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org/JPCB Article

Deciphering the Role of the Non-ice-binding Surface in the

Antifreeze Activity of Hyperactive Antifreeze Proteins
Prasun Pal, Sandipan Chakraborty,* and Biman Jana*
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ABSTRACT: Antifreeze proteins (AFPs) show thermal hysteresis through specific

interaction with the ice crystal. Hyperactive AFPs interact with the ice surface through
Downloaded via UNIV DEGLI STUDI DI PERUGIA on May 22, 2023 at 07:45:35 (UTC).

a threonine-rich motif present at their ice-binding surface (IBS). Ordering of water

around the IBS was extensively investigated. However, the role of non-IBS in ice
growth inhibition is yet to be understood completely. The present study explores the
nature of hydration and its length-scale evaluation around the non-IBS for hyperactive
AFPs. We observed that the hydration layer of non-IBS is liquid-like, even in highly
supercooled conditions, and the nature of hydration is drastically different from the
hydration pattern of non-AFP surfaces. In similar conditions, the hydration layer
around the IBS is ice-like ordered. Non-IBS of the hyperactive AFP exposes toward the
bulk and is able to maintain the liquid-like character of its hydration water up to 15 Å.
We also find that the amino acid compositions and their spatial distribution on the
non-IBS are markedly different from those of the IBS and non-AFP surfaces. These
results elucidate the combined role of IBS and non-IBS in ice-growth inhibition. While
IBS is required to adsorb on ice efficiently, the exposed non-IBS may prevent ice nucleation/growth on top of the bound AFPs.

1. INTRODUCTION
Understanding the water-ice phase transition and its
modulation by external perturbation is an active area of
research. The process is a central event in many natural
atmospheric phenomena such as snowfall and atmospheric
icing as well as in the cryo-industry. Nature develops a unique
tool to modulate ice growth as a survival strategy for many
organisms living in extremely cold conditions.1,2 These
organisms produce antifreeze proteins (AFPs) that can depress
the freezing point of water in a noncolligative manner.2 Certain
AFPs are capable of depressing freezing points up to 5−6 °C
or even higher, and they are termed as hyperactive AFPs.3,4
They are either characterized by β-helical topology,5 a
flattened cylindrical structure,6 or a solenoid architecture.7
The ice binding surface (IBS) of different hyperactive AFPs is
topologically as well as sequentially similar and commonly
characterized by the Threonine-X-Threonine (T-X-T) motif
where X can be any amino acid. The degree of repeat is
different for different hyperactive AFPs, which in turn alters
the extent of ice binding ability. In general, measurement of
thermal hysteresis (TH) activity is an age-old technique used
to quantify the efficacy of AFP and provides evidence of ice
binding. Mutations of residues at the IBS lower the TH activity
and alter the growing ice morphology, evident from ice-etching
techniques, which infer that the IBS interacts with the ice
surface. Recent simulation studies show that the perfect spatial
arrangements of strategically located amino acid repeats make
the IBS suitable for ice interactions.8−15 In fact, both the IBSs
for AFPs and INPs (ice nucleating proteins) favorably interact
with the ice crystal.16−18 However, due to the size limitation,
the IBS of AFP is not suitable for ice nucleation.19 The large
size of the IBS of INPs stimulates ice nucleation since it can
form stable ice nuclei that can initiate ice growth. Very
recently, the notion got further credence from the experimental
report, which showed that the fish type III AFP and beetle
TmAFP can induce ice nucleation above the homogeneous
freezing temperature in solution, probed by using a customized
microfluidic device that employs nanoliter droplets.19 Ice
nucleation temperature is strongly correlated with the critical
size of ice nuclei, and a larger protein surface increases the
temperature where ice nucleation is triggered.19
Although the IBSs of both AFPs and INPs17 are suitably
designed to interact with the ice surface, the specific character
of non-IBS might play a determining role in ice-growth
inhibition by AFPs. According to the adsorption-inhibition
model, IBS interacts with the ice plane, and the non-IBS plane
exposes to the bulk solvent. Ice grows in between two bound
AFPs, not on the non-IBS, which accounts for the generation
of the curved surface within the hysteresis gap.13 Using HD-
VSFG spectroscopy and molecular dynamics simulation, it has
Received: February 12, 2020
Revised: May 15, 2020
Published: May 19, 2020

© 2020 American Chemical Society https://dx.doi.org/10.1021/acs.jpcb.0c01206

4686 J. Phys. Chem. B 2020, 124, 4686−4696
The Journal of Physical Chemistry B
been shown that non-AFP also binds to the ice surface but
with a less affinity than the AFPs.20 Importantly, non-AFPs are
incapable to retard ice growth and engulfed within a growing
ice crystal as an impurity.20 However, AFPs can retard ice
growth, and there is no ice growth on the non-IBS exposed
toward the bulk water. Free energy calculation reveals that the
binding free energy for the interaction of IBS with the ice
surface is more favorable than the interaction between non-IBS
and ice surfaces.9 Liu et al. demonstrated that, for the solid
surface-tethered AFP, the non-IBS exposed to the bulk
solution impedes ice nucleation, whereas exposed IBS
promotes ice nucleation.21
Many molecular dynamics simulations suggested that water
is more ordered around the IBS than around the non-IBS,
which rationalizes the preferential ice interaction by IBS.
