to the heavy-atom
mass center of the AFGP8, and to pull the entire AFGP8 away
from the ice toward the aqueous medium. The projection of
the distance between the heavy-atom mass center of the
AFGP8 and the ice crystals onto the x-axle was selected as the
coarse variable to describe the pulling process. The force
constant was 1000 kJ mol−1 nm−2. For each binding model, 30
parallel 75 ns SMD simulations were performed. The Jarzynski
equality38 was applied to determine the final free-energy
profiles. All molecular assemblies investigated are shown in
Table 1. Additional methodology and simulation details are
provided in the Supporting Information.
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Figure 3. (A−C) Time evolution of the amount of ice for each binding mode of AFGP8, averaged over multiple runs at different temperatures with
error bars, compared with that in the ice−water assembly without AFGP8. Error bars are shown as the shaded areas around the curves. Since the
ice−water assembly has completely frozen in 110 ns and the amount of ice remains stable in 300 ns, the subsequent simulation is not carried out.
(D−I) Snapshots of each binding mode of AFGP8 at different temperatures.
■ RESULTS AND DISCUSSION
Diversity of Binding Modes between AFGP8 and Ice.
Our simulation results indicate that AFGP8 can bind to ice
both reversibly through its hydrophobic methyl groups (called
peptide binding) and irreversibly through its hydrophilic
disaccharide moieties (called saccharide binding). Four out
of five 1.5 μs parallel simulations of assembly 2 (saccharides
initially facing ice) at 269 K resulted in saccharide binding and
one in peptide binding. Similarly, four out of five 1.5 μs parallel
simulations of assembly 3 (peptide initially facing ice) at 269 K
resulted in peptide binding and one in saccharide binding.
Finally, a total of five saccharide bindings and five peptide
bindings were obtained at 269 K. The simulation snapshots of
multiple parallel simulations (at 269 K) of assemblies 2 and 3
at 1.5 μs are shown in Figures S3 and S4. Compared with the
pure ice-water assembly without AFGP8 in Figure S5, it is
found that the presence of AFGP8 can significantly inhibit the
growth of ice crystals. We found that AFGP8 can bind to ice
crystals through multiple conformations, which can be
classified into two modes: saccharide binding and peptide
binding. The most representative saccharide binding is
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depicted in Figure S3A, and peptide binding is shown in
Figure S4C.
Figure 2A,B shows an enlarged view of the corresponding
binding modes. In saccharide binding, AFGP8 is anchored in
ice through freezing the disaccharide moieties, while the rest of
the protein “floated” in water, as depicted in Figure 2A. In stark
contrast, in peptide binding, AFGP8 binds to ice through the
methyl groups of the peptide and disaccharides, as shown in
Figure 2B. As a result, methyl groups can be adsorbed on the
ice surface, which has been reported in previous studies.17,39
We further conclude that the dominant conformation of the
peptide backbone is the PPII helix in peptide binding mode,
which is in good agreement with the result obtained in ref 17.
Compared with peptide binding, the conformation of the
AFGP8 is more stretched in saccharide binding. A detailed
description is provided in the Supporting Information.
To explore the performance of inhibiting ice growth by these
two binding approaches and the effect of temperature on it,
additional simulations for the same molecular systems
(assemblies 2−4) at 268 and 263 K were carried out, and
the time evolution of the average amount of ice at different
temperatures was monitored (see Figure 3). Details of all the
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Figure 4. Representative configurations of AFGP8 bound to the prism plane of ice. (A−D) Saccharide binding. In each panel, the top and bottom
snapshots correspond to the front and bottom views, respectively. The peptide backbone is shown in NewCartoon representation.
