Solution conformation of C-linked antifreeze glycoprotein analogues and modulation of ice recrystallization

Article abstract of DOI:10.1021/ja904169a

Antifreeze glycoproteins (AFGPs) are a unique class of proteins that are found in many organisms inhabiting subzero environments and ensure their survival by preventing ice growth in vivo. During the last several years, our laboratory has synthesized func

Full text of DOI:10.1021/ja904169a

Published on Web 10/13/2009
Solution Conformation of C-Linked Antifreeze Glycoprotein
Analogues and Modulation of Ice Recrystallization
Roger Y. Tam, Christopher N. Rowley, Ivan Petrov, Tianyi Zhang,
Nicholas A. Afagh, Tom K. Woo, and Robert N. Ben*
Department of Chemistry, D’Iorio Hall, 10 Marie Curie, UniVersity of Ottawa,
Ottawa, ON, Canada K1N 6N5
Received May 22, 2009; E-mail: rben@uottawa.ca
Abstract: Antifreeze glycoproteins (AFGPs) are a unique class of proteins that are found in many organisms
inhabiting subzero environments and ensure their survival by preventing ice growth in vivo. During the last
several years, our laboratory has synthesized functional C-linked AFGP analogues (3 and 5) that possess
custom-tailored antifreeze activity suitable for medical, commercial, and industrial applications. These
compounds are potent inhibitors of ice recrystallization and do not exhibit thermal hystersis. The current
study explores how changes in the length of the amide-containing side chain between the carbohydrate
moiety and the polypeptide backbone in 5 influences ice recrystallization inhibition (IRI) activity. Analogue
5 (n ) 3, where n is the number of carbons in the side chain) was a potent inhibitor of ice recrystallization,
while 4, 6, and 7 (n ) 4, 2, and 1, respectively) exhibited no IRI activity. The solution conformation of the
polypeptide backbone in C-linked AFGP analogues 4-7 was examined using circular dichroism (CD)
spectroscopy. The results suggested that all of the analogues exhibit a random coil conformation in solution
and that the dramatic increase in IRI activity observed with 5 is not due to a change in long-range solution
conformation. Variable-temperature 1H NMR studies on truncated analogues 26-28 failed to elucidate the
presence of persistent intramolecular bonds between the amide in the side chain and the peptide backbone.
Molecular dynamics simulations performed on these analogues also failed to show persistent intramolecular
hydrogen bonds. However, the simulations did indicate that the side chain of IRI-active analogue 26 (n )
3) adopts a unique short-range solution conformation in which it is folded back onto the peptide backbone,
orienting the more hydrophilic face of the carbohydrate moiety away from the bulk solvent. In contrast, the
solution conformation of IRI-inactive analogues 25, 27, and 28 had fully extended side chains, with the
carbohydrate moiety being exposed to bulk solvent. These results illustrate how subtle changes in
conformation and carbohydrate orientation dramatically influence IRI activity in C-linked AFGP analogues.
Introduction
binds to the surface of ice and causes a localized freezing point
depression via the Kelvin effect.3 While direct evidence
Biological antifreezes (BAs) are peptide-based structures that
have the ability to prevent the seeding of ice crystals in vivo to
protect against cryoinjury and death.1 BAs have been grouped
into two classes: antifreeze proteins (AFPs) and antifreeze
glycoproteins (AFGPs).2 Unlike AFPs, AFGPs share a great
deal of structural homology across the many different species
of fish in which they can be found. Nonetheless, both classes
of BAs possess the same distinct antifreeze properties, namely,
thermal hysteresis (TH) and ice recrystallization inhibition (IRI).
With respect to TH, the mechanism of action is regarded as an
adsorption inhibition phenomenon whereby the BA irreversibly
supporting this mechanism has been obtained,4,5 the nature of
the protein or carbohydrate interaction with ice remains a source
of speculation.6,7 In the case of AFPs, there is evidence implying
that hydrophobic interactions may be the dominant force
modulating the binding of the AFP to ice.7 However, in AFGPs,
evidence suggests that hydrophilic interactions are the dominant
driving force for adsorption onto the ice lattice. Subsequently,
various models that rationalize ice binding on the basis of
complementarity of specific hydroxyl groups with the ice lattice
have emerged.4 Recently, the importance of hydration and its
role in the AFP/AFGP mechanism of action has been
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9
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A R T I C L E S
Tam et al.
Figure 1. Structures of native AFGP-8 (1) and C-linked AFGP analogues 2 and 3.
examined.8-10 These results support the hypothesis that the
mechanism of action for TH and IRI may be different9-12 and
that IRI may not be dependent on the ice-binding ability of the
solute but on the ability of the solute to disturb the three-
dimensional hydrogen-bonding network of the ice-water
interface.9,10
indicate that 3 exhibits little or no in vitro cytotoxicity and can
penetrate cells and also inhibit apoptotic cell death.15
Several structural analogues of 3 have been prepared and
assessed for antifreeze activity to better understand how the
spatial relationship between the carbohydrate moiety and the
polypeptide backbone affects the IRI activity.13 The results of
this study reveal that extension of the carbon spacer by even
one carbon atom dramatically decreases the ability of these
analogues to inhibit ice recrystallization. In view of these results,
it is surprising that C-AFGP analogue 2 displayed IRI activity.