However, it is noteworthy that the presence of a preordered
water layer around the IBS is not common to all
AFPs.8−12,22−24 The presence of “ice-like” water around the
IBS of hyperactive TmAFP was not observed, probed either by
the 17O magnetic relaxation dispersion (MRD) method or
molecular dynamics simulation.25,26 Similar observation was
also reported by Meister et al. for another hyperactive AFP,
DAFP-1, using vibrational sum-frequency generation spectros-
copy.27 Duboué-Dijon and Laage, on the other hand, reported
IBS induced short-range enhancement of the water structure
and a greater slowdown of water reorientation dynamics.28 On
the other hand, the presence of ordered water population
around the IBS for many hyperactive,29 type II,30 and type
III31 AFPs is crystallographically resolved. Detailed inves-
tigations and thermodynamic analysis reveal the role of
hydrophobic hydration of IBS residues in the stabilization of
the ordered water layer in the low-temperature region.10,32−34
The implication of this ordered water layer on ice binding has
been elucidated at the atomic level for type I,8 II,11 III,12 and
insect AFPs.9,10 While hydration around the IBS is primarily
explored to interpret its role in ice recognition, the hydration
pattern around the non-IBS is comparatively less explored.
Previous computer simulations depicted that the ordering of
water around the non-IBS is significantly lower than that
around the IBS.22,23 Kuffel et al. showed that the ordering of
solvation water around the IBS and non-IBS is distinct. In
terms of structural ordering, there are some similarities
between the solvation water around the IBS and ice.35 On
the other hand, structural characterization of hydration water
probed by the third-order Steinhardt parameter (q3) showed
that the order of water around the non-IBS is bulk-like.26
Duboué-Dijon and Laage reported that the average asphericity
of hydration water around the non-IBS is almost identical to
that of the non-AFP.28 However, Kozuch et al. developed a
neural network-based antifreeze activity prediction platform
where it has been observed that the short hydrogen bond
lifetime of hydration water around the non-IBS is an important
parameter dictating the antifreeze activity.36 Using computer
simulation, it has recently been shown that water density is
higher around the non-IBS plane than around the IBS.37
However, there is no such measurable difference between the
hydration shell thickness and densities between AFPs and non-
AFPs.37 Despite all these efforts, a comprehensive picture of
hydration pattern around the non-IBS, its temperature, and
length-scale dependency is missing, which is required to
understand how the hydration around the non-IBS is different
from the hydration pattern around IBS and non-AFP surfaces.
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The present study aims to explore the hydration of non-IBS
to understand how it is different from the hydration of IBS,
non-AFP, and bulk water. Differential water ordering around
the non-IBS, IBS, non-AFP, and bulk water has been probed to
understand their temperature and length-scale dependencies.
Results obtained from the study provide a crucial foundation
to comprehend the understanding on the role of non-IBS in
the antifreeze activities for hyperactive AFPs.
2. METHODS
All the simulations were performed using the GROMACS
2016.438,39 package using the AMBER-ILDN40,41 force field
with the TIP5P42 water model. The Visual Molecular
Dynamics (VMD) Package was used for visualization
purposes.43
2.1. Preparation of the Systems. Structures of hyper-
active insect AFPs and non-AFP were obtained from the
protein structural repository, PDB IDs 1EZG6 (TmAFP),
1M8N44 (SbwAFP), and 2BM445 (non-AFP). The non-AFP
considered in the study belongs to a similar fold like the
hyperactive AFPs such that the effects of size and geometry are
minimal. The non-AFP is a dimer, and a monomeric unit was
considered in the study. The C-terminal end (residues 120−
181) does not adopt the β-role topology; thus, it was excluded
from the study. All the heteroatoms and crystallographic water
molecules were also excluded from the simulations. The
threonine-rich flat β-sheet of these hyperactive AFPs was
considered as the IBS, and other residues that were on the
opposite surfaces were considered as non-IBS in our study.
Details of the residues considered as IBS and non-IBS for the
two AFPs (TmAFP and SbwAFP) and residues considered for
the non-AFP are shown in Table S1 (Supporting Information).
Mutated TmAFP was constructed by mutating all the
threonine residues at the IBS to glycine.
2.2. Simulation Details. All proteins were initially
minimized in vacuo using the steepest descent algorithm to
remove any bad contacts. Then, each of them was solvated in a
box containing TIP5P water molecules. The dimension of the
simulation box containing TmAFP was 6.24 × 5.92 × 7.99 nm3
filled with 9230 water. Two Na+ ions were added to make the
system charge neutral. The dimension of the solvated box for
SbwAFP was 6.81 × 6.99 × 8.39 nm3 with 12,455 water
molecules. Three Cl− ions were added to neutralize the charge
of the system. The box dimension of the solvated non-AFP was
7.14 × 7.21 × 10.38 nm3 with 16,668 water molecules. Four
Na+ ions were added to neutralize the charge of the system.
The box dimension of the mutated TmAFP was 6.24 × 5.89 ×
7.99 nm3 containing 9175 water molecules. Two Na+ ions were
added to neutralize the charge of that system. The triclinic box
was so chosen that the protein atoms were at least 2 nm apart
from the box edges. Long-range electrostatic interactions were
evaluated by the particle mesh Ewald46 method with a cutoff of
1.0 nm and a grid spacing of 0.16 nm. A cutoff of 1.0 nm was
applied to calculate the van der Waals (vdW) interactions.
