From the Division of Structural Biology and
Biochemistry, Hospital for Sick Children, Toronto, Ontario M5G 1X8,
Canada, the § Department of Biochemistry and
Department of Laboratory Medicine and Pathobiology, University
of Toronto, Toronto, Ontario M5G 1L5, Canada, and the
** Department of Biological Sciences, National University of
Singapore, Singapore 119 260, Singapore
Received for publication, January 5, 2003
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ABSTRACT |
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Antifreeze proteins (AFPs) are found in
many marine fish and have been classified into five biochemical
classes: AFP types I-IV and the antifreeze glycoproteins. Type I AFPs
are Fish antifreeze proteins are diverse in structure and have been
grouped into five biochemical classes based on their structural characteristics: antifreeze proteins
(AFPs)1 types I-IV and
antifreeze glycoproteins (AFGPs) (1-3). Although diverse in structure,
all AF(G)Ps act by what is known as the adsorption inhibition mechanism
(2, 4-6), where the AF(G)Ps lower the observed freezing point in a
non-colligative manner creating a hysteresis between the equilibrium
melting point and the observed freezing point. The degree of thermal
hysteresis is used as a measure of AF(G)P activity.
Type I AFPs are Ala-rich, partially amphipathic single The wflAFPs and wfsAFPs share a high level of structural identity even
though the wfsAFPs have approximately half the activity of wflAFP-6
(9). Both wflAFPs and wfsAFPs possess two 11-residue motifs of the
structure TaaXAXXAAXX (where the first
Thr is always conserved, uppercase A is a conserved Ala, lowercase a is
almost always Ala, and X can be one of several amino acids),
with wflAFP-6 having a third full motif at the N terminus (see Fig. 1).
Furthermore, the wflAFPs possess complete ice-binding motifs (IBMs),
LTAAN (8), whereas the wfsAFPs have incomplete IBMs of the form ATAAA, and this difference in IBMs has been proposed to be the cause of the
lower thermal hysteresis found in wfsAFPs (12). However, the
introduction of two proposed IBMs (KT-D, and DT-K) into wfsAFP-2 did
not produce any improvement in activity (12). Furthermore, the
introduction of the LTAAN IBM into wfsAFP-2 only increased the
activity by ~15%, yet lowered the helical content from 80 to
60%.2
The IBM hypothesis is based on earlier beliefs that wflAFP-6 requires
regularly spaced polar groups to match the arrangement of water
molecules on the ice surface for the formation of hydrogen bonds (5,
13-16). However, recent study of wflAFP-6 has suggested that the major
ice-binding surface resides on the hydrophobic face of the polypeptide,
with major contribution from conserved Ala residues. Furthermore, the
driving forces behind ice binding are van der Waals interactions and
the hydrophobic effect (17-21). The major sequence variations between
wflAFP-6 and the wfsAFPs are in the N and C termini (see Fig. 1). Most
notably, wflAFP-6 has the N-terminal sequence DTASDA, whereas all
wfsAFPs begin with MDAP (9). Furthermore, the MDAP sequence can be
found in the longhorn sculpin skin-type AFP (22), and in the minor serum AFPs of the shorthorn and grubby sculpins (23, 24), all of which
have ~50% activity of wflAFP-6. The region of highest variability in
type I AFPs lies in the C-terminal regions (7, 9).
This study examines the contributions of the N- and C-terminal
sequences to the antifreeze activity of the liver- and skin-type AFPs.
Polypeptides were synthesized where the N- and C-terminal sequences of
two representative AFPs, wflAFP-6 and wfsAFP-2, were systematically
exchanged, then assayed for antifreeze activity and Polypeptide Synthesis and Purification--
Polypeptides were
synthesized by continuous flow Fmoc chemistry on NovaSyn KA 100 resin
(25) using a Biolynx 4170 automated peptide synthesizer (Amersham
Biosciences, Montreal, Quebec, Canada) by the Advanced Protein
Technology Centre, Hospital for Sick Children (Toronto, Ontario,
Canada). A 20% piperidine solution in dimethyl formamide was used for
removal of the Fmoc protection group. For each gram of resin (0.1 nmol
substitution), a 4× excess of Fmoc amino acid activated with
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate and diisopropylethylamine (1:1:2, mol/mol/mol) (26)
was used for the coupling reaction, with a reaction time of 1 h.
Peptide-resin conjugates were washed with dimethyl formamide, diethyl
ether, and dried under reduced pressure. Dry peptide-resin conjugates
were cleaved with 20 ml of trifluoroacetic acid containing 4 ml of
thioanisole, 0.4 ml of m-cresol, 2 ml of 1,2-ethanedithiol, 3 ml of ethylmethylsulfide, and 4 ml of bromotrimethylsilane at 0 °C
for 1 h. Peptides were extracted, dissolved in 0.1%
trifluoroacetic acid, and desalted on a Sephadex G10 column. Crude
polypeptide preparations were further purified by reverse-phase HPLC
using a Jupiter 10-µm C4 300 Å (250 mm × 21.20 mm)
column (Phenomenex, Torrance, CA) using an acetonitrile gradient in
0.1% trifluoroacetic acid. Homogeneity of peptides was analyzed by
C18 reverse-phase HPLC, amino acid analysis, and
electrospray ionization mass spectrometry. All polypeptides were
synthesized with free N termini and amidated C termini except where indicated.
