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INTRODUCTION |
Type I AFP1 from winter
flounder, represented by the abundant serum isoform HPLC-6 (1) is a
remarkably long free-standing
-helix (2). Its helicity can be
attributed to several structural features, which include an abundance
of alanine, an extensive capping network at both termini (3), and an
internal salt bridge (1, 4). From ice etching studies, it was shown
that this AFP binds to the {20-21} hexagonal bipyramidal planes of
ice along the
01-12
direction (5). It has been suggested that
the critical connection between structure and function in this protein
is that putative ice-binding residues (Thr, Asx, and Leu) are aligned and regularly spaced along one face of the helix (5, 6). Each residue
(e.g. Thr-2, Thr-13, Thr-24, and Thr-35) is spaced 11 amino
acids apart (16.5 Å), which closely matches the 16.7-Å distance
between repeating features of the ridge and valley topology along the
01-12
direction of the {20-12} binding plane. Adsorption of
type I AFP to these lattice binding sites through a precise distance
and geometry match involving H-bonding and van der Waals interactions
(5, 7) leads to inhibition of ice growth by the Kelvin effect (8, 9).
In the process, seed ice crystals are constrained to form hexagonal
bipyramids with a c:a axial ratio of 3.3:1, which matches
the ratio predicted from the adsorption planes revealed by ice etching
studies (5, 6).
Several attempts have been made to model the helix in contact with the
ice surface (3, 7, 10, 11). As a result, a common concern is that the
number and strength of the potential interactions between ice and AFP
are barely sufficient for tight binding (3, 7). One solution proposed
is that all four ice-binding threonines share the same rotamer
configuration and bind to ice in a "zipper-like fashion" (11). A
similar hypothesis, stemming directly from the x-ray structure, is that
tight binding relies on the simultaneous docking of similarly aligned
and constrained ice-binding side chains that together form a flat
ice-binding surface (3). The requirement of side-chain rigidity for
tight binding seems to be at odds with the NMR solution structure for type I AFP, which failed to find any evidence that the ice-binding side
chains were locked into a specific common rotamer, even at 3 °C
(12). To investigate this, and the related issue of the length of the
ice-binding site, we set out to construct a minimized type I AFP that
was sufficient for binding to ice. These experiments have shed new
light on the mechanism of AFP binding to ice. Antifreeze proteins do
not necessarily bind to a preformed site on ice but instead help to
shape the site to which they bind. This principle may be of general
relevance to mineralization and demineralization processes.
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EXPERIMENTAL PROCEDURES |
Peptide Synthesis, Purification, and Mass Analysis--
All
peptides were prepared by solid-phase peptide synthesis using a
benzhydrylamine hydrochloride resin on a Labortec SP 640 peptide
synthesizer as described previously (13). The peptides were cleaved
from the resin by reaction with HF (10 ml/g resin) containing 10%
anisole for 1 h at
5 °C to 0 °C. The crude peptides were
purified by reversed-phase high performance liquid chromatography on a
SynChropak RP-4 preparative C4 column (250 × 21.2-mm internal diameter, 6.5-µm particle size, 300-Å pore size) (SynChrom,
Lafayette, IN) with a linear AB gradient of 0.1% B/min with a flow
rate of 5 ml/min, where solvent A is 0.05% trifluoroacetic acid in
water and solvent B is 0.05% trifluoroacetic acid in acetonitrile. For amino acid analysis, peptides were hydrolyzed in 6 N HCl
containing 0.1% phenol for 1 h at 160 °C in sealed evacuated
tubes. Amino acid analysis was performed on a Beckman model 6300 amino
acid analyzer (Beckman, San Ramon, CA). Mass analysis of the peptides was performed on a Fisons VG Quattro triple quadropole mass
spectrometer (Manchester, UK) fitted with an electrospray ionization
source operating in the positive ion mode. A number of 10-µl
injections of the peptide samples (usually in aqueous acetonitrile
containing 0.05% trifluoroacetic acid at an approximate concentration
of 50 pmol/µl) were made into a carrier solution composed of
water/acetonitrile (1:1, v/v) containing 0.05% trifluoroacetic acid at
a rate of 10 µl/min into the electrospray source. The quadropoles
were scanned from 600 to 1400 mass over charge ratio at 10 s/scan. Data
were acquired in the multi-channel acquisition mode with 10-15 scans typically being summed to produce a spectrum.