multiple parallel simulations are provided in the Supporting
Information. It can be seen that in the absence of AFP, the
ice−water system has completely frozen in 110 ns, while in the
presence of AFGP8, regardless of its final binding modes, water
is still not completely frozen in 1.5 μs, indicating that AFGP8
can inhibit ice growth through both its peptide and the
saccharide moieties. As shown in Figure 3A, compared to
peptide binding, in saccharide binding, although ice grows
faster in the initial stage (<600 ns), the growth becomes
slightly slower in the final stage (>750 ns). It can be seen from
the average results of five simulations at 269 K (see Figure 3A)
that although the saccharide binding contributes slightly more
to the inhibition of ice growth, there is no significant difference
between the two binding modes seen from the overlapped
errors bars. At 268 K (see Figure 3B), however, the inhibition
performance of saccharide binding is significantly higher. See
also Movies S1−S4 in the Supporting Information. At 263 K,
since the temperature is significantly lower than the TH
activity of the protein, AFGP8 is completely frozen in ice
crystals in only 150 ns, regardless of its initial orientation, as
shown in Figure 3F.
Reversible and Irreversible Bindings. By analyzing all
the trajectories of peptide binding, we found that the
hydrophobic methyl groups of the peptide and disaccharides
can interact with the growing ice crystals in a very short time,
probably due to the action of the solvated water around the
peptide (see Figure S11). However, such binding mode is
found to be reversible. AFGP8 can rapidly exchange between
its different bound conformations with ice through adsorption
and desorption events. The molecular mechanism of reversible
binding is similar to that reported in ref 17, and more details
obtained in this study are provided in the Supporting
Information.
In the case of saccharide binding, the disaccharide group
anchors itself in the ice crystal by hydrogen bonding with ice
through its multiple hydroxyl groups, and the anchored
disaccharide group is then further partially overgrown by the
advancing ice front. In other words, AFGP8 is partly frozen in
the grown ice, resulting in an irreversible binding. In eight
simulations of saccharide binding at 269 and 268 K, once the
disaccharide moiety was frozen in ice, it was never observed to
dissociate from the latter, while the rest of AFGP8 remained
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exposed to the aqueous solution, as delineated in Figure 4A−
D. The irreversible binding process can also be seen from
Movies S1 (269 K) and S3 (268 K). Since the mechanism of
reversible binding has been reported in detail in the study of
Mochizuki and Molinero,17 only the molecular mechanism of
irreversible binding is mainly discussed below.
Free-Energy Calculations Reveal the Molecular
Mechanism of Irreversible Binding. To estimate the ability
of saccharides/peptide to bind to ice crystals, biased
simulations are generally considered. However, a biased
simulation of the binding process is thwarted due to processes
involving water freezing. In this study, the reverse process, that
is, the unbinding of AFGPs from ice crystals was investigated
utilizing SMD.38 Details are provided in the section of free-
energy calculations in the Supporting Information. To this end,
two molecular assemblies (5 and 6 in Table 1) were built. The
initial structures are from the conformations in Figure 2A,B.
To improve computational efficiency, excess ice in the lower
layer was removed, while a thick layer of water was added on
top of the original box to leave enough space for pulling (see
Figure S1). The ice in the model was restrained at its initial
position to prevent it from melting. The temperature of the
SMD simulation is set to 270.8 K to prevent the original ice
around the frozen disaccharide group from melting at the
initial stage of pulling and to prevent the water from freezing
throughout the pulling process (see Figure S2). The bound
AFGP8 (including saccharide-bound and peptide-bound
AFGP8) was pulled from the ice crystals toward the aqueous
medium in 30 independent nonequilibrium experiments, with
a constant pulling speed of 0.3 Å/ns.
The work distribution observed at four different values of
Δd overall obeys a normal law (see Figure S12), indicating that
the calculation has achieved convergence. The free energy
profile underlying the extraction process was calculated on the
basis of the Jarzynski equality.38 As depicted in Figure 5, the
free energy barrier by pulling the saccharide-bound AFGP8 out
of the ice is much greater than that of pulling the peptide-
bound AFGP8 out, indicating that it is thermodynamically
much more favorable for AFGP8 to combine with the ice
crystals through the saccharide moieties. This difference in
binding ability shows that peptide binding may be reversible,
consistent with the simulation results reported by Mochizuki
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Figure 5. Free-energy profiles describing the pulling processes of the
AFGP8 from ice crystals to aqueous medium obtained from thirty 75
ns SMD simulations.