Measurement of Antifreeze Activity--
Synthetic polypeptides
were assayed for antifreeze activity using a Clifton Nanolitre
Osmometer (Clifton Technical Physics, Hartford, NY) as previously
described (27). Briefly, purified and lyophilized polypeptides were
re-dissolved in 0.1 M ammonium bicarbonate (pH 7.9). Serial
dilutions were made for each polypeptide and thermal hysteresis
readings taken for each dilution. Measurements at each dilution were
averaged from readings from four to six different wells in the sample
plate. Concentration was determined by amino acid analysis on the two
highest diluted samples. Amino acid analysis was performed by the
Advanced Protein Technology Centre, Hospital for Sick Children.
Percentage of activity was determined by comparison of thermal
hysteresis values for each polypeptide at concentrations of 0.5-3
mM at intervals of 0.5 mM, and error bars
represent ±1 standard deviation of the average percent activity
reported. From results obtained for Skin(wt), results from different
assay groups were reproducible with a 5% experimental error.
Circular Dichroism Spectroscopy--
Circular dichroism
spectroscopy was performed using an AVIV 62A DS circular dichroism
spectrometer (AVIV Instruments, Inc., Lakewood, NJ) equipped with a
thermal control unit in a 1-mm quartz cuvette. Polypeptides were
dissolved in 0.1 M ammonium bicarbonate (pH 7.9), and
assayed at two concentrations in the range of 10 or 50 µM. Spectra were collected with 1-nm bandwidth and 1 nm between data points at 0 and 40 °C using an averaging time of 6 s from 250 to 190 nm. Thermal denaturation was performed using the same
parameters at 222 nm with temperature equilibrium time of 30 s/°C.
All spectra were the result of subtraction of data collected on 0.1 M ammonium bicarbonate alone. The fraction helix (fH) for each polypeptide at indicated
temperatures was calculated by the method of Luo and Baldwin (28) from
mean molar residue ellipticity observed at 222 nm
(q222) using Equation 1.
Pairwise Comparison (Table I)--
In column 1 of Table I, the
percentage of activity of the polypeptide containing the wfsAFP-2
region was subtracted from the value for the polypeptide containing the
wflAFP-6 region. Positive values indicate higher activity in the
presence of wflAFP-6 components, and negative values indicate higher
activity for the wfsAFP-2 components. Each column 1 value was assigned
a label: A1-A4, B1-B4, and C1-C4. In column 3, an analysis similar
to that in column 1 was performed on the helical contents for each
analogue, with each resulting value assigned labels a1-a4, b1-b4, and
c1-c4. Statistical analysis of the data was achieved by performing
standard t tests assuming equal variances between the two
mean values obtained from the activity levels determined from 0.5 to 3 mM at intervals of 0.5 mM after normalization
to the activity level of Liver(wt). An Pairwise Analysis of the Increased Activity of wflAFP-6
Components and the Dependence on the Characteristics of the Remainder
of the Polypeptide (Table II)--
In Table II, the values in each
column were the result of the calculation shown. For example, in column
1, row A, the A1 value was derived from the difference in activity when
[S]-Nterm was replaced by [L]-Nterm (A1 value, Table I) with the
remaining components composed of wflAFP-6 components. The A3 value was
derived from the difference in activity when the same exchange was
performed, but with wfsAFP-2 components comprising the remainder of the
polypeptide. Thus, the first value presented in Table II, column 1, row
A is the difference between the increased activity associated with the
replacement of [S]-Nterm by [L]-Nterm when the remainder of the
polypeptide was composed of wflAFP-6 components as opposed to
wfsAFP-2 components. All other values determined in Table II follow
the same methodology. Statistical analysis was performed as described
for Table I.
A comparison of wflAFP-6 and wfsAFP-2 sequences demonstrated that
the region of highest identity between the two polypeptides occurs in
the central region (wflAFP-6 core; L-core and wfsAFP-2 core; S-core),
with no identity at the termini (see Fig.
1A). For wflAFP-6, the
N-terminal sequence, [L]-Nterm, was defined as DTASDA, and the
C-terminal sequence, [L]-Cterm, was defined as AR. Correspondingly,
in wfsAFP-2, the N-terminal sequence, [S]-Nterm, was defined as MDAP,
and C-terminal sequence, [S]-Cterm, defined as KAGAAR.