Circular Dichroism Spectroscopy--
CD spectra were measured on
a Jasco J-500C spectropolarimeter (Jasco, Easton, MD) equipped with a
Jasco DP-500N data processor as described previously (14). A Lauda
water bath (model RMS, Brinkmann Instruments, Rexdale, Ontario, Canada)
was used to control the temperature of the cell. CD spectra were the
average of four scans obtained by collecting data at 0.1-nm intervals
from 255 to 190 nm. The buffer used was 50 mM
KH2PO4, 50 mM KCl (pH 7.0).
Antifreeze Activity and Photomicroscopy--
Thermal hysteresis,
defined as the temperature difference (°C) between the melting point
and the non-equilibrium freezing point of a solution, was measured
using a nanoliter osmometer (Clifton Technical Physics, Hartford, NY),
as described by Chakrabartty and Hew (4). For the purpose of
comparison, an ice crystal growth rate of more than 0.2 µM/s is defined as having reached the solution freezing
point (15). This operational definition was introduced to standardize
thermal hysteresis measurements, when it became apparent that some AFP
mutants exhibit slow ice crystal growth prior to a well defined
freezing end point, and others grow so rapidly that a distinguishable
end point is not observed. All measurements were made in 0.1 M NH4HCO3 (pH 7.9) and 0.02% (w/v)
sodium azide. Ice crystal morphology was observed through a Leitz
Dialux 22 microscope and recorded by a Panasonic CCTV camera linked to
a JVC Super VHS video recorder. Still images were obtained from a
Silicon Graphics INDY terminal using IRIS Capture version 1.2. For ice
growth rate analysis, samples were held at a fixed degree of
undercooling and images were captured at 0- and 10-min time points.
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RESULTS |
Peptide Design--
Type I AFP is an ideal protein for
minimization because there are few structural restraints to making it
smaller by simply shortening the helix. The initial minimized 15-amino
acid AFP structure (15KE in Fig.
1) was formed by removing the two central 11-amino acid repeats (residues 13-34 inclusive) and reinstating the
internal salt bridge into the remaining repeat on the opposite side of
the helix to the threonines. By removing 22 residues (exactly two
repeats), the critical 16.5-Å spacing between the remaining two
threonines could be maintained, but only if the peptide retained its
-helicity. The latter concern was the key structural restraint in
the minimization exercise. By shortening the protein from the center,
the helix stabilizing N- and C-cap structures were not disturbed.
However, these features, together with the intrachain salt bridge
between Lys-7 and Glu-11, were not enough to make the peptide fully
helical, even at 1 °C (Fig. 2) (see
below).

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Fig. 1.
Sequence of the WT AFP and peptide
analogs. Peptides were amidated at the C terminus and contained a
free N terminus unless otherwise stated. Peptide 15KE refers
to the prototype peptide resulting from the deletion of 22 amino acids
from WT. Variations were designed to increase the helical conformation
of this peptide. These included acetylation of the free N terminus
(Ac-15KE), a reverse orientation of the salt bridge
(15EK), and inclusion of a lactam bridge constraint
(15EKlac). Purity of the peptides was determined by HPLC and
mass spectrometry. A and B, two views of a
molecular model of 15EKlac showing the orientation of the lactam bridge
relative to the two Thr side chains (A, end-on view;
B, horizontal view). The N and C termini of the peptide are
labeled N and C, respectively.
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Fig. 2.
CD spectra of wild-type and minimized
antifreeze peptides. CD spectra were run in 50 mM
KH2PO4, 50 mM KCl (pH 7.0) at
1 °C. Peptides were WT ( ), 15KE ( ), Ac-15KE ( ), 15EK ( ),
and 15EKlac ( ).