Each of the solvated systems was initially energy-minimized
with 5000 steps by using the steepest descent algorithm. The
minimized solvated protein was then equilibrated in the
canonical (NVT) ensemble for 1 ns by employing restraining
forces with a force constant of 1000 kJ mol−1 nm−1 to the
protein (backbone) only, while the water molecules were
allowed to move freely. Final production simulations of 50 ns
were performed with the equilibrated solvated protein in the
NVT ensemble at different temperatures (240, 250, 260, 273,
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and 300 K) where the protein backbone was restrained. The
temperature was controlled by coupling with a thermostat with
a time constant set to 0.1 ps using the v-rescale algorithm.47 To
check the ordering of the bulk, we also performed simulation
of a box of 512 TIP5P water molecules with a dimension of 2.5
× 2.5 × 2.5 nm3 at five different temperatures using the same
simulation parameters mentioned above. Additional simula-
tions for another non-AFP, ubiquitin (PDB ID: 1UBQ), were
performed at the mentioned five different temperatures using
the same protocol mentioned above. The simulation box
dimension was 7.06 × 7.23 × 7.66 nm3, containing 7755
TIP5P water molecules.
2.3. Analysis. Structural analysis of water around the
protein surface was performed on the last 30 ns of the
production simulation for each system by calculating the four-
body order parameter (F4)48 based on the H−O···O−H
torsional angle of the hydrogen-bonded water dimer and with
the modified Steinhardt bond-order parameter (<q6>).49
The nature of ordering of water was probed with the four-
body order parameter called the F4 order parameter. One
needs to calculate the H−O···O−H torsional angle (ϕ) of a
different temperatures (240, 250, 260, 273, and 300 K). The
TIP5P water model has been used in the study since the
melting point (Tm) of the water model is close to the melting
temperature of ice.52 We have used the AMBER-ILDN force
field for simulation, which is widely used in biomolecular
simulation. We have further validated the force field by
assessing its ability to maintain the inward orientations of the
OH groups of the threonine ladder of the IBS, as observed in
the crystal structure, by calculating the root-mean-square
deviation from the crystallographic orientation. The observed
fluctuation is only around 0.04 nm (Figure S1, Supporting
Information), which indicates that the crystallographic
orientation of the OH groups is maintained throughout the
simulation. We have defined the first hydration shell by
considering the water molecules within 0.56 nm from each
surface of the protein. The distance corresponds to the entire
first peak in the radial distribution function of water with
respect to heavy atoms of the protein. The radial distribution
function of water around the TmAFP surfaces at 260 K is
shown in Figure S2 (Supporting Information). Evident from
the figure, the water density is higher around the non-IBS than

÷÷÷÷÷÷÷◊
hydrogen-bonded water dimer. By definition, hydrogen
farthest from the partner oxygen atom of the water molecule
in the water dimer was used to define the OH vectors to
calculate the H−O···O−H torsion angle (ϕ).
F4 = ⟨cos 3ϕ⟩
F4 can distinguish between ice, liquid, and clathrate. The
F4 values are ∼0.7 for clathrate water, ∼0.0 for liquid water,
and ∼ −0.3 for hexagonal ice.
Another order parameter used to probe ordering of water
was the modified Steinhardt bond order parameter, <q6>. It is
a local bond-order parameter that measures the local structure
around IBS, in agreement with Nutt and Smith, which
indicates a more liquid-like water structure around the non-
IBS. Notably, ice is more ordered but less dense than liquid
water.22 Further water structure analysis using O−O−O angle
distribution reveals more ordering of water around the IBS in
(1) comparison to the non-IBS (Figure S3, Supporting Informa-
33,48,50 tion), as observed by Kuffel et al.35 These observations validate
our simulation methodology, which is then used to explore the
hydration patterns around different AFP and non-AFP surfaces
in critical detail.
3.1. Water Ordering around the IBS Is Considerably
Different from That around the Non-IBS for Hyper-

ÄÅ ÉÑ
around a particle51 and defined as (here, l = 6) active AFPs. The Steinhardt bond-order parameter, <q6>, has
ÅÅ 4π ÑÑ
ÅÅ 2Ñ
|⟨qlm(i)⟩| ÑÑÑ
been used to probe the water ordering around the IBS and
⟨ql(i)⟩ = ÅÅ
ÅÅ 2l + 1 ÑÑ
+l
non-IBS. The parameter is widely used to differentiate between
ÅÇ ÑÖ
∑ liquid water and ice. Upon going from liquid-like water to a
m =−l (2) highly ordered water structure, the <q6> value increases.

ÅÄÅ ÑÉÑ
where Evident from Figure 1A, ordering of water around the IBS is

ÅÅ ÑÑ
⟨qlm(i)⟩ = ÅÅÅ Å qlm(j)ÑÑÑÑ
Nneighs(i)

ÅÅ Nneighs(i) + 1 ÑÑ
1
ÅÅÇ ÑÑÖ
∑
j=0 (3)
Nneighs(i)
1
qlm(i) = ∑ Ylm(θij , ϕij)
Nneighs(i) j=1 (4)
Ylm (θij, ϕij) is the spherical harmonics, and θij and ϕij are the
polar and azimuthal angles of the bond vector connecting the
ith water molecule to the jth member of its Nneighs(i) measured
in an arbitrary laboratory frame of reference, respectively. The
sum goes over all neighboring oxygen atoms of water Figure 1. (A) Distribution of the <q6> order parameter of water
molecules, Nneighs(i), that are within 3.5 Å from the oxygen within the first solvation shell around the IBS and non-IBS of TmAFP
atom of water(i). The calculation includes also the oxygen upon lowering the temperature from 300 to 240 K. The left inset
atom of that water(i) itself. figure represents changes of the average <q6> values upon lowering
the temperatures. The right inset figure represents the distribution of
3. RESULTS the <q6> order parameter for bulk water at different temperatures. (B)
Distribution of the F4 order parameter of the hydration water around
The ordering of water around the IBS and non-IBS planes of the IBS and non-IBS of TmAFP upon lowering the temperature from
two hyperactive AFPs (TmAFP and SbwAFP) has been probed 300 to 240 K. The left inset figure represents changes of average F4
by using molecular dynamics simulation. For comparison, we values for hydration water around the IBS and non-IBS upon lowering
have also considered a non-AFP with similar topology. the temperatures, and corresponding bulk distribution is shown in the
Simulations of each system have been performed at five right inset figure.