-helical, partially amphipathic, Ala-rich polypeptides. The
winter flounder (Pleuronectes americanus) produces two type
I AFP subclasses, the liver-type AFPs (wflAFPs) and the skin-type AFPs
(wfsAFPs), that are encoded by distinct gene families with
different tissue-specific expression. wfsAFPs and wflAFPs share a high
level of identity even though the wfsAFPs have approximately half the
activity of the wflAFPs. Synthetic polypeptides based on two
representative wflAFPs and wfsAFPs were generated to examine the role
of the termini in antifreeze activity. Through systematic exchange of N
and C termini between wflAFP-6 and wfsAFP-2, the termini were determined to be the major causative agents for the variation in
activity levels between the two AFPs. Furthermore, the termini of
wflAFP-6 possessed greater helix-stabilizing ability compared with
their wfsAFP-2 counterparts. The observed 50% difference in activity
between wflAFP-6 and wfsAFP-2 can be divided into ~20% for
differences at each termini and ~10% for differences in the core.
Furthermore, the N terminus was determined to be the most
critical component for antifreeze activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical
polypeptides found in several sculpins and righteye flounders (2, 7,
8). The winter flounder (Pleuronectes americanus) produces
two subclasses of type I AFPs, the liver-type (wflAFPs) and the
skin-type AFPs (wfsAFPs), which are encoded by distinct gene families
(9). wflAFP-6 (formerly known as HPLC-6) is the major winter flounder
plasma AFP and is produced in the liver as a prepro-precursor, and then
secreted into circulation, where it is then processed into the mature
polypeptide of 37 residues (10, 11). Conversely, the wfsAFPs have a
wider tissue distribution and are produced as mature intracellular
polypeptides (9).
-helical
content. The results demonstrated that the difference in activity was
caused primarily by sequence variations at the N and C termini.
Furthermore, the N and C termini of wflAFP-6 were able to confer higher
overall helical content and greater thermal stability over their
skin-type AFP counterparts.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Temperature and chain length dependences of
(Eq. 1)
C and
H in Equation 1 are defined by Equations 2 and 3.
(Eq. 2)
T is the temperature in °C, and
Nr is the chain length in residues. Where data
are presented as percentages, fH was multiplied by 100. Values reported at 0 °C are averages of three to five independent readings.
(Eq. 3)
value of 0.05 and a null
hypothesis of no difference between the indicated mean values were used.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Synthetic polypeptide analogues of wflAFP-6
and wfsAFP-2 used to determine the role of N and C termini in
antifreeze activity. All polypeptides were synthesized with free N
termini, and amidated C termini except where indicated by
-OOH. The putative Asp-2/Lys-6 (D2-K6) salt
bridge (see "Discussion") is indicated by
double-arrowhead lines. IBMs are
indicated with the complete IBM of wflAFP-6 (LTAAN) indicated
above its sequence, and the incomplete IBM of wfsAFP-2
(ATAAA) indicated below its sequence. A,
wild-type wflAFP-6 and wfsAFP-2 sequences. wflAFP-6 sequence is in
uppercase text, whereas wfsAFP-2 sequence is in
lowercase text. The polypeptides were divided
into three regions: N terminus, core region, and C terminus. Identical
residues between wflAFP-6 and wfsAFP-2 are highlighted in
gray. Corresponding wflAFP-6 and wfsAFP-2 N and C termini
are indicated. B, sequence of polypeptide analogues. The
wflAFP-6 analogue is designated Liver(wt), and the wfsAFP-2
analogue is designated Skin(wt). Uppercase and
lowercase notation is maintained to distinguish the original
source of each region of the polypeptides.
The synthetic polypeptides were designed to examine the effects of
N-terminal exchange, C-terminal exchange, and simultaneous exchange of
both termini. In the N-terminal exchange series, polypeptides consisting of wild-type sequences for wfsAFP-2 and wflAFP-6, Skin(wt) and Liver(wt), were also assayed. The Skin(wt) activity was ~50% of
Liver(wt) (Fig. 2D), in
agreement with previous results (9). For ease of comparison, all
activity levels were expressed as a percentage of Liver(wt) activity.
Skin(wt) was used as an internal control to allow comparison of results
between the three groups of polypeptides. Fig. 2D summarizes
the observed activity levels for each polypeptide, and Table
I (column 1) details the difference in
activity between polypeptides that varied at a single region. In each
instance (Table I, column 1) differences in activity levels were
determined to be statistically significant.
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The Exchange of N- or C-terminal Sequences-- Lack of C-terminal amidation in the wfsAFP-2 C terminus ([L]Skin-OOH) did not significantly affect activity (Fig. 2A). The conversion of the wflAFP-6 N or C terminus to the corresponding wfsAFP-2 sequences resulted in a loss of ~27% of activity (Fig. 2D; Table I, column 1, values A1 and C1). In wfsAFP-2, replacement of the N or C terminus with those found in wflAFP-6 resulted in a gain of ~15% in activity (Fig. 2D; Table I, column 1, values A3 and C3).
When [S]-Nterm was replaced by [L]-Nterm, the magnitude of the increased activity was ~14% greater when the remaining polypeptide contained L-core and [L]-Cterm (Table II, column 1, A1-A3 value). When the remaining polypeptide was composed of a mixture of components, the increase in activity associated with the same exchange was ~4% higher in the presence of S-core and [L]-Cterm as opposed to L-core and [S]-Cterm (Table II, column 1, A4-A2 value). Similarly, for the exchange of [S]-Cterm with [L]-Cterm, the increased activity was ~11% higher when wflAFP-6 components were present (Table II, column 1, C1-C3 value). Furthermore, the increased activity associated with this exchange was ~7% higher when [L]-Nterm and S-core were present as opposed to the opposite composition (Table II, column 1, C4-C2 value).