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Two minor adjustments were made in an attempt to increase the
stability of the peptide. One was acetylation of the N terminus (peptide Ac-15KE in Fig. 1). Doig et al. (16)
have reported that the unfavorable effect of protonation at the N
terminus is approximately 0.5 kcal/mol and that acetylation of the free
amino group can improve helix stability. This can be attributed to
elimination of the positive charge and possibly to hydrogen bond
formation between the acetyl CO and an unsatisfied main chain NH group. The other adjustment was to reverse the order of the ion pair residues
Lys-7 and Glu-11 to Glu-7 and Lys-11 (peptide EK15 in Fig.
1) in order to orient the salt bridge more favorably with the
helix macrodipole (17, 18).
Previously it has been shown that incorporation of a lactam bridge
between Glu and Lys spaced four residues apart dramatically increased
the helical content of small amphipathic peptides relative to their
uncyclized linear peptides bearing the same sequence (13). The modified
peptide 15EK was ideally suited for lactam bridge synthesis because of
the orientation of the ion pair, Glu-7 and Lys-11. Thus, the third
modification of the prototypical minimized type I AFP (15EK) was the
formation of a Glu-7 to Lys-11 i,i+4 lactam bond in place of
the salt bridge. It was thought that this covalent link might be the
most effective way of maintaining the correct 16.5-Å spacing between
the two remaining threonines.
CD Spectroscopy--
The CD spectra of type I AFP (WT) and
minimized AFPs in benign conditions at 1 °C are shown in Fig. 2. The
CD spectra of the wild-type protein are characteristic of a highly
helical molecule with minima at 222 and 208 and a maximum at 192 nm.
Typically, the molar ellipticity at 222 nm ([
]222)has
been used to measure the helical content in peptides (19). For the
wild-type protein, this has been previously reported to be
36,100
deg·cm2·dmol
1 at 1 °C (6). All
minimized peptides displayed significantly less
-helical character
than the full-length AFP. However, it is unlikely that a peptide of 15 residues in length would be as helical or as stable as a 37-residue
protein due to the length dependence of the [
]222
signal. For the minimized peptides stabilized by a salt bridge, the CD
spectra resemble a mixture of helical and random conformations. The CD
spectra of 15KE and 15EK are nearly superimposable, and their
[
]222 values at 1 °C are quite similar (
19,450
deg·cm2·dmol
1 and
18100
deg·cm2·dmol
1), yielding estimates for
helical content of 62% and 58%, respectively. However, acetylation of
the N terminus resulted in a lower helical content (42%), as measured
by the 222-nm signal. This may be due to acetylation disrupting the
N-cap. All three peptides were essentially 100% helical in the
presence of 50% TFE (data not shown). Introduction of a lactam bridge
in place of the salt bridge greatly increased the helical content of
the peptide to 90% ([
]222 =
28,100
deg·cm2·dmol
1). This is the only
minimized peptide for which the CD spectrum is similar to that of the
wild-type protein with a minimum at 222 nm that exceeds the value at
208 nm, suggesting the peptide adopts an
-helical conformation. Once
again, this peptide was highly helical in 50% TFE (data not
shown).
Thermal denaturation profiles of all minimized AFP peptides are shown
in Fig. 3. For the peptides stabilized by
salt bridges, the [
]222 signal sharply declined and
leveled off at
7000 deg·cm2·dmol
1. The
denaturation profile of EK-lac was somewhat broader, indicating a less
cooperative transition. Approximately 50% of the helicity observed at
1 °C was lost at 52 °C. (In contrast, the wild-type protein had a
Tm of 22 °C.) This stability has been observed previously for other constrained peptides and has been attributed to
residues within the covalent constraint being unable to fully denature.

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Fig. 3.
Thermal denaturation profiles of minimized
peptides. The molar ellipticity at 222 nm ([ ]222)
was measured for each of the 15-mers as a function of temperature.
Peptides were identified by the same symbols used in Fig. 2 (see
inset).