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higher than that around the non-IBS. The <q6> distribution
around the non-IBS is liquid-like (shown in the inset).
Interestingly, upon lowering the temperature, the hydration
water layer around the IBS progressively becomes more
ordered as the peak of <q6> distribution shifts from ∼0.52 at
300 K to ∼0.55 at 240 K. However, the non-IBS surface resists
such ordering of water to some extent upon lowering the
temperature. The peak of <q6> distribution changes from
∼0.39 to ∼0.40 upon lowering the temperature from 300 to
240 K. This clearly signifies that the hydration water around
the non-IBS retains a liquid-like character even in significant
supercooled conditions.
On the other hand, the F4 order parameter is used to
differentiate between different states of water. The F4 values
are ∼0.7 for clathrate water, ∼0.0 for liquid water, and −0.3 for
hexagonal ice.53 Previously, F4 was successfully probed to
uncover the functional transition from clathrate to ice-like
ordering in water upon lowering the temperature for a
hyperactive AFP adsorbed on the ice surface.9 Upon lowering
the temperature progressively from 300 to 240 K, a gradual
shift in the F4 distribution toward the more negative values has
been noticed, which indicates more ice-like ordering of the
hydration water around the IBS (Figure 1B). Marked
differences have been observed in the behavior of hydration
water around IBS and non-IBS upon lowering the temperature.
Lowering the temperature from 300 to 260 K, the F4
distribution of hydration water around the non-IBS is almost
unchanged and remains liquid-like (Figure 1B). Further
lowering the temperature from 260 to 240 K, there is a
minute shift in the F4 distribution toward the negative values
for hydration water around the non-IBS. The peak value is ∼
−0.03 at 240 K for non-IBS, while for IBS, it is −0.112 at the
same temperature. Thus, while non-IBS resists the ice-like
ordering of the hydration water, IBS promotes the ice-like
ordering, which is clearly evident from the average F4 value
upon lowering the temperature (Figure 1B, inset).
We have further evaluated the contrasting behavior of
hydration water around the IBS and non-IBS for another
hyperactive AFP from Spruce budworm (SbwAFP) with the
aid of both <q6> and F4 order parameters to address the
generality of the observations, and results are shown in Figure
2. We have observed a similar pattern of hydration around the
IBS and non-IBS upon lowering the temperatures (Figure 2),
Figure 2. (A) Distribution of the <q6> order parameter of hydration
water around the IBS and non-IBS of SbwAFP upon lowering the
temperature from 300 to 240 K. The inset figure represents the
average <q 6> values at different studied temperatures. (B)
Distribution of the F4 order parameter of hydration water around
the IBS and non-IBS of SbwAFP upon lowering the temperature from
300 to 240 K. The inset figure represents the average F4 values of
water around the IBS and non-IBS at different temperatures.
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as observed in the case of TmAFP. However, the degree of
change is different. Here, we have also noticed that water
around the IBS is more ordered than that around the non-IBS,
evident from the distribution of <q6> (Figure 2A). Upon
lowering the temperature, water around the non-IBS surface
remains more liquid-like than the hydration water of IBS and
bulk, which is evident from both the <q6> and F4 distributions
(Figure 2A,B).
Similar contrasting behavior of hydration water around the
IBS and non-IBS is evident for both TmAFP and SbwAFP,
which indicates that the feature is probably common across
different hyperactive insect AFPs. The observed results support
the observation of Liu et al., where it has been shown that ice
nucleation is facilitated on the exposed IBS surface, whereas
exposed non-IBS retards ice nucleation.21 However, a critical
question still remains unexplored. Is this behavior of the
hydration water around the non-IBS similar to the hydration
pattern of non-AFPs?
3.2. Behavior of the Hydration Water around the
Non-IBS of Hyperactive AFP Is Different from That of
Non-AFP. We have considered a non-AFP that possesses
similar β-roll topology as observed in the case of TmAFP. Also,
the sizes of the AFP and non-AFP are very similar, and both of
them contain 7 β-rolls. This consideration minimizes the
effects of geometry and excluded the volume effect on
observed quantities.
Interestingly, changes of both F4 and <q6> distributions for
hydration water around the non-AFP surfaces are markedly
different from the behavior of water around the non-IBS of
TmAFP upon lowering the temperature. <q6> distribution
reveals that water around the non-AFP is more ordered than
that around the non-IBS at all the studied temperatures for
TmAFP (Figure 3A). A similar observation has also been
Figure 3. (A) Distribution of the <q6> order parameter of water
within the first solvation shell around the non-IBS of TmAFP and
non-AFP surfaces upon lowering the temperature from 300 to 240 K.
(B) Distribution of the F4 order parameter of hydration water around
the non-IBS of TmAFP and the surfaces of non-AFP upon lowering
the temperature from 300 to 240 K. Corresponding changes in
average <q6> and F4 values are shown in the inset.
noticed for SbwAFP when the temperature-dependent
behavior of its hydration shell has been compared with the
hydration behavior around the non-AFP surfaces (Figure S4,
Supporting Information). Notably, the degree of ordering of
water around the non-AFP is markedly less than that of the IBS
of both the hyperactive AFPs. To further assess the generality
of the observation, we choose another globular non-AFP,
namely, ubiquitin, a widely studied model protein. Comparison
of hydration shell ordering around ubiquitin and non-IBS of
TmAFP upon lowering the temperature is shown in Figure S5
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(Supporting Information). We observed a similar behavior as
noted above. Thus, non-AFPs do not favor ice formation like
the IBS of an AFP. Potentials of mean force calculations also
reveal that the IBS of AFP possesses a higher binding affinity
with the ice surface than the non-AFPs,20 which is in line with
the observation obtained from this study.