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Simultaneous Exchange of N- and C-terminal Sequences-- The [L]Skin[L] polypeptide was similar to a previously studied wflAFP-6 mutation, ATAAA, which was reported to have ~85% of the activity of a wild-type analogue as determined by the activity curves presented by Loewen et al. (29). The ATAAA analogue differed from [L]Skin[L] at positions 8 and 30, where ATAAA possessed Ala instead of Lys. Loewen et al. (29) observed lowered solubility for ATAAA, which affected their analysis of the analogue; however, no solubility issues were observed with [L]Skin[L] for the concentrations used here. If the experimental error of ~5% between assays is considered, then the combined effects of N- and C-terminal exchange on thermal hysteresis levels appeared to be somewhat additive. For [L]Skin and Skin[L], activity was increased in each case to ~64%, and ~67% of Liver(wt) (Fig. 2D). In combination, the exchange of both N- and C-terminal sequences, [L]Skin[L], resulted in activity of ~89%. Similarly, the 54% level of activity for [S]Liver[S] was close to the level of activity expected from the combined reductions for each individual exchange.
The simultaneous exchange of both termini was also evaluated as a single exchange in the core region. Thus, replacement of L-core in wflAFP-6 with S-core (essentially a replacement of complete IBMs with incomplete IBMs) resulted in a loss of ~11% of activity (Table I, column 1, value B1). The corresponding exchange in wfsAFP-2 resulted in a gain of ~3% in activity (Table I, column 1, value B3). The magnitude of the effect of the S-core to L-core exchange was greater by ~7% in the context of the wflAFP-6 termini than for the wfsAFP-2 termini (Table II, column 1, B1-B3 value). In the mixed-termini case, the magnitude of the exchange was more prominent by ~3% when the termini were composed of [L]-Nterm and [S]-Cterm then when composed of [S]-Nterm and [L]-Cterm (Table II, column 1, B4-B2 value).
Pairwise Comparison of the Effects of the N Terminus, Core Region, and C Terminus on Antifreeze Activity-- A pairwise comparison of activity levels between polypeptides that differed at a single region was performed to address the experimental error involved in each individual assay. Each region of wflAFP-6 demonstrated a higher level of activity than the corresponding region of wfsAFP-2. The [L]-Nterm provided an average improvement of ~20% (± 6%), L-core an improvement of ~7% (± 4%), and [L]-Cterm an improvement of ~22% (± 5%) in activity over the corresponding wfsAFP-2 components (Table I, column 1). Only in the evaluation of the core region did the error significantly impact the average value determined; nevertheless, the general trend was toward higher activity when the L-core was present as opposed to the S-core. However, as demonstrated in Table II, the magnitude of the increased activity associated with a wflAFP-6 region over that of the corresponding wfsAFP-2 region was dependent on the identities of the remaining regions.
The Magnitude of the Increase in Activity Associated with a wflAFP-6 Component Is Dependent on the Identity of the Remaining Regions-- In Table II (columns 3 and 5), a comparison of the values in Table I (column 1) was performed to determine the impact of the identities of the other regions that were present during the three exchanges. The presence of [L]-Cterm during exchange at the N terminus produced a ~9% higher improvement of activity compared with when the [S]-Cterm was present (Table II, column 3, A), and a ~5% higher improvement of activity was observed in the presence of L-core compared with S-core (Table II, column 5, A). Thus, a 4% higher improvement of activity (Ax1-Ax2 value) was observed when the wflAFP-6 component was present at the C terminus compared with when it was present within the core. Similarly, the improved activity of L-core over S-core demonstrated a 5% higher increase in the presence of [L]-Nterm over [S]-Nterm (Table II, column 3, B). For this same exchange, a slightly higher increase in activity was noted in the presence of [L]-Cterm when [S]-Nterm was present. However, the observed increase in activity when [L]-Nterm was present was not statistically significant (Table II, column 6, B). In this exchange, the improvement of activity associated with L-core was higher when wflAFP-6 components were present at the N terminus as compared with when they were present at the C terminus (Bx1-Bx2 value). The improvement of activity with [L]-Cterm in place of [S]-Cterm demonstrated a 9% higher enhancement in the presence of [L]-Nterm (Table II, column 5, C). For this exchange, the increased difference in activity levels when L-core was present was not statistically significant when [L]-Nterm was also present; however, the result was significant in the presence of [S]-Nterm (Table II, column 6, C). For this exchange, the improvement in activity was higher when wflAFP-6 components were present at the N terminus compared with when they were present in the core (Cx1-Cx2 value). All trends indicated that the improvement of activity associated with each wflAFP-6 component was always greater when other regions of wflAFP-6 were present. Furthermore, the location of the other wflAFP-6 components demonstrated differing levels of influence on the increased activity associated with each exchange, with the order of dependence for other regions being N terminus > C terminus > core. This order of dependence was also apparent in the values determined in column 1 (Table II). For example, in the A4-A2 value (Table II, column 1, A), the degree of activity enhancement was higher for the S-core and [L]-Cterm combination than for L-core and [S]-Cterm because the effect of the C terminus took precedence. This dependence appeared to have a "long range" effect, as the increased activity associated with [L]-Nterm over [S]-Nterm was more dependent on the identity of the opposite terminus, not the core region that was adjacent to it. The dependence of activity levels on the identity of the opposite terminus was also observed with the C terminus exchange.