Furthermore, this suggests that the amide bond in the side chain
of 2 may be an important structural feature in this structural
class of ice recrystallization inhibitors.
Our laboratory has undertaken the task of rationally designing
and synthesizing functional carbon-linked AFGP (C-AFGP)
analogues possessing custom-tailored antifreeze activity.9,11,13
In particular, we are interested in designing compounds that
have potent IRI activity but no TH activity. Such a class of
compounds are of interest as novel cryoprotectants. The notion
that a potent inhibitor of ice recrystallization has cryoprotective
abilities in vivo is based on the fact that the majority of cellular
damage during cryopreservation occurs as a result of ice
recrystallization.14 Toward this end, we have prepared a series
of analogues (Figure 1) possessing a high degree of IRI activity.
To date, C-AFGP analogues 2 and 3 remain as two of the most
potent inhibitors of ice recrystallization developed in our lab.11,13
Furthermore, in vitro studies performed in our laboratory
Critical features of hydrated AFP16 and AFGP17-20 structures
(such as intramolecular and intermolecular hydrogen-bonding
interactions) have previously been studied by molecular dynam-
ics (MD) simulations in an attempt to further elucidate the
factors responsible for antifreeze activity. Using MD simulations
(performed in the presence of water), along with circular
dichroism (CD) spectroscopy and variable-temperature (VT) 1H
NMR, this work explores how a decrease in the length of the
amide-containing side chain in analogues of 2 influences IRI
activity and how this activity is modulated by subtle localized
changes in the conformation of the glycoconjugate.
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M. C.; Davies, P. L.; Sonnichsen, F. D. Biochemistry 1997, 36, 14652–
14660. (b) Leinala, E. K.; Davies, P. L.; Jia, Z. Structure 2002, 10,
619–627. (c) Baardsnes, J.; Davies, P. L. Biochim. Biophys. Acta 2002,
1601, 49–54. (d) Siemer, A. B.; McDermott, A. E. J. Am. Chem. Soc.
2008, 130, 17394–17399.
Materials and Methods
Solid-Phase Synthesis of Full Glycopeptide Polymers 6
and 7 and Monomeric Model Systems 26-28. All of the
glycoconjugates were synthesized using conventional Fmoc solid-
phase synthesis on an APEX 396 using acid-labile Wang resin.21
Purification of the glycopeptides was performed via HPLC using
reversed-phase chromatography (Polaris C-18 RP column; mobile
(8) Nutt, D. R.; Smith, J. C. J. Am. Chem. Soc. 2008, 130, 13066–13073.
(9) Czechura, P.; Tam, R. Y.; Murphy, A. V.; Dimitrijevic, E.; Ben, R. N.
J. Am. Chem. Soc. 2008, 130, 2928–2929.
(10) Tam, R. Y.; Ferreira, S. S.; Czechura, P.; Chaytor, J. L. J. Am. Chem.
Soc. 2008, 130, 17494–17501.
(11) Eniade, A.; Purushotham, M.; Ben, R. N.; Wang, J. B.; Horwath, K.
Cell Biochem. Biophys. 2003, 38, 115–124.
(12) (a) Knight, C. A. Nature 2000, 406, 249–251. (b) Sidebottom, C.;
Buckley, S.; Pudney, P.; Twigg, S.; Jarman, C.; Holt, C.; Telford, J.;
McArthur, A.; Worrall, D.; Hubbard, R.; Lillford, P. Nature 2000,
406, 256. (c) Pudney, P. D. A.; Buckley, S. L.; Sidebottom, C. M.;
Twigg, S. N.; Sevilla, M.-P.; Holt, C. B.; Roper, D.; Telford, J. H.;
McArthur, A. J.; Lillford, P. J. Arch. Biochem. Biophys. 2003, 410,
238–245.
(15) Liu, S.; Wang, W.; von Moos, E.; Jackman, J.; Mealing, G.; Monette,
R.; Ben, R. N. Biomacromolecules 2007, 8, 1456–1462.
(16) Nada, H.; Furukawa, Y. J. Phys. Chem. B 2008, 112, 7111–7119.
(17) Lane, A. N.; Hays, L. M.; Feeney, R. E.; Crowe, L. M.; Crowe, J. H.
Protein Sci. 1998, 7, 1555–1563.
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Crowe, J. H. Biophys. J. 2000, 78, 3195–3207.
(13) Liu, S.; Ben, R. N. Org. Lett. 2005, 7, 2385–2388.