Changes in the F4 values reveal progressive ordering of
hydration water around the non-AFP surfaces with the
lowering of temperature (Figure 3B, Figures S4B and S5B),
which is unlike the behavior of hydration water around the
non-IBS surface. Thus, the behavior of water around the non-
IBS is unique for AFPs, and it resists water ordering into an
ice-like form even in highly supercooled conditions, which
might have implications on the ice-growth inhibition ability of
AFPs.
3.3. Degree of Differential Water Ordering around
the IBS and Non-IBS: Effect of Temperatures. We have
then probed the effect of temperature on the discriminatory
behavior of water ordering induced by the IBS and non-IBS of
AFPs. Also, the changes of water ordering around the non-AFP
surface upon lowering the temperature have been considered
for comparison (Figure 4A,B). 300 K temperature has been
Figure 4. Changes in average (A) F4 and (B) <q6> order parameters
of water within the first solvation shell around the IBS, non-IBSs of
two AFPs (TmAFP and SbwAFP), and a non-AFP at different
temperatures.
considered as the reference temperature, and the degree of
water ordering has been probed by subtracting the average F4
and <q6> values at 300 K from the respective average values at
a particular temperature. A coherent picture has been evident
either probed by F4 or <q6>. The non-IBSs of TmAFP and
Figure 5. (A) Changes in the average F4 order parameter values of different hydration shells around the IBS, non-IBS of TmAFP, and non-AFP at
260 K. (B) Changes of ΔF4 (F4 at 300 K to F4 at 260 K) in different hydration layers around the IBS, non-IBS of TmAFP, and nonAFP and
compared with the bulk values.
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SbwAFP resist ice-like ordering of hydration water very
efficiently up to 260 K. Notably, the Tm of TIP5P water is
∼274 K.52 A considerable reduction in F4 values and
concomitant increase in <q6> values of hydration water at
260 K around the IBS for both the AFPs have been noticed.
This indicates the ice-like ordering of hydration water around
the IBS. The hydration water around the non-AFP stays in
between non-IBS and IBS of the AFP, which indicates that the
non-AFP cannot resist ice-like ordering of its hydration water
upon lowering the temperature; however, it is also less efficient
to promote ice-like ordering of the hydration water in
comparison to the IBS.
Upon lowering the temperature up to 240 K, there is a
marginal ordering in the hydration water around the non-IBS
for both the AFPs, evident from the minute decrease in F4 and
increase of <q6> values. However, there is a large increase in
water ordering around the IBS for both the AFPs in the 260−
240 K temperature region, evident from a large decrease in F4
and an increase in <q6> values. The magnitude of the decrease
in F4 values indicates a favorable ice-nucleating condition
templated by the IBS; however, the area of the IBS of the two
AFPs used in this study is too small to initiate any significant
heterogeneous ice nucleation. In this low-temperature region,
the hydration shell around the non-IBS of both the AFPs still
retains its liquid-like character.
3.4. Long-Range Effects of Non-IBS-Induced Water
Ordering in Hyperactive AFPs. We have then probed the
long-range effects of the IBS and non-IBS-induced water
ordering and compared with the non-AFP-induced water
ordering using the F4 order parameter. We have shown here
the results obtained at 260 K (Figure 5A). Results obtained at
300 and 273 K are largely similar to that of 260 K and
therefore shown in the Supporting Information (Figure S6).
Results obtained at 260 K for SbwAFP are also shown in Figure
S7 (Supporting Information), which also show a similar trend.
We have considered water within 5.6 Å from the protein
surface as the first solvation shell, which corresponds to the
entire first peak in the g(r) plot of the protein surface and
water oxygen (Figure S2). Thereafter, we have considered a 2
Å slab to define the next hydration layer. At 260 K, the order of
water around the IBS is maximum (∼ −0.045) and least
around the non-IBS (∼ −0.009), and non-AFP (∼ −0.025) is
in between these two extreme cases. The corresponding bulk
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F4 value is highly negative (∼ −0.066) at 260 K. This is
expected since it was previously reported that protein
modulates the ordering of water strongly within the first
solvation layer;54,55 therefore, these water molecules are less
responsive to external stimuli like temperature.
Interestingly, not only the different degree of water ordering
modulation by various surfaces is evident in the first hydration
shell, but also it is percolated up to many layers beyond the
first hydration shell. Figure 5A reveals that the F4 values of
hydration water around the IBS approach more closely the
bulk values upon extending the solvation layer length scale in
comparison to the non-IBS and non-AFP surfaces. Distance-
dependent behavior of hydration water around the non-IBS
plane is very unique and retains a more liquid-like character
than other surfaces. At a particular temperature, the degree of
ordering in the successive 2 Å hydration layer retains this
differential feature of water ordering around different surfaces;
however, the value decreases continuously upon going further
from the different protein surfaces. Effects of protein surfaces
nullify at ∼17 Å (Figure 5A). However, the degree of changes
in water ordering in the successive hydration layer is different
for different protein surfaces, and this has been probed with
ΔF4, as shown in Figure 5B. Changes of water ordering around
the IBS saturate within the few layers beyond the first
hydration shell. The effect of non-AFP surfaces on protein
hydration also decays sharply within 10 Å as the changes are
almost saturating (Figure 5B). Long-range modulation of water
ordering is clearly evident around the non-IBS, which extends
up to 15 Å. Thus, non-IBS is not like the non-AFP surfaces; it
is distinct and unique, which can modulate water ordering and
dynamics in a different way.