-Helical Content of Polypeptides at 0 °C--
Each
polypeptide was assayed for its
-helical content utilizing circular
dichroism (CD) spectroscopy. Spectra (data not shown) were recorded at
0 and 40 °C in 0.1 M ammonium bicarbonate (pH 7.9) at
two different concentrations. At 0 °C, all polypeptides displayed
spectra indicative of high
-helical content with typical negative
minima at 222 and 208 nm, and positive maximums at approximately 192 nm. At 40 °C, the spectra were indicative of helix-random coil mixed
structures and were identical at two different concentrations. Helical
content determinations indicated Liver(wt) to be nearly entirely
-helical (~95%), which was in good agreement with previous structural studies of wflAFP-6 (8, 30, 31), and Skin(wt) was found to
have a helical content of ~82% (Fig.
3).
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An initial inspection of the results (Fig. 3A) for exchanges to wflAFP-6 demonstrated that the effects on helicity for each exchange appeared to be additive. The decreases in helical content between Liver(wt) and [S]Liver (10.3%), and between Liver(wt) and Liver[S] (8.9%), gave a combined decrease of 19.2% that would predict a helical content of 75.3% for [S]Liver[S], which was close to the observed value of 77.9%. However, for wfsAFP-2, replacement of the C terminus alone (Skin[L]) produced ~100% helical content (Fig. 3A), whereas [L]Skin demonstrated ~90% helical content.
Pairwise Comparison of the Effects of the N Terminus, Core Region,
and C Terminus on -Helical Content--
The differences in helical
content between polypeptides that differed at a single region are
summarized in Table I, column 3. Several of the differences were found
not to be statistically significant in this comparison; however, in
three of these instances, the p values were close to the
value of 0.05 used in the analysis (Table I, column 4, a2, b1, and c2).
Overall, the results of the pairwise comparison of the effects of
exchanges on helical content were not as consistent as for the activity
analysis, because of the aberrant values found for Skin[L] (Table I,
column 3). However, if the results were evaluated without Skin[L]
(Table I, column 5 in parts A and B), the [L]-Nterm imparted ~8.7%
higher helical content than the [S]-Nterm. Conversely, the L-core
sequence was ~4.5% less helical than the corresponding S-core
region. Although the experimental error would preclude forming
conclusions based on the assay of individual polypeptide comparisons,
the general trend observed in the pairwise comparison indicated that
the [L]-Nterm induced a higher helical content than the [S]-Nterm,
whereas the S-core induced a higher helical content than the
L-core.
For the C-terminal exchange, the [L]-Cterm always produced higher
helical content than [S]-Cterm. Only in the presence of [S]-Nterm
and L-core was the increased helical content (Table I, column 3, c2
value) associated with [L]-Cterm found to be statistically
insignificant. However, a dependence on the nature of the core sequence
for the increased helicity associated with [L]-Cterm was observed, as
helical content was ~10% higher when the S-core was present (Table
I, column 6). When the L-core was present, the [L]-Nterm demonstrated
a greater effect on enhanced helicity in the presence of the
[L]-Cterm (8.9 6.3% = 2.6%). Conversely, when the S-core
was present, the [S]-Nterm provided higher enhancement of helicity in
the presence of the [L]-Cterm (16.5%
20.3% =
3.8%). In both
instances, the difference values were based on a single scenario,
unlike earlier comparisons where trends could be discerned because of
multiple values from independent comparisons. However, these results
may indicate that the dependence on the nature of the core region is
further enhanced when the core region is associated with its native N
terminus. An analysis of the helicity values obtained for the
N-terminal exchange demonstrated that the same relationship existed for
the N terminus. The effect of higher helical content associated with
[L]-Nterm was greater in the presence of L-core, but when either core
was present, the increased helical content was higher when each core
was associated with its own C terminus (Table I, column 3, A).
Correlation of Activity Levels and Helical Content--
When
comparing all analogues studied, activity levels increased as helical
content increased (Fig. 4A).
However, for all analogues, a rigorous direct correlation between
helical content and activity was not observed (Fig. 4A,
overall linear regression line). If the L-core and S-core analogues are
analyzed separately, the analogues containing L-core demonstrated a
very good direct correlation between activity and helical content
(R2 = 0.990), whereas the S-core containing
analogues still demonstrated a low level of direct correlation.