(14) For examples of cellular damage caused by ice recrystallization, see:
(a) Wang, T.; Zhu, Q.; Yang, X., Jr.; DeVries, A. L. Cryobiology
1994, 31, 185–192. (b) Mazur, P. C. Science 1970, 168, 939–949. (c)
Mazur, P. C. Am. J. Physiol.: Cell Physiol. 1984, 247, C125–C142.
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(21) Fields, G. B.; Fields, C. G. J. Am. Chem. Soc. 1991, 113, 4202–4207.
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Solution Conformation of C-Linked AFGP Analogues
A R T I C L E S
phase: 0.1:99.9 TFA/water to 0.1:25:74.9 TFA/acetonitrile/water
gradient over 30 min).
2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS) at a concentration
of 20 µg/mL was used as a chemical shift internal standard.26
Molecular Dynamics Simulations. Localized solution conforma-
tions of the truncated glycopeptides 25-28 and native AFGP-8 in water
were computed via MD simulations using the AMBER 9 program.27
The N-acetylated monomers 25-28 were defined as nonstandard
amino acids within the AMBER antechamber program. The GLYCAM
model developed by Woods and co-workers28 was used to assign
partial charges to the carbohydrate. In C-AFGPs 25-28, the galactose
moiety is linked to the alkyl side chain through a methylene unit instead
of an anomeric oxygen; thus, C1 was not constrained to its GLYCAM
charge. RESP fitting29 was performed using electrostatic potentials
generated from two Hartree-Fock/6-31G*-minimized conformations,
where the ΦR/ΨR angles of the amino acids were constrained to the
R-helix (ΦR ) -60°, ΨR ) -40°) and ꢀ-strand (ΦR ) -120°, ΨR )
140°) conformations.30 The peptides were minimized using the
generalized Born implicit solvent model with a 12 Å nonbonded cutoff,
annealed at 600 K for 100 ps, and then equilibrated at 300 K for 100
ps. The equilibrated system was solvated in a TIP3P31 water box with
a 16 Å distance between the box edge and the peptide, adding
2500-3000 water molecules. The solvated peptide was then equili-
brated for 1 ns (using a 1 fs time step) in the NPT ensemble at 300 K
and 1 bar with a thermostat frequency of 5.0 ps-1 and a barostat
relaxation time of 2.0 ps. A 10 ns trajectory with frames recorded
every 1 ps was generated for each tripeptide monomer starting with
the equilibrated structures and utilizing the same simulation parameters
as the equilibration step. Free-energy profiles of rotations around the
alkyl chain linker (Ψs, ꢁ1-4) were generated from 324 MD simulations
sampling the full 360 degrees of rotation for the angles, which were
restrained in 20° increments using a harmonic restraint with a spring
constant of 10 kcal mol-1 deg-1. Intramolecular hydrogen bonding
between the galactose and the backbone was examined using the ptraj
program in the AMBER suite.
Ice-Recrystallization Inhibition Assay. Samples were assayed
for recrystallization inhibition (IRI) activity using the “splat-cooling”
method, as described previously.22 A total of three images of the
resulting ice wafer were photographed through a Leitz compound
microscope equipped with an Olympus 20× (infinity-corrected)
objective with a Nikon CoolPix digital camera. Sample analysis
for ice crystal sizes was performed using the mean elliptical method.
In this method, the ten largest ice crystals were chosen from the
field of view (FOV) in each image. Selection of these crystals was
arbitrary in that they were chosen after a visual inspection of the
image. The two-dimensional surface area of each of these ten
crystals was then calculated via approximation of the crystal as an
elliptical area. The major and minor elliptical axes were defined
by the two largest orthogonal dimensions across the ice grain
surface. The surface area of each ice grain was then calculated using
the formula A ) πab, in which A is the area and a and b are the
lengths of the major and minor elliptical axes, respectively. Totaling
all of the individual measurements for each FOV produces a value
for the average grain surface area that is termed the mean largest
grain size (MLGS). The error was calculated as the standard error
of the mean (SEM), and t tests were performed to the 95%
confidence level.
Thermal Hysteresis Assay. Nanoliter osmometry was performed
using a Clifton nanoliter osmometer (Clifton Technical Physics,
Hartford, NY), as described by Chakrabartty and Hew.23 All of
the measurements were performed in doubly distilled water. Ice
crystal morphology was observed through a Leitz compound
microscope equipped with an Olympus 20× (infinity-corrected)
objective, a Leitz Periplan 32X photo eyepiece, and a Hitachi KP-
M2U CCD camera connected to a Toshiba MV13K1 TV/VCR
system. Still images were captured directly using a Nikon CoolPix
digital camera.