3.5. Synergism between the IBS- and Non-IBS-
induced Hydration Water Ordering Is Not Evident in
Hyperactive AFPs: Observations from the Mutational
Studies. The designing principle of IBS is naturally selected
for hyperactive AFPs. There is a T-X-T motif at the IBS;
however, the degree of repeat varies in different hyperactive
AFPs. TmAFP contains seven such repeats (A/T/Q-X-T). We
have mutated the threonine residues at the IBS to glycine
residues and then probed water ordering at both the IBS and
non-IBS. Our objective is to understand the effects of
disruption of water ordering around the IBS on the hydration
pattern of the non-IBS.
We have considered two temperatures to compare the
hydration patterns of the wild-type and mutated TmAFP, 273
and 260 K. Distributions of <q6> (Figure 6A) and F4 (Figure
6B) order parameters for the hydration water at two different
temperatures around the IBS and non-IBS are shown. For IBS,
Figure 6. Distributions of (A) <q6> and (B) F4 order parameters of
water molecules within the first solvation shell around the IBS and
non-IBS of wild-type and mutated TmAFP at 273 and 260 K.
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the distribution of <q6> shifts toward lower values, and the
distribution of F4 shifts toward zero. These results indicate that
mutations indeed reduce the ice-like water ordering around the
IBS. On the other hand, the distribution of <q6> and F4 order
parameters for hydration water around the non-IBS remains
essentially unchanged upon mutation at both the temperatures.
This signifies that modulation of water ordering around the
IBS and non-IBS is probably uncorrelated.
3.6. Comparisons of Amino Acid Composition and Its
Spatial Distribution on the Surfaces of IBS, Non-IBS,
and Non-AFPs. Electrostatic surface potential calculations
clearly depict that the IBS of both the AFPs is flat and
hydrophobic in comparison to the non-IBS and non-AFP
surfaces. Non-IBS of TmAFP is slightly negatively charged.
There are two non-IBS planes of SbwAFP. One of them is less
charged, but the flat and periodic patterns of residues are
missing.
The other non-IBS is more charged. Both positively and
negatively charged regions are evident, and the regions are
segregated. On the other hand, there is no such pattern in the
charge distribution on any of the non-AFP surfaces (Figure 7).
Regions of the positively and negatively charged regions are
less segregated. To understand the minute differences in amino
acid compositions of various surfaces of both AFPs and non-
AFPs, the amino acid distribution pattern has been calculated
for six different hyperactive AFPs and 40 representative non-
homologous structurally uncorrelated non-AFPs from four
different classes (All α, All β, α + β, and α/β). The results are
shown in Figure 8. Details of the selected AFP and non-AFP
proteins are enlisted in Table S2 (Supporting Information).
Amino acids are grouped into four different classes
according to Qiao et al.56 Serine, threonine, asparagine,
glutamine, cysteine, glycine, proline, and tyrosine have been
grouped as polar neutral; arginine, histidine, and lysine have
been grouped as positively charged; aspartic and glutamic acids
are clubbed as negatively charged; alanine, isoleucine, leucine,
methionine, phenylalanine, tryptophan, and valine have been
considered as hydrophobic amino acids. Hydrophobic and
polar neutral amino acids are abundant at the IBS of
hyperactive AFPs (Figure 8A). The decomposition of the
distribution at the individual amino acid level reveals that
threonine residues are mostly present among all the amino
acids with a polar neutral side chain. It is well known that the
ice-binding motif of the hyperactive AFPs is T-X-T, where T is
a threonine residue. It has been shown recently that threonine
plays a critical role in stabilizing the anchored clathrate water
array present at the IBS of hyperactive AFPs, which help the
AFP to adsorb on the ice surface.9,10 Hydrophobic amino acids
and the methyl side chain of threonine residues induce water
ordering at low temperatures that mediates ice adsorption
guided by water structure complementarities between the
ordered water layer and interfacial water of a particular ice
surface where the AFP specifically binds.
On the other hand, different polar residues are abundant on
the surface of the non-IBS without any particular preference
toward any particular amino acid. Also, there is a significantly
higher presence of charged amino acids than the IBS, and the
frequencies of hydrophobic amino acids are less on the non-
IBS than on the IBS. However, the number of residues with
either charge is considerably lower on the non-IBS than on the
non-AFP surfaces. Spatial distributions of amino acids from
different groups on the surfaces of TmAFP (Figure 8C) and
SbwAFP (Figure S8A) are shown. On the other hand, spatial
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Figure 7. Cartoon representations of the (A) TmAFP, (B) SbwAFP, and (C) non-AFP are shown. (D) The electrostatic surface potentials of
different planes of TmAFP, SbwAFP, and non-AFP are shown. Blue and red colors are used to indicate the positively and negatively charged
regions.
Figure 8. (A) Normalized distributions of different types of amino
acids present at the IBS and non-IBS of six AFPs and 40 non-AFPs are
shown. (B) Normalized distribution of each of the 20 amino acids
present at the IBS and non-IBS of six AFPs and 40 non-AFPs is also
depicted. The front view of the spatial distributions of different types
of amino acids on the (C) TmAFP and (D) a non-AFP with similar
topology, a pentapeptide repeat protein from M. tuberculosis (PDB ID:
2BM4), is shown. The orange color signifies a polar uncharged amino
acid, the blue color represents a positively charged amino acid, the red
color represents a negatively charged amino acid, and the violet color
signifies a hydrophobic amino acid side chain. Proline, cysteine, and
glycine are rendered in green color.
distributions of amino acids on four structurally uncorrelated
non-AFPs are shown in Figure 8D and Figure S8B−D. In
addition to the pentapeptide repeat protein from Mycobacte-
rium tuberculosis, which shows topological similarities with
TmAFP, three other model globular non-AFPs, namely,
ubiquitin, lysozyme, and barnase, have been considered. In
the case of non-AFPs, the protein core is composed of
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hydrophobic amino acids, and amino acids of different natures
are randomly distributed on the solvent-exposed surfaces.