Examination of the residuals for the linear regression for all
analogues demonstrated that a reasonably good correlation between
activity and helical content existed until the polypeptides achieved a
helical content of ~90%, beyond which the correlation became very
poor.
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Comparison of Observed -Helical Content with Predicted
Values Based on a Simple Two-state Helix Prediction
Method--
In Table III, the
predicted helical contents were derived at by using a simple two-state
helix prediction method where each polypeptide component was isolated
and assigned a total number of helical residues likely to be found in
each region. For wflAFP-6, helix predictions were based on the
assumption that each region ([L]-Nterm, L-core, and [L]-Cterm) was
100% helical based on structural data (8, 31). For wfsAFP-2, the
assignments were based on current theories of helix capping (see
"Discussion" for rationale behind assignment of helical and
non-helical residues). In the [S]-Nterm, 3 of 4 residues were assumed
to be non-helical (all residues preceding Pro4), the S-core was assumed
to be 100% helical, and in [S]-Cterm, 4 of 6 residues were assumed
to be non-helical (all residues following and including Gly-36). The
predicted helical contents for each polypeptide was then simply arrived
at by determining the total number of residues in a helical
conformation under the assumption that each region was independent of
effects caused by the identity of the other regions.
Examination of all polypeptides that contained the S-core demonstrated
a remarkably high correlation of the predicted values with the observed
values, except for Skin[L], which was underestimated by ~11%. In
polypeptides that contained L-core, including Liver(wt), the helical
content was consistently overestimated by ~5%.
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Thermal Denaturation of Polypeptides-- The polypeptides were subjected to thermal denaturation from 0 °C to 60, 64, or 65 °C, and their helical contents were assayed by CD spectroscopy at 222 nm (data not shown). The thermal denaturation of each polypeptide was fully reversible, but demonstrated very broad transitions over the temperature ranges used. Because of the broad nature of the unfolding process, standard practices of curve fitting for thermal denaturation data (32, 33) could not be performed on data collected here without producing significantly large values of error (data not shown). Thus, only a qualitative assessment of unfolding is presented by evaluating the helical fraction (fH) of each polypeptide during thermal denaturation.
The two wild-type polypeptides displayed partial cooperativity in helix unfolding as evidenced by "S-shaped" denaturation profiles and a slight degree of thermal stability as temperatures increased (Fig. 3B). At temperatures below ~25 °C, Liver(wt) demonstrated enhanced helical content over Skin(wt). All polypeptides demonstrated some degree of cooperative unfolding except for [S]Liver and [S]Liver[S], which lacked S-shaped unfolding profiles and any indications of thermal stability (Fig. 3C). For [L]Skin, there was only a slight increase in helical content at 0 °C compared with Skin(wt), but an increase in thermal stability was observed (Fig. 3D). For Skin[L], the unfolding process maintained only a slight cooperative nature (Fig. 3D). In [L]Skin[L] (Fig. 3D), thermal stability was enhanced as observed for [L]Skin. Overall, [L]Skin and [L]Skin[L] demonstrated the highest thermal stability of all the polypeptides assayed.
Thr to Ser Mutation in [L]Skin--
To determine the cause of
the increased thermal hysteresis observed for [L]Skin, Thr-2 was
mutated to Ser, [L(T2S)]Skin (Fig. 5A), which retained the -OH
group necessary for the formation of the hydrogen-bonding network in
the N terminus that stabilizes the helix structure (8), but removed the
-methyl group of Thr that is involved in ice-binding (17, 18, 20,
21). Thus, this mutation was designed to determine whether the
increased activity of [L]Skin was the result of an enhanced N-cap
function leading to greater helix stability (see "Discussion") or
of the introduction of a new, or more efficient, ice-binding surface. The activity of [L(T2S)]Skin was slightly less than Skin(wt) (Fig. 5B); however, no loss of helical content for [L(T2S)]Skin
at 0 °C was observed (Fig. 5C). Thus, the improved
activity of the [L]Skin mutation was because of the introduction of a
more efficient ice-binding surface centered around the
-methyl group
of Thr-2, and not because of the increased helical content of the
entire polypeptide afforded by the exchange of [S]-Nterm by
[L]-Nterm.
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DISCUSSION |
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Taking into account the results of individual exchanges, the pairwise comparison, and earlier studies,2 we conclude that the ~50% difference in antifreeze activity between wflAFP-6 and wfsAFP-2 is derived from (i) average contributions of ~20% by both the N (DTASDA versus MDAP) and C termini (AR versus KAGAAR), and (ii) a 10% contribution by the core region (complete IBM (LTAAN) versus incomplete IBM (ATAAA)). Furthermore, the magnitude of change in antifreeze activity generated by the exchange of a wfsAFP-2 component with a wflAFP-6 component was dependent on the characteristics of the other polypeptide regions. The region that imparted the greatest influence on activity between wfsAFP-2 and wflAFP-6 was the N terminus, because of its own ability to modulate activity levels and its influence on the activity of other regions of the polypeptide. The termini of wflAFP-6 demonstrated a greater ability to impart both higher helical character and thermal stability as compared with the wfsAFP-2 termini. However, the wflAFP-6 core region exhibited lower helix propensity than the wfsAFP-2 core region. Thus, the activity level of each region is likely related to a complex interplay between its own inherent ice-binding ability, helix propensity, and the helix propensity imparted upon the entire polypeptide by each region.