and Molinero,17 while the saccharide binding may be
irreversible, in agreement with the experimental results of
Meister et al.27
To understand the mechanism of irreversible binding, we
further analyzed the processes of saccharide binding and
unbinding. Figure 6A−C shows the binding process where a
disaccharide group anchored to the ice surface by hydrogen
bonding with the ice at the interface and then frozen by the
advancing ice front. It can be, therefore, speculated that the
hydrogen bonding between disaccharides and ice constitutes
the basis of anchoring, which will be discussed in detail below,
and the coverage of the newly grown ice around is another
important factor leading to the irreversible binding. Figure
6D−F shows the unbinding process simulated by SMD. It is
reasonable to believe that the free-energy barrier (see Figure
5A) to be overcome to pull AFGP from ice mainly comes from
disrupting not only the hydrogen bonds between the
disaccharide group and ice but also part of the hydrogen
network of the ice that prevent the anchored disaccharide
moiety from entering the water environment. The higher free-
Figure 6. (A−C) Configurations obtained from the 1500 ns trajectory of the simulation of the saccharide binding corresponding to Figure 2A. The
disaccharide group (A,B) anchored into the ice lattice mediated by hydrogen bonds, (C) frozen and partially overgrown by the advancing ice front.
(D−F) Configurations obtained from the 75 ns trajectory of one of the 30 SMD simulations of the unbinding of the bound structure in Figure 2A.
The liquid water molecules are shown as blue dots.
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energy barrier makes the saccharide binding irreversible. It is
worth mentioning that a transition from peptide binding to
saccharide binding was observed in 1 out of 10 1.5 μs parallel
simulations at 269 K, but the opposite process was not
observed, which further supports that the former is reversible
and the latter is irreversible.
Dual Role of Saccharide Moieties in Irreversible
Binding. Acting as an Anchor. As shown in Figure 2A, one
disaccharide is irreversibly frozen in ice to play an anchoring
role. We counted the number of hydrogen bonds formed
between this disaccharide and water molecules (including ice
and free water molecules) when the entire AFGP8 is exposed
to the solution (0−300 ns) and is adsorbed on ice crystals
(1200−1500 ns), separately. As shown in Figure 7A−C, the
number of hydrogen bonds and the distributions of average
distance and angle do not vary, indicating that water molecules
that solvate the disaccharide group in aqueous solutions can be
replaced by those in hexagonal ice. We further calculated the
hydrogen bond lifetime between the disaccharide and water
molecules, as shown in Figure 7A. When the disaccharide is
combined with ice crystals, the hydrogen bond lifetime
increases significantly, indicating that the disaccharide group
is frozen in ice, as shown in Figures 3D and 6C. Front and
bottom views of snapshots of sugar chains binding to ice
crystals are shown in Figure 7D,E, respectively, in which the
hydrogen bonds formed by hydroxyl hydrogen atom and water
molecule are represented by blue lines, and the hydrogen
bonds formed by hydroxyl/carbonyl oxygen atom and water
molecule are represented by magenta lines. It can be seen from
Figure 7D that the hydrogen bonds formed by galactose group
and water molecule and the hydrogen bonds formed between
H2O molecules in hexagonal ice are highly coincident, which is
conducive to its substitution of water molecules to form
hydrogen bonds with ice crystals. Although the coincidence
degree of hydrogen bonds of N-acetyl-D-galactosamine group
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Figure 7. Anchoring role in saccharide binding. (A) Amount and lifetime of hydrogen bonds formed between disaccharide and water molecules,
when the AFGP8 is exposed to the solution (0−300 ns) or adsorbed on ice crystals (1200−1500 ns). Distributions of (B) average distance and (C)
average angle at 0−300 ns and 1200−1500 ns, corresponding definitions are shown in the inset of (C). Final snapshot of 1.5 μs saccharide binding
simulation at 269 K, front view (D) and bottom view (E).