Results and Discussion
Circular Dichroism. CD spectra of the glycopolymers 3, 5-7,
23, 24, and AFGP-8 (1) were obtained using a Jasco model J-810
automatic recorder spectropolarimeter interfaced with a Dell
computer. All of the measurements were performed in quartz cells
with 0.1, 0.5, or 1.0 cm path lengths. Spectra were obtained with
a 1.0 nm bandwidth time constant of 2 s and a scan speed of 50
nm/min. Eight scans were added to improve the signal-to-noise ratio,
and baseline corrections were made against each sample. All of
the spectra were recorded between 190 and 300 nm, and all of the
CD experiments were performed in doubly distilled H2O at pH 7.4.
Data obtained from CD spectroscopy were converted into molar
ellipticities (deg cm2 dmol-1). Glycopeptide secondary structures
were estimated using the deconvolution software CD Pro. The data
from each spectrum were analyzed using three different deconvo-
lution programs: SELCON3, CDSSTR, and CONTINLL. Of these
three programs, SELCON3 and CONTINLL gave the most
consistent results. IBASIS 5 was used as the set of reference
proteins; it contains 37 proteins with R-helix, ꢀ-structure, polypro-
line II, and unordered conformations with optimal wavelengths of
185-240 nm.24
Our laboratory recently reported the synthesis of C-linked
AFGP analogue 2.11 Assessment of its antifreeze activity
revealed an extremely small TH gap (0.02 °C) and moderate
IRI activity. In contrast, C-linked AFGP analogue 3 possessed
no TH activity but was a very potent inhibitor of ice recrys-
tallization. In fact, this analogue was equipotent to native AFGP
8 from Gagus ogac in its ability to inhibit ice recrystallization.13,15
However, increasing the length of the spacer between the
carbohydrate moiety and the polypeptide backbone in 3 by
inserting additional carbons abolished the IRI activity.13 In
contrast, C-AFGP analogue 2 containing a six-atom side chain
was still an inhibitor of ice recrystallization, suggesting that the
amide bond may be an important structural feature for IRI in
this particular class of compounds. In order to validate this
hypothesis, structural analogues of 2 (compounds 5-7) with
the same polymer length as 3 (i.e., four repeating tripeptide
units) but different side-chain lengths were designed and
1
Variable-Temperature NMR Studies. VT H NMR studies
were performed on truncated glycopolymers25 26-28 on a Varian
Inova 500 MHz spectrometer at temperatures ranging from 0 to 50
°C in 5 °C increments with a 95:5 mixture of H2O and D2O as the
solvent. An appropriate water suppression program was run, and
(26) Hoffman, R. E.; Davies, D. B. Magn. Reson. Chem. 1988, 26, 523–
535.
(27) (a) Case, D. A.; et al. AMBER 9; University of California: San
Francisco, 2006. (b) Case, D. A.; Cheatham, T. E.; Darden, T.; Gohlke,
H.; Luo, R.; Merz, K. M.; Onufriev, A.; Simmerling, C.; Wang, B.;
Woods, R. J. J. Comput. Chem. 2005, 26, 1668–1688.
(28) Woods, R. J.; Dwek, R. A.; Edge, C. J.; Fraser-Reid, B. J. Phys. Chem.
1995, 99, 3832–3846.
(22) Knight, C. A.; Hallet, J.; DeVries, A. L. Cryobiology 1988, 25, 55–
60.
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Chem. 1993, 97, 10269–10280. (b) Cieplak, P.; Cornell, W. D.; Bayly,
C.; Kollman, P. A. J. Comput. Chem. 1995, 16, 1357–1377.
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W.; Yang, R.; Cieplak, P.; Luo, R.; Lee, T.; Caldwell, J.; Wang, J.;
Kollman, P. J. Comput. Chem. 2003, 24, 1999–2012.
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2000, 287, 243–251. (b) Sreerama, N.; Woody, R. W. Anal. Biochem.
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A R T I C L E S
Tam et al.
Figure 2. Retrosynthetic analysis of C-Linked AFGP analogues 4-7.
Scheme 1. Synthesis of Glycoconjugate Building Blocks 10 and 11a
a Reagents and conditions: (a) CDI (1.2 equiv), BnOH (1.3 equiv), DCM, 21 °C, 14 h; (b) TFA/DCM (1:1), 0.1 M, 21 °C, 5 h (17), 14 h (18); (c)
PhI(OCOCF3)2 (1.1 equiv), EtOAc/CH3CN/H2O (2:1:1), 0.02 M, then pyridine, pH 4, 21 °C, 4 h (19), 16 h (20); (d) 16 (0.83 equiv), HBTU (0.92 equiv),
DCM, then DIPEA (2.5 equiv), 21 °C, 16 h; (e) Pd/C (5 wt %), H2, EtOH, 1 atm, 21 °C, 6 h.
synthesized (Figure 2). In view of the precedented relationship
between glycopeptide length and antifreeze activity,11,32,33 it was
necessary to synthesize lysine-containing derivative 4 compris-
ing only four repeating tripeptide units instead of six in order
to enable direct comparison of the IRI activities.34
Synthesis of C-linked AFGP Analogues 4-7. C-Linked AFGP
analogues 4-7 were prepared using standard Fmoc-based solid-
phase peptide synthesis protocols (Figure 2).9,11,21 Building
blocks 8-11 were synthesized by coupling the orthogonally
protected Fmoc-lysine derivatives 12-15 with C-linked pyra-
nose derivative 16.9,35 Amino acid derivatives 12 and 13 were
prepared from commercially available L-lysine and L-ornithine,
respectively; however, the unnatural amino acid derivatives of
L-diaminobutanoic acid (14) and L-diaminopropanoic acid (15)
were prepared via a modified Hofmann rearrangement36 with
orthogonally protected L-glutamine and L-asparagine, respec-
tively (Scheme 1).