The thermal hysteresis levels associated with type I AFPs have been shown to be positively correlated with helical content (34), which is likely the result of the ability of ice-binding residues to adopt the proper configuration for ice recognition. This positive correlation of activity with helical content was confirmed with the analogues studied here (Fig. 4). However, the results clearly demonstrated that other factors as well as helical content affect activity levels, especially after relatively high helical content (>~90%) is achieved, an example of which was demonstrated by the [L(T2S)]Skin mutation. Furthermore, a more direct correlation was observed for analogues containing L-core than for S-core, indicating that the activity of analogues containing L-core were more sensitive to the helical content.
From current theories of helix capping, it can be predicted that Gly-36
of wfsAFP-2 occupies the C' position in a C-cap, or at least acts as a
helix terminator, and Pro-4 is constrained to occupy the N1 position of
the N-cap under the conventional notation for residues at helix termini
(35). If all intervening residues adopt -helical (
,
) angles,
the predicted helical content of Skin(wt) would be ~80%, which is
very close to the observed value of ~82% (see Fig. 3A).
Thus, from this analysis, the helix propensity of the S-core is
equivalent to, or more likely, greater than (see below) the helix
propensity of the L-core (at 0 °C), even though the CD measurements
consistently demonstrated a lower overall helical content at 0 °C
for wfsAFP-2. The nearly identical helical content of [S]Liver[S]
to Skin(wt) at 0 °C (~78% versus ~82%) could be
interpreted in the same manner as for Skin(wt). However, the lack of
thermal stability of [S]Liver[S] compared with Skin(wt) (see Fig.
3C) would suggest that the L-core possesses a lower helical
propensity than the S-core, which would be expected because of the
higher Ala content in the S-core (36). Furthermore, the L-core
consistently demonstrated a lower helical content than S-core (Table I,
column 2, B). The assessment of a slightly lower than 100% helical
propensity for L-core led to an overestimation of the helical content
for polypeptides containing L-core in the simple two-state helix
prediction model (see Table III). The analysis presented in Table III
was not an attempt to fully describe the structural characteristics of
type I AFPs, but was used merely to illustrate that, in direct
comparison, L-core was in fact not 100% helical whereas S-core
maintains high helical content under several differing contexts. Thus,
the S-core contributed more overall helical character than L-core when
present in type I AFPs. Furthermore, the lower helix propensity of
L-core compared with S-core manifested itself in the correlation
between helical content and activity (Fig. 4), where the activity
levels were more directly dependent on helical content for analogues
containing L-core than for those containing S-core. Thus, although
L-core improved the activity compared with S-core, the presence of
complete IBMs lowered the helical propensity within the core, which led
to the observed higher correlation between activity and helical content
for L-core-containing polypeptides.
Helix capping refers to patterns of hydrogen bonding and hydrophobic
interactions present at the termini of -helices that satisfy
residues that do not possess main-chain amide hydrogen to carbonyl
oxygen hydrogen bonding partners of the form i
i + 4 that are found in the core of the helix (35). As
evident from the crystal structure, such helix termini capping networks exist in wflAFP-6 (8). Furthermore, helix-capping ability would lead to
a lower nucleation barrier for helix formation and, thus, greater
thermal stability and helical content for the entire helix, because it
has been suggested that helix caps can exert a significant effect on
helix nucleation (37). Thus, inherent in a higher propensity for helix
cap formation is a greater ability to impart helical character to the
entire polypeptide. The ability of [L]-Nterm to impart higher thermal
stability and its consistent demonstration of the ability to impart
higher helical content to the entire polypeptide at 0 °C may
indicate a greater N-capping propensity for [L]-Nterm compared with
[S]-Nterm. In native winter flounder AFPs, the higher helix-inducing
ability of the [L]-termini as compared with the corresponding
[S]-termini may be necessitated by the lower helix propensity of the
L-core caused by the presence of the more active complete IBMs. Thus,
when L-core replaced S-core in [S]Liver[S], only a ~3% increase
in activity was noted (see Table I) because the [S]-termini were not
able to promote a high enough helical content within the core region to
produce the full antifreeze activity of L-core. Conversely, nearly the
full magnitude of the effect on activity for the core exchange was
observed in [L]Skin[L], because all components in this polypeptide
were able to produce maximal helical content.
In wfsAFP-2, Lys-6, which is normally found i + 4 to Asp-2,
was not transferred along with [S]-Nterm in the various exchanges. The Asp-2/Lys-6 pair (see Fig. 1, D2-K6) may be necessary to
form a salt bridge to stabilize the helix by forming an N-cap.