Figure 8. Perturbation of water by saccharide moieties. (A) Distribution of F4 order parameters of water around the unbound saccharide moieties
under saccharide binding compared with that under peptide binding. (B,C) Snapshots of saccharide binding simulation at 268 K. Part of the frozen
water molecules melt due to the perturbation of the unbound saccharide moieties.

with hexagonal ice is not high, it can also combine with ice
crystals.
Perturbing Water Structure and Inhibiting Ice Growth. It
should be pointed out that although the disaccharide group in
Figure 7D,E is frozen in ice, the rest part of the AFGP remains
in liquid water (see Figure 2A). The perturbation of liquid
water by the hydrophilic saccharide moieties is considered to
prevent water from freezing. 23,28 However, after one
disaccharide moiety is frozen, will the perturbation perform-
ance of the rest of the disaccharide moieties be affected? To
this end, we compared the perturbation of water by the flexible
saccharide moieties under the two binding modes. As shown in
Figure 8A, the distribution of the F4 order parameter40 for the
free water molecules within 5 Å around the saccharide moieties
was calculated. For saccharide binding, only the unbound
saccharide moieties were considered, for peptide binding, all
saccharide moieties were included. The F4 order parameter
calculates the H−O···O−H torsional angle (ϕ) of a hydrogen-
bonded water dimer and is defined as follows:
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F4 = cos 3
F4 can distinguish between ice and liquid, ∼0.0 for liquid
water, and ∼ −0.5 for hexagonal ice. The result shows that
when the saccharides bind to ice, there is a slight shift in the F4
distribution toward zero, indicating that the perturbation effect
on water molecules becomes stronger. This increased
perturbation may be due to the fact that the contact area
between AFGP8 and the ice crystals is larger than that when
the peptide moiety binds to ice, and only one disaccharide
group is anchored in ice, while the others have faster kinetic
properties. To better reveal the perturbation effect on water
molecules by saccharide moieties, the root-mean-square
deviations (rmsds) of the unfrozen part of AFGP8 and the
entire AFGP8 were calculated using the trajectory obtained
from assembly 2 (see Figure S9 in the Supporting
Information). The greater volatility of the rmsd indicates the
higher flexibility of AFGP8, showing that the perturbation
effect on water molecules by saccharide moieties becomes
stronger, when at least one disaccharide moiety is frozen in the
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grown ice. Moreover, the perturbation by saccharide moieties
can melt the frozen water molecules at the interface, as shown
in Figure 8B,C, which can also be seen from the time evolution
of the amount of ice (Figure S10) and from Movies S1 and S3
in the Supporting Information. In these movies, the free part of
the AFGP is observed to constantly surf on the newly grown
ice, causing the latter to partially melt.
■ CONCLUSIONS
Water freezing is a seemingly ubiquitous natural phenomenon,
the mechanism of which has been verified for nearly a
century.41 There are still many controversies, such as the
antifreeze mechanism of AFGPs. In this contribution, our
simulations reveal that AFGP8 can bind reversibly and
irreversibly to the prism plane of ice crystals through its
hydrophobic methyl groups and the hydrophilic saccharide
moieties, respectively. This result indicates that different from
other AFPs, AFGP8 has multiple antifreeze mechanisms.