To prepare the building blocks of the unnatural amino acids
10 and 11, commercially available trityl-protected glutamine
(17) and asparagine (18) were reacted with 1,1-carbonyldiimi-
dazole (CDI) in the presence of benzyl alcohol to afford the
corresponding benzyl esters in 91 and 85% yield, respectively.
Deprotection of the trityl protecting group using trifluoroacetic
acid (TFA) furnished formamides 19 and 20 in quantitative
yields. A modified Hofmann reaction using bis(trifluoroacetoxy-
iodo)benzene (PIFA) produced the diaminobutanoic and diami-
(35) Eniade, A.; Murphy, A. V.; Landreau, G.; Ben, R. N. Bioconjugate
Chem. 2001, 12, 817–823.
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J. K.; Boutin, R. H. J. Org. Chem. 1984, 49, 4272–4276. (b) Zhang,
L.; Kaufmann, G. S.; Pesti, J. A.; Yin, J. J. Org. Chem. 1997, 62,
6918–6920. (c) Schmuck, C.; Geiger, L. Chem. Commun. 2005, 772–
774.
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(33) Rao, B. N.; Bush, C. A. Biopolymers 1987, 26, 1227–1244.
(34) Eniade, A.; Ben, R. N. Biomacromolecules 2001, 2, 557–561.
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Solution Conformation of C-Linked AFGP Analogues
A R T I C L E S
Figure 3. RI activity of 5.5 µM solutions of native AFGP-8 (1), C-linked AFGP analogues 3-7, 23, and 24, and the PBS control solution.
nopropanoic amino acid derivatives 14 and 15 in 64 and 72%
yield, respectively. These were immediately purified and coupled
to pyranose derivative 16 to furnish the respective glycocon-
jugates 21 and 22 in 70 and 80% yield. Selective deprotection
of the benzyl ester was accomplished by hydrogenolysis under
atmospheric pressure in 6 h to furnish building blocks 10 and
11 in 90-93% yield (based on recovered starting materials).
Evaluation of Antifreeze Activity of C-Linked AFGP
Analogues 4-7. C-Linked AFGP analogues 4-7 were assessed
for thermal hysteresis activity. All of the analogues failed to
demonstrate any TH activity using a Clifton nanoliter osmom-
eter. Ornithine analogue 5, possessing a three-carbon side chain
between the polyamide backbone and the amide bond, exhibited
weak dynamic ice shaping and produced single ice crystals with
hexagonal morphology, indicating a weak interaction with the
ice surface.4,5 Analogues 6 and 7 did not show any dynamic
ice shaping, suggesting that there is no interaction with the ice
lattice or the quasi-liquid layer of ice.
Glycoconjugates 4-7 were also assessed for their ability to
inhibit ice recrystallization (Figure 3). In these measurements,
native AFGP-8 (1) was used as a positive control for IRI activity,
while phosphate buffered saline (PBS) represented a negative
control. It has been previously shown that a sample concentra-
tion of 5.5 µM is optimal within the dynamic range for this IRI
assay.9,13 Consequently, all of the samples were tested at a
concentration of 5.5 µM. The vertical axis in Figure 3 represents
the mean largest ice crystal size, where larger values indicate
larger ice crystals and less inhibition of ice recrystallization.
Our results show that lysine analogue 4 comprising only four
repeating tripeptide units exhibited no TH or IRI activity. This
was not surprising, as glycopolymer 4 contains two fewer
repeating tripeptide units than 2.11 However, shortening the side
chain of 4 by one carbon atom (to give ornithine analogue 5)
resulted in a dramatic increase in IRI activity. This trend is
consistent with speculation that positioning the carbohydrate
moiety in close proximity to the polyamide backbone (akin to
native AFGP 8 and C-linked AFGP analogue 3) may impose
constraints on the orientation of the carbohydrate moiety in the
glycopeptide, resulting in increased activity. However, we were
surprised that analogues with even shorter side chains than 5
(i.e., 6 and 7) exhibited no IRI activity despite having apparently
closer proximities to the polypeptide backbone. We subsequently
hypothesized that the IRI activity of 5 might be the result of a
dramatic change in solution conformation. Consequently, the
solution conformations of analogues 4-7 were studied using
CD and VT 1H NMR spectroscopy along with MD simulations.