Furthermore, the C terminus of wfsAFP-2 may be involved in a complex
C-cap structure, because C-capping residues usually reside outside the helix proper and often require a Gly residue at C' to allow for positive (,
) angles (35). Although the lower capping
ability of [S]-Nterm may suggest that the Asp-2/Lys-6 salt bridge is
necessary in wfsAFP-2, the improved thermal stability of [L]Skin over
Skin(wt) would suggest that [L]-Nterm still possessed a superior
ability to impart higher helix propensity to the entire polypeptide.
Furthermore, the inherent thermal stability of the S-core suggests that
the helix-nucleating capabilities of the termini are not as critical for S-core. The putative Asp-2/Lys-6 salt bridge may also explain the
seemingly aberrant results observed for Skin[L] in that some form of
interaction between regions caused the three residues preceding Pro-4
to adopt a helical conformation. Further work is necessary to
investigate the validity of the proposed Asp-2/Lys-6 salt bridge.
The "long range" dependence of the increased activity associated with each [L]-termini on the identities of the opposing termini was likely related to the relative helix propensity of the remainder of the polypeptide. Thus, the order of the activity dependence (N terminus > C terminus > core) was a reflection of the importance of each region on the overall helix propensity. The placement of the core last in the order does not imply that the helix propensity of the core is inconsequential to activity. The low level of influence was observed because both L-core and S-core were highly helical, and exchanges between the two likely did not significantly influence the character at the termini or significantly change the nature of the hydrophobic binding face. However, relatively distant termini likely affected the activity through their ability to induce helical content to the overall polypeptide through helix capping, thereby enhancing the ice-binding sites in other portions of the polypeptide.
In wflAFP-6, the first 11-residue motif occurs in the N terminus. A
study of a minimized peptide consisting of the N and C termini of
wflAFP-6 (38) and mutagenesis studies performed on the first and last
Thr residues of wflAFP-6 (21) have suggested a role for the N terminus
in activity. The Thr Ser mutation in [L(T2S)]Skin clearly
demonstrated that Thr-2 is involved in antifreeze activity. However, in
a previous study, it was found that the first four residues of wflAFP-6
could be deleted with no effect on activity (27). It should be noted
that there are no reports of a single mutation (T2S) in wflAFP-6 (7),
and further work is necessary to identify the cause of these opposing results.
The magnitude of the detrimental effect of replacing [L]-Nterm with [S]-Nterm was greater than the magnitude of the enhancement for the opposite exchange. This may reflect the fact that, in [S]Liver, the loss of activity was the result of a combination of lower helical content afforded by [S]-Nterm, which affected the ice-binding sites of the remainder of the polypeptide, and more importantly, the loss of a highly active ice-binding site within [L]-Nterm itself. In contrast in the opposite exchange, Skin(wt) to [L]Skin, the increased activity was only the result of the introduction of a more efficient ice binding site, and the improved helicity only minimally affected the remainder of the ice binding sites, because of the inherently high helical content associated with S-core. At the C terminus, the low level of evolutionary conservation (7) indicates that this region is less likely to be critical for biological activity. The increased activity with [L]-Cterm over [S]-Cterm may be caused in part by the presence of a more efficient capping structure, which resulted in a lower entropic cost to ice binding, as no sacrifice of conformational entropy at the C terminus was necessary upon ice binding. Nevertheless, the results also suggest that [L]-Cterm may have an enhanced ice binding ability over [S]-Cterm, in a similar manner as described for the N termini.
The data obtained in this study have demonstrated that there is a
complex relationship between the helical propensities of the different
regions of type I AFPs (N terminus, core, and C terminus) and the level
of antifreeze activity. Thus, the conclusions presented here should be
used as a first step in more in-depth studies of the determinants of
antifreeze activity that reside in the N and C termini of other type I AFPs.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Frank D. Sönnichsen for critical review of an original draft of the manuscript, Dr. Nam-Chiang Wang for peptide synthesis, Rey Interior for amino acid analysis, Dr. Avijit Chakrabartty for assistance with CD spectroscopy, Dr. Daniel S. C. Yang for helpful discussions, and Cora Young for assistance with preparation of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the Canadian Institutes of Health Research (formerly the Medical Research Council, Canada) (to C. L. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of an Ontario graduate scholarship.
To whom correspondence should be addressed: Dept. of Biological
Sciences, National University of Singapore, Singapore 119260, Singapore. Tel.: 65-6874-2692; Fax: 65-6779-2486; E-mail:
dbshead@nus.edu.sg.
Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M300081200
2 Q. Lin, W. Low, and C. L. Hew, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: AFP, antifreeze protein/polypeptide; AFGP, antifreeze glycoprotein; wflAFP, winter flounder liver-type AFP; wfsAFP, winter flounder skin-type AFP; HPLC, high performance liquid chromatography; IBM, ice-binding motif; CD, circular dichroism; wt, wild-type; [L]-Nterm, liver-type N terminus; [L]-Cterm, liver-type C terminus; L-core, liver-type core; [S]-Nterm, skin-type N terminus; [S]-Cterm, skin-type C terminus; S-core, skin-type core; Fmoc, N-(9-fluorenyl)methoxycarbonyl.
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