Furthermore, we find that AFGP8 remains highly flexible when
bound to ice, whether via methyl groups or sugar side chains,
and thus, has the potential to adjust its conformation to adapt
and bind to irregular ice crystals, which may account for the
superior ability to inhibit ice crystal recrystallization.17 We
further studied the strength of these two binding modes by
free-energy calculations and suggested that the saccharide
binding is more stable than the peptide binding. In saccharide
binding, one or two disaccharide side chains are anchored into
the ice lattice mediated by hydrogen bonds and frozen in ice,
resulting in an irreversible binding. In peptide binding, AFGP8
adsorbs to the ice surface through weak hydrophobic
interactions of methyl groups with the ice crystals, resulting
in a reversible binding. In addition, when one disaccharide
moiety is frozen, significant perturbation of water by the other
disaccharide moieties not only effectively prevents the
surrounding water from freezing but also induces melting of
the advancing ice fronts. This irreversible binding mechanism
may explain the size effect of the AFGPs on ice growth
inhibition. The experimental study observes that the larger
AFGP isoform exhibits more excellent performance in
inhibiting ice growth than the smaller one.27 We can speculate
that this is because the large isoforms (AFGP1-6) have more
unfrozen saccharide moieties compared to the small ones
(AFGP7-8) and can thereby perturb the water molecules over
long distances. In this contribution, we propose that both
reversible and irreversible binding are of paramount
importance for the ability of AFGP8 to inhibit ice growth
and provide the molecular mechanism for the irreversible
binding of AFGP8 to ice.
■
*
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jpcb.2c06183.
Methods, results of multiple parallel simulations, analysis
of water molecules in the hydration layer, test of the
temperature for SMD simulations, PPII helix conforma-
tions analysis, molecular mechanism of the reversible
binding, time evolution of rmsd of the unfrozen part of
AFGP8 and entire AFGP8, work distribution from the
30 irreversible stretching simulations, and influence of
initial orientation of AFGP on simulation results (PDF)
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Movie S1 describing the irreversible binding at 269 K
(MP4)
Movie S2 describing the reversible binding at 269 K
(MP4)
Movie S3 describing the irreversible binding at 268 K
(MP4)
Movie S4 describing the reversible binding at 268 K
(MP4)
■ AUTHOR INFORMATION
Corresponding Authors
Xueguang Shao − Research Center for Analytical Sciences,
Haihe Laboratory of Sustainable Chemical Transformations,
Tianjin Key Laboratory of Biosensing and Molecular
Recognition, State Key Laboratory of Medicinal Chemical
Biology, College of Chemistry, Nankai University, Tianjin
300071, China; orcid.org/0000-0001-5027-4382;
Email: xshao@nankai.edu.cn
Wensheng Cai − Research Center for Analytical Sciences,
Haihe Laboratory of Sustainable Chemical Transformations,
Tianjin Key Laboratory of Biosensing and Molecular
Recognition, State Key Laboratory of Medicinal Chemical
Biology, College of Chemistry, Nankai University, Tianjin
300071, China; orcid.org/0000-0002-6457-7058;
Email: wscai@nankai.edu.cn
Authors
Weijia Zhang − Research Center for Analytical Sciences, Haihe
Laboratory of Sustainable Chemical Transformations,
Tianjin Key Laboratory of Biosensing and Molecular
Recognition, State Key Laboratory of Medicinal Chemical
Biology, College of Chemistry, Nankai University, Tianjin
300071, China; orcid.org/0000-0001-9200-6036
Han Liu − Research Center for Analytical Sciences, Haihe
Laboratory of Sustainable Chemical Transformations,
Tianjin Key Laboratory of Biosensing and Molecular
Recognition, State Key Laboratory of Medicinal Chemical
Biology, College of Chemistry, Nankai University, Tianjin
300071, China; orcid.org/0000-0003-0194-0646
Haohao Fu − Research Center for Analytical Sciences, Haihe
Laboratory of Sustainable Chemical Transformations,
Tianjin Key Laboratory of Biosensing and Molecular
Recognition, State Key Laboratory of Medicinal Chemical
Biology, College of Chemistry, Nankai University, Tianjin
300071, China; orcid.org/0000-0003-0908-0046
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jpcb.2c06183
Author Contributions
†
W.Z. and H.L. contributed equally.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This study was supported by the National Natural Science
Foundation of China (22073050 and 22174075), the Natural
Science Foundation of Tianjin, China (20JCYBJC01480), and
the Haihe Laboratory of Sustainable Chemical Transforma-
tions (ZYTS202105).
https://doi.org/10.1021/acs.jpcb.2c06183
J. Phys. Chem. B 2022, 126, 10637−10645
The Journal of Physical Chemistry B
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