Assessing the Solution Conformation of C-linked Analogues
4-7 Using CD and VT NMR Spectroscopy. The solution con-
formation of native AFGP has been extensively studied using
a variety of spectroscopic17-19,37-42 and computational17-20,43
techniques. Previous spectroscopic studies have shown incon-
sistent results. Vacuum UV CD37 and H NMR experiments
have shown the dominant conformation to be a threefold left-
handed helix, whereas quasi-elastic light scattering (QELS)
studies39 have suggested an extended coil; other dynamic light
scattering (DLS),40 CD,40,41 and 13C NMR42 studies have
suggested that AFGP-8 predominantly exists as a random coil
in solution.
The solution conformations of C-linked AFGP analogues 3,
5-7, 23, and 24 were analyzed using CD spectroscopy (Figure
4). Previous studies have demonstrated that the long-range
solution conformation of native AFGP does not change as a
function of temperature. Consequently, all of the CD measure-
ments were performed at 22 °C.40 The results presented in Figure
4 suggest that all of the glycopolymers possess similar solution
conformations on the time scale of CD spectroscopy and that
the predominant solution conformation is that of a random coil.
This finding was consistent with recent CD and NMR studies
38
1
(37) Bush, C. A.; Feeney, R. E.; Osuga, D. T.; Ralapati, S.; Yeh, Y. Int. J.
Pept. Protein Res. 1981, 17, 125–129.
(38) (a) Bush, C. A.; Feeney, R. E. Int. J. Pept. Protein Res. 1986, 28,
386–397. (b) Bush, C. A.; Ralapati, S.; Matson, G. M.; Yamasaki,
R. B.; Osuga, D. T.; Yeh, Y.; Feeney, R. E. Arch. Biochem. Biophys.
1984, 232, 624–631.
(39) Ahmed, A. I.; Feeney, R. E.; Osuga, D. T.; Yeh, Y. J. Biol. Chem.
1975, 250, 3344–3347.
(40) Bouvet, V. R.; Lorello, G. R.; Ben, R. N. Biomacromolecules 2006,
7, 565–571.
(41) (a) Filira, R.; Biondi, L.; Scolaro, B.; Foffani, M. T.; Mammi, S.;
Peggion, E.; Rocchi, R. Int. J. Biol. Macromol 1990, 12, 41–49. (b)
Franks, F.; Morris, E. R. Biochem. Biophys. Acta 1978, 540, 346–
356. (c) Raymond, J. A.; Radding, W.; DeVries, A. L. Biopolymers
1977, 16, 2575–2578.
(42) Berman, E.; Allerhand, A.; DeVries, A. L. J. Biol. Chem. 1980, 255,
4407–4410.
(43) Tachibana, Y.; Fletcher, G. L.; Fujitani, N.; Tsuda, S.; Monde, K.;
Nishimura, S. Angew. Chem., Int. Ed. 2004, 43, 856–862.
9
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A R T I C L E S
Tam et al.
Figure 4. CD spectra and deconvolution data for native AFGP-8 and C-linked analogues 3, 5-7, 23, and 24 dissolved in doubly distilled water at 22 °C.
Solution conformation populations were estimated using IBASIS 5.
of AFGP,40-42 which indicated that the long-range conformation
of these C-linked AFGP analogues does not appear to be very
different than that of native AFGP-8. More importantly, this
result suggests that the apparent loss of IRI activity observed
for 6, 7, 23, and 24 is not due to a dramatic change in long-
range solution conformation. However, while CD spectroscopy
can provide useful insight into the solution structure of a protein,
it cannot provide information about the spatial relationship
between the carbohydrate residue and the polypeptide backbone
or verify the existence of nonbonding interactions between the
carbohydrate moiety and the polypeptide backbone. Such
interactions may be important for antifreeze activity, as it has
been suggested that they may orient the disaccharide of native
hydrogen bonds with temperature coefficients, Cierpicki and
Otlewski45 examined the temperature coefficients of 793 amide
bonds from 14 proteins in H2O/D2O, and by comparison with
existing X-ray and NMR data, they determined that values more
positive than -4.6 ppb/°C are indicative of the amide proton
being involved in an intramolecular hydrogen bond 70-98%
of the time.