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Thermal Hysteresis Activity Measurements--
All peptides were
assayed for thermal hysteresis activity using a nanoliter osmometer
(4), which also provided an opportunity to check for interactions
between protein and ice, as revealed by the shaping of ice crystals.
None of the minimized peptides was able to depress the freezing point
below the melting point at concentrations of 50-100 mg/ml (~50
mM). Even the slightest undercooling caused the seed ice
crystal to grow steadily. In the presence of 15KE and 15EK, the ice
crystal took the shape of a rounded disk indistinguishable from that
formed in the presence of buffer alone (Fig.
4A, panels b and
d). With Ac-15KE, there was a hint of a hexagonal shape to
the disk, suggestive of peptide binding to the ice (Fig. 4A,
panel c). In contrast, 15EKlac was very active in shaping
the ice into an incomplete hexagonal bipyramid, which generally lacked
one or both apices (Fig. 4A, panels e and f). When the c:a axis ratio was
measured by extrapolation from 10 representative crystals, it ranged
from 3.5:1 to 3.1:1, with an average value of 3.3. This average value
is precisely that obtained with full-length, wild-type AFP (6, 15),
which is the ratio predicted for expression of the {20-21}
pyramidal planes. This value was maintained even as the ice crystal
grew considerably over a time course of 1-10 min, indicating a
persistent influence of the peptide over the ice surfaces expressed. In
one sequence of time-lapse video microscopy (Fig. 4B), the
ratio varied from 3.4 at 1 min to 3.3 at 5 and 10 min, during which
time the volume of the crystal increased manyfold. In another series
(data not shown), the ratio was 3.20 at 1 min and 3.17 at 10 min. Thus, a single ice-binding repeat that was constrained to be
-helical was
sufficient to define the {20-21} pyramidal plane but could not
prevent ice-crystal growth. The fact that 15EKlac generated a faceted
ice crystal without causing non-colligative depression of the freezing
point could be explained by transient (reversible) binding of the
peptide to the ice surface.

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Fig. 4.
Ice crystal morphology. A, ice
crystals formed in the presence of WT (a), 15EK
(b), Ac-15-KE (c), 15KE (d), and
15EKlac (e and f). The peptide nomenclature is
that used in Fig. 1. B, time lapse analysis of ice crystal
growth in the presence of 15EKlac, where a, b,
and c were recorded at 1, 5, and 10 min, respectively.
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DISCUSSION |
The high helical content of wild-type AFP is thought to reflect
the importance of maintaining the polypeptide backbone in this
conformation, such that the spacing of the threonines and other
ice-binding residues matches the ice lattice (3, 5, 7, 11). Although
all the minimized AFP peptides that were stabilized by a salt bridge
contained a certain amount of helical structure as determined by CD,
their helical content was substantially less than that of the wild-type
protein or of that induced in 50% TFE. The overall conformation of
these peptide is most likely an ensemble where sections of the peptide
adopt fully helical, partly helical, and random conformations. This is
consistent with the findings of Merutka et al. (20), where a
small peptide that was even more helical than the 15-mers in Fig. 1 (as
determined by CD) contained a significant population of non-helical
conformers (as determined by NMR). For the lactam-bridged peptide, the
internal lactam bridge biases the peptide to adopt a more helical
structure. This, in turn, should lower the change in conformational
entropy between peptides bound to ice and those free in solution. For the non-lactam-bridged peptides, it is likely that the free energy of
binding to ice is insufficient to compensate for the loss of conformational entropy upon immobilization of the peptide backbone into
a fully helical conformation.
The significance of the interaction between the minimized,
lactam-bridged type I AFP and ice lies in the insight it provides about
the adsorption phase of the adsorption-inhibition mechanism of action
of AFPs. This insight helps resolve two paradoxes. One is that AFPs
bind to planes of ice that are not extensively expressed in the absence
of the antifreeze. The other is that the Thr residues of type I AFP are
rarely optimally configured for immediate binding to ice because of
side chain rotation (12). A solution to these paradoxes is illustrated
for type I AFP by the model shown in Fig.