There can be the presence of small microdomains, but clearly,
different types of amino acids are not segregated. In contrast,
the non-IBS surfaces of both TmAFP and SbwAFP are
characterized by a segregated pattern of different domains of
polar and charged amino acids. Charged amino acids are
absent on the exposed non-IBS surface, which faces the bulk
when the IBS of AFP is bound to the ice surface; rather, they
exist at the interface between the IBS and non-IBS. Clearly, the
amino acid composition and its spatial distribution of non-IBS
are markedly different from those of the IBS. Also, it is unique
and not comparable to the surfaces of other non-AFPs with
varied topology. Notably, there is enormous structural diversity
among non-AFPs. We have considered four structurally
distinct representative non-AFPs and observed a unanimous
trend among them; however, further consideration of many
other non-AFPs is needed to consolidate the observations.
4. DISCUSSION
Ice growth inhibition by AFP is a multidimensional problem
and yet to be understood completely. Most of the studies focus
on the role of IBS on ice binding. A highly ordered water
population around the IBS was previously probed with the aid
of both experiments and computer simulations.8−12,20,57
Recent free energy simulations further elucidated the role of
these ordered water layers in ice binding for different classes of
AFPs.8−12 However, this preordering of hydration water is an
essential criterion for ice recognition or not still remains highly
debatable.8−12,22−24 Recently, the presence of an ordered water
layer during the adsorption of a hyperactive AFP on the ice
surface has been probed using HD-VSFG spectroscopy.20 This
study also showed that the non-AFP protein can adsorb/bind
on the ice surface but with a less affinity than the AFP.20 Apart
from this distinction, AFP and non-AFP showed different
behaviors in terms of ice nucleation and growth upon lowering
the temperature. It has been suggested that, upon lowering the
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temperature, ice grows on top of the non-AFP, and it retains as
an impurity in the growing ice slab.20 However, AFP retards
ice growth on top of it within the thermal hysteresis limit,
which is in accordance with the adsorption-inhibition
model.13,20 These observations imply a different nature and
degree of water ordering around the non-IBS of AFP.
However, the hydration pattern of non-IBS is very little
understood. The order of the hydration shell around the non-
IBS and its temperature and length-scale dependence are
mostly unexplored. Using a combined approach of molecular
dynamics simulations at varied temperature range and
sequence analysis, we have evaluated the nature of water
ordering around the IBS and non-IBS and compared it to the
hydration of non-AFPs. We have observed that water is highly
ordered around the IBS of AFP compared to non-IBS and non-
AFP surfaces at all temperatures. This is in accordance with
many previous studies.8−12,22,23 The water around the IBS is
ice-like ordered, and the population of ice-like water increases
upon lowering the temperature for both of the hyperactive
AFPs studied here. Temperature-dependent studies demon-
strate that the non-IBS surface resists water ordering up to
significantly low-temperature regions. The water around the
non-IBS remains liquid-like up to 260 K, and after that, there is
a very minute increase in ordering of water upon further
lowering of temperature. Notably, we have used TIP5P water
in our study. The Tm of the water model is 274 K.52 Thus, non-
IBS retains the liquid-like nature of its hydration water even in
significantly supercooled conditions. This feature is markedly
different from the hydration around the IBS. Also, the nature
of the hydration shell structure is not similar to the hydration
water around the non-AFP surfaces where we found an
increment in the ordering of water upon lowering the
temperature. However, the degree of ice-like ordering in the
hydration shell of non-AFP is significantly lower than that of
the IBS. Differences in the degree of hydration shell ordering
around the different surfaces upon lowering the temperature
are summarized in Figure 9. Here, we represent the ordering in
terms of two order parameters, F4 and <q6>.
The differential pattern of hydration shell ordering around
the IBS and non-IBS might stem from the difference in their
Figure 9. 3D plot of average F4 and <q6> values of the first hydration
shell water around the different protein surfaces (IBS, non-IBS, and
non-AFP) upon lowering the temperatures. Bulk values at the
respective temperature are also shown.
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amino acid compositions and its spatial distributions, which is
markedly different. Previously, it was shown that different types
of amino acids modulate the water orientation differently, and
the length scale of such an influence widely varied among
different classes of amino acids.56 Negatively charged amino
acids orient 98% hydration water dipole toward the protein
surface, and such effects percolated up to 16 Å, and positively
charged amino acids orient 94% water up to 12 Å.56 Water
reorientation abilities of neutral polar and hydrophobic amino
acids were also evident.56 Thus, different residues alter water
orientations differently. Notably, the nature of water
reorientation ability and its length scale may change in the
low-temperature region, but the details are completely
unexplored. The unique composition and spatial distribution
of neutral polar and charged residues at the non-IBS might be
responsible for the observed temperature-resistant liquid-like
hydration pattern around the surface. A notable observation is
the long-range effects of non-IBS-induced water reorganiza-
tions. At least for hyperactive AFPs, we have observed that
non-IBS retains a significant population of liquid-like water in
highly supercooled conditions up to 15 Å, while the effect of
IBS and non-AFP surface induced water ordering decays
around 10 Å. Notably, bulk water also adopts a more ice-like
character upon lowering the temperature, but the degree of
ordering is lower than that of the hydration water around the
IBS, as evident from the <q6> values at different temperatures.