Truncated monomeric tripeptide model systems (26-28) were
used in our analysis, since it has previously been suggested that
AFGPs are structurally segmented into semirigid tripeptide
units.20 Furthermore, the use of such model systems in similar
experiments is well-precedented and greatly simplifies the
analysis.25 As a positive control, native AFGP-8 was also
analyzed;47 temperature coefficients of the C2 N-acetylgalac-
tosamine N-H proton ranged from -4.6 to -5.5 ppb/°C for
glycoconjugate residues that are adjacent to only alanines and
AFGP-8 relative to the backbone.20,25,38 For instance, VT H
1
NMR studies by Mimura et al.25 suggested the existence of
intramolecular hydrogen bonds between the amide proton of
N-acetylgalactosamine (GalNHAc) and the carbonyl oxygen of
threonine in model systems of native AFGP-8. It was suggested
that this hydrogen-bonding interaction significantly restricted
the orientation of the carbohydrate moiety relative to the
polypeptide backbone and facilitated interaction with the ice
lattice. While C-AFGP analogues 4-7 do not possess a C2
acetamide residue in the carbohydrate, we hypothesized that a
similar interaction might exist between the side-chain amide
and the polypeptide backbone in analogue 5. If so, the
carbohydrate residue might be ideally positioned to interact with
the ice lattice, and this favorable interaction may not be present
17,25
1
correlated well with previous H NMR data for AFGPs.
1
Figure 5 shows the partial H NMR spectra of monomer
model systems 26-28. Amide protons were assigned using two-
dimensional (2D) NMR correlation spectroscopy (COSY) (see
the Supporting Information for the full 1H and 2D COSY NMR
data). The temperature coefficients for the three monomers
ranged from -6.3 ( 0.6 to -7.9 ( 0.9 ppb/°C, which is
significantly more negative than the threshold of -4.6 ppb/°C,
suggesting that no strong intramolecular hydrogen bonds persist
between the side chains and the peptide backbone on the NMR
time scale. Furthermore, these results do not show any significant
trends between temperature coefficient of each monomer and
the IRI activity of the respective polymer. This implies that the
change in IRI activity as a function of side-chain length in 5-7
1
in the other analogues. To investigate this possibility, VT H
NMR studies were performed on 26-28, model systems for
C-AFGP analogues 5-7. In this technique, amide protons that
participate in intramolecular hydrogen bonds exhibit a smaller
change in chemical shift as the temperature is increased.44-46
This temperature-dependent change in chemical shifts is known
as the temperature coefficient and is represented by dδ/dT, where
δ is the chemical shift and T is the temperature (in °C or K).
To quantitatively correlate the strength of intramolecular
(44) Ohnishi, M.; Urry, D. W. Biochem. Biophys. Res. Commun. 1969,
36, 194–202.
(45) Cierpicki, T.; Otlewski, J. J. Biomol. NMR 2001, 21, 249–261.
(46) Baxter, N. J.; Williamson, M. P. J. Biomol. NMR 1997, 9, 359–369.
(47) Native AFGP-8 was generously donated by AquaBounty Farms Inc.
and has the amino acid sequence described in ref 17.
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Solution Conformation of C-Linked AFGP Analogues
A R T I C L E S
1
Figure 5. VT H NMR (500 MHz) spectra and temperature coefficients (ppb/°C) of amide protons of truncated monomer model systems 26-28 in 95:5
H2O/D2O with DSS as an internal standard.
Figure 6. (A) Atom and torsion angle definitions used for model AFGP analogue tripeptides 25-28. (B) Summary of the statistical analysis of the MD
simulations, showing % intramolecular hydrogen-bonding occupancy, average CR-C1 distance (Å), and free energy of isomerization from chair (4C1) to
skew-boat (1S3) conformations of tripeptides 25-28 and native AFGP-8.
is not due to strong intramolecular hydrogen bonds between
the side chain and the polypeptide backbone as previously
hypothesized. However, this result does not rule out the
possibility that the carbohydrate moiety of 5 adopts a unique
spatial orientation relative to the polypeptide backbone. Con-
sequently, we modeled the aqueous-phase conformational
dynamics of C-AFGP analogues 4-7 and their respective model
systems 25-28 using MD simulations.
significant structural differences among the conformations
adopted by 25-28. It should be noted that Corzana and co-
workers48 recently reported that a direct intramolecular hydrogen
bond between the N-H donor of an N-acetylgalactosamine and
the threonine peptide backbone carbonyl acceptor in an AFGP
analogue was minimal in the solution conformation and that
the unique conformation of the N-acetylgalactosamine-threo-
nine glycoconjugate was strongly influenced by the formation
of water bridges between the carbohydrate and the amino acid.
Our MD simulations did reveal that strong intramolecular
hydrogen-bond interactions existed between the O4 and O6
hydroxyls of galactose and that the amount of hydrogen bonding
varied as a function of side-chain length in C-AFGP analogues
25-28 (Figure 6B). Persistent intramolecular hydrogen bonds
existed between O4 and O6 for analogues 25, 27, and 28 (none
of which possesses potent IRI activity), while lower percentages
of hydrogen bonding (weaker bonds) were observed to exist
Molecular Dynamics Simulations of C-linked AFGP Analogues
25-28. Monomeric tripeptides 25-28 were modeled in the
1
presence of water to corroborate the VT H NMR studies and
determine whether key conformational differences between RI-
active (5) and -inactive (4, 6, 7) C-linked AFGP analogues exist.