5. In the absence of AFP, an ice crystal
will typically grow as a rounded disk (Fig. 5A) as water
molecules add to the prism surfaces (a-axial growth) in
preference to the two basal planes (c-axial growth). The
crystal surface may appear microscopically smooth but will be uneven at
the submicroscopic to atomic level on all but the basal planes. The
irregular addition of water to a prism surface of the ice lattice
creates a small section of the pyramidal plane {20-21} (Fig.
5B). This section, involving just five layers of water,
already expresses the characteristic surface properties of the AFP's
adsorption plane, including the 16.7-Å spacing along the
01-12
direction, and is sufficient to initiate antifreeze binding.

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Fig. 5.
Graduated binding of type I AFP to ice.
A, type I AFP, represented by the green helix
with projecting i,i+11 threonyl side chains, is
in close proximity to the prism surface of a seed ice crystal.
B, random addition of water to the ice lattice creates a
{20-21} surface (illustrated by red oxygen atoms) that
is large enough to bind one repeat of the helix bordered by Thr-13 and
Thr-24. C, residency of this helical section on the ice
surface, however brief, leads to shaping of the {20-21} pyramidal
plane, as demonstrated with 15EKlac. With wild-type AFP, growth of ice
in the a direction during shaping results in additional
contacts with the helix that further increase its residency on the ice
and lead to complete and irreversible binding.
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It is clear from solution NMR studies that all four threonines in type
I AFP are free to occupy different rotamer configurations, although the
middle two have a preference for
60 °C 55% of the time (12).
Although the chances of all four threonines being in the same
orientation at any one instant is quite small, the evidence from
minimized AFP binding to ice shows that this is a not a precondition
for initial contact, because dynamic ice shaping occurs with a single
repeat. It is quite likely, therefore, that two neighboring threonines
(especially the middle two) will be found with the same rotamer
configuration. A single ice-binding repeat would then be the smallest
unit of the AFP that theoretically could define both the plane and
direction of AFP binding to ice. That it does this in practice is
demonstrated by the shaping of ice crystals by 15EKlac into truncated
hexagonal bipyramids with the 3.3:1 c:a axis ratio typical
of full-length type I AFP. Thus, it is not necessary for all four
threonines to touch down on the ice surface to secure that plane for
binding.
The model suggested by these studies is that type I AFP binding to ice
is unlikely to be an "all-or-nothing" event. Instead, it is best
described by an induced-fit mechanism, where touchdown by any two
i,i+11 threonines with the same rotamer
configuration (representing a single ice binding repeat) is sufficient
to stabilize the smallest facet of ice that will bind repetitively.
Even transient occupancy of this ice surface would allow ice to advance
relative to the occupied region, particularly by water addition to the prism surfaces (Fig. 5, B and C). If, during this
period, the advance is sufficient to engage a third threonine
(representing a second ice-binding repeat), which might have had to
rotate its side chain to provide the 16.5-Å spacing, then the binding
would be stronger. Should dissociation have occurred prior to the
engagement of the second repeat, the net effect would have been to
shape the ice closer to the {20-21} plane, thereby making it
easier for another type I AFP molecule to engage. Either through a
dissociation-reassociation process or by side-chain reorganization
while on the ice surface, the eventual outcome is that all repeats
would make contact (Fig. 5C). At this point, binding would
be virtually irreversible because dissociation would require the
simultaneous breaking of all the AFP-ice interactions. This shaping of
the ice surface, coupled with selection for AFPs with ideal rotamers,
is a dynamic, synergistic process where an improved binding site leads
to more efficient binding, which further improves the binding site.
Although type I AFP may be unusual in having somewhat flexible
ice-binding residues, ice-site shaping by an induced-fit mechanism is a
principle that could apply to other AFPs.
We thank Paul Semchuk for peptide synthesis
and purification, Dr. Robert Parker for amino acid analysis, and Dr.
Michael Kuiper for assistance with the figures.