It is pertinent to mention that the perturbations to the F4 and
<q6> values upon lowering temperature may be different on
protein surfaces compared to the bulk. These two parameters
move in the opposite direction with the increment in ice-like
water population: the F4 value decreases, while the <q6> values
increase. However, how these two parameters behave upon
different conditions and perturbations is worthy to investigate
in more details. Notably, the value of an order parameter for
hydration water around different surfaces at a particular
temperature reveals that the ice-like ordering is found to be
still significantly higher in the hydration shell around the IBS
than around other surfaces. Notably, Duboué-Dijon and
Laage28 studied the hydration layer ordering around a
hyperactive antifreeze protein using the TIP4P/2005 water
model at 300, 260, and 235 K and noticed that the water
dynamics around the IBS is slower than the entire AFP
hydration shell, probed by the reorientation dynamics
retardation factor. This observation implies that the non-IBS
hydration shell shows different temperature sensitivity from the
IBS. However, mean asphericity calculation did not reveal such
temperature-dependent behavior. Thus, the choice of appro-
priate order parameter is crucial to elucidate subtle changes in
the hydration behavior. Kuffel et al.58 also showed the
difference in hydration shell behavior around the IBS and
non-IBS planes in a wide range of temperatures, 240−300 K,
using the translational and rotational diffusion coefficients as
order parameters. Using the surface pair correlation function,
Nutt and Smith22 showed a significant increase in water
ordering around the IBS upon lowering the temperature from
300 to 220 K, but the changes around the non-IBS is
insignificant.
Our study clearly highlights the role of non-IBS in the
antifreeze activity of hyperactive AFPs. IBS is designed to
interact with the ice surface in a strong and specific manner,
while the non-IBS-induced liquid-like ordering of its hydration
shell may prevent the ice nucleation and subsequent growth on
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top of the bound AFPs. Thus, the combined effect of IBS and
non-IBS might be operative to inhibit ice growth.
5. CONCLUSIONS
We have shown that the IBS and non-IBS of the hyperactive
AFPs are unique in terms of amino acid compositions and their
spatial distributions, which enable the non-IBS plane to control
the organization of its hydration layer in a liquid-like
arrangement. Non-IBS is able to maintain the liquid-like
character of its hydration layer even in highly supercooled
conditions, and the effects are percolated to many layers
beyond the first solvation shell, up to 15 Å. The behavior of
hydration shell ordering around the non-IBS is also markedly
different from the non-AFP surfaces both in terms of
temperature and length-scale behavior. Non-AFP surfaces do
not possess the ability to retain the liquid-like character of its
hydration shell as efficiently as the non-IBS. Thus, combined
effects of IBS and non-IBS might orchestrate the ice-growth
inhibition ability of hyperactive AFPs. The present study
elucidates the intrinsic differences of the hydration layer
ordering around different AFP and non-AFP surfaces and their
temperature and length-scale dependency. Therefore, the study
provides a direct correlation between the nature of surfaces
and observed ordering in the hydration layer. The observations
obtained from the present study lay the foundation to interpret
the hydration behavior in a more complex scenario where AFP
is adsorbed on a heterogeneous ice/water interface during ice-
growth inhibition. The role of amino acids spatial distributions
in controlling the water structure in highly supercooled
conditions paves the way for designing highly effective,
nontoxic AFP mimetics with potential industrial applications.
■
*
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jpcb.0c01206.
Residues considered in the study for IBS and non-IBS
for two different AFPs and non-AFP, details of the AFPs
and non-AFPs considered for residue analysis, root-
mean-square deviation of threonine OH groups at the
IBS of TmAFP with respect to the crystallographic
orientation throughout the simulation timescale, radial
distribution function of water (oxygen) around the two
surfaces (IBS and non-IBS) of TmAFP at 260 K,
distribution of the O−O−O angle of water molecules
within the first solvation shell around the IBS and non-
IBS of SbwAFP at 273 K, distributions of the <q6> and
F4 order parameters of water within the first solvation
shell around the non-IBS of SbwAFP and a non-AFP
upon lowering the temperature from 300 to 240 K,
distributions of the <q6> and F4 order parameters of
water within the first solvation shell of the non-IBS of
TmAFP and a non-AFP (ubiquitin) upon lowering the
temperature from 300 to 240 K, changes in the average
F4 order parameter values of hydration shells with
different thicknesses around the IBS, non-IBS of
TmAFP, and non-AFP at 273 and 300 K, changes in
the average F4 order parameter values of different
hydration shells around the IBS, non-IBS of SbwAFP,
and non-AFP at 260 K, spatial distribution of different
types of amino acids on the SbwAFP and three non-
AFPs (PDF)
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■
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AUTHOR INFORMATION
Corresponding Authors
Sandipan Chakraborty − Amity Institute of Biotechnology,
Amity University, Kolkata 700135, India;
Email: sandipanchakraborty.13@gmail.com
Biman Jana − School of Chemical Sciences, Indian Association
for the Cultivation of Science, Kolkata 700032, India;
orcid.org/0000-0001-7684-1963; Phone: +91 33 2473
4971; Email: pcbj@iacs.res.in; Fax: +91 33 2473 2805
Author
Prasun Pal − School of Chemical Sciences, Indian Association for
the Cultivation of Science, Kolkata 700032, India
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jpcb.0c01206
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This research is supported by the Department of Science and
Technology SERB grant EMR/2016/001333 for funding. P.P.
acknowledges Mr. Sridip Parui for his help during the
development of the order parameter code. P.P. also thanks
Department of Science and Technology (DST), India for
fellowship.
■
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