The presence of intramolecular hydrogen bonding was examined
using AMBER’s ptraj program and failed to identify any
persistent intramolecular hydrogen bonds between the amide
protons and other hydrogen bond acceptors (see the Supporting
Information for full analysis). This result correlates with the
results of the VT NMR studies described previously. While the
existence of weak intramolecular hydrogen bonds between
the galactose residue and the backbone were found, the
occupancy of these interactions was too low to account for any
(48) (a) Corzana, F.; Busto, J. H.; Jime´nez-Ose´s, G.; Asensio, J. L.; Jime´nez-
Barbero, J.; Peregrina, J. M.; Avenoza, A. J. Am. Chem. Soc. 2006,
128, 14640–14648. (b) Corzana, F.; Busto, J. H.; Jime´nez-Ose´s, G.;
Garc´ıa de Luis, M.; Asensio, J. L.; Jime´nez-Barbero, J.; Peregrina,
J. M.; Avenoza, A. J. Am. Chem. Soc. 2007, 129, 9458–9467.
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A R T I C L E S
Tam et al.
Figure 7. (A) Free-energy profiles for the ꢁ1 torsional angle in 25-28. (B) Free-energy profile for the ꢁ2 and ꢁ3 torsional angles in 26. Global minima at
(ꢁ2 ) -52.8°, ꢁ3 ) -57.6°) for 26 (white box) and the higher-energy trans conformation (red box) are indicated. (C) Free-energy profiles for the Ψs
torsional angle in 25-28.
between O4 and O6 in analogue 26, which is a potent inhibitor
of ice recrystallization. A similar trend was also observed when
we examined the two carbohydrate residues of the native
AFGP-8 system. This result implies that the interaction of O4
and O6 with water molecules adjacent to the glycoprotein (as
opposed to interaction with each other) may be an important
requirement for IRI activity. Furthermore, the relative stereo-
chemistry of the O4 hydroxyl has previously been shown to be
an important factor in modulating the hydration environment
of the carbohydrate, and our laboratory has verified that the
degree of hydration is directly related to the IRI activity.9,10,49,50
The ΦR/ΨR torsional distributions for 25-28 were calculated
and suggest that these four peptides adopt similar polypeptide
backbone conformations (see Figure S-1 in the Supporting
Information). Thus, we investigated the relationship between
the orientations of the carbohydrate moiety and the backbone
as a function of IRI activity. This was done by examining the
torsion angles in the side chain [ꢁ1-ꢁn (n ) 1-4) and Ψs] and
the average distance from CR of the glycosylated amino acid
residue to C1 of the carbohydrate (Figures 6B and 7).
The orientation of the side chain relative to the backbone
depends on the rotation with respect to the ꢁ1 torsional angle.
To quantify this interaction, free-energy profiles for rotation with
respect to the ꢁ1 torsional angle were calculated for 25-28
(Figure 7A). The energy barrier for rotation with respect to ꢁ1
is greater for 26 than for 25, 27, and 28, indicating that this
rotation is much more restricted in 26.
Analysis of the MD trajectories revealed the tendency of the
ꢁ2 and ꢁ3 angles in the alkyl chain of 26 to adopt a [gauche(-),
gauche(-)] {denoted as [g(-),g(-)]} conformation and that the
ꢁ3 and ꢁ4 angles in the longer alkyl chain of 25 adopt the (trans,
trans) [denoted as (t,t)] conformation (Figure 7B). The [g(-),g(-)]
torsion angles for ꢁ2 and ꢁ3 of 26 cause the carbohydrate to be
oriented almost parallel to the backbone, forming a hydrophobic
pocket with the alkyl chain and the peptide backbone. Water
molecules appear to be excluded from this pocket, increasing
the conformational stability. This is consistent with the increased
energy barrier for rotation with respect to ꢁ1 in 26. This
hydrophobic pocket is not observed in 25, 27, or 28, and
consequently, the alkyl side chains in these analogues extends
in a trans fashion into the solution, resulting in greater freedom
of rotation with respect to the ꢁ1 torsional angle and less
conformational stability. Restraining the ꢁ3 and ꢁ4 torsional
angles of 25 to [g(-),g(-)] allows this analogue to adopt a
conformation in which a hydrophobic pocket similar to that in
26 is formed, but at an increase of 1-2 kcal mol-1 in free
energy.
The orientation of the carbohydrate residue relative to the
backbone was determined on the basis of the Ψs torsional angle
in the C-glycosidic bond (Figure 7C). Rotation with respect
to Ψs exposes different faces of the carbohydrate to the solvent.
The hydrophobic contact formed in 26 favors Ψs in the range
60 to 180°. For Ψs in the range -180 to 0°, the free energy of
26 is 0.5-2.0 kcal mol-1 higher than for 25, 27, and 28. Thus,
as a result of the different side-chain conformations attributed
to their varying lengths, the orientation of the carbohydrate
relative to the peptide backbone in 26 is significantly different
than in the other three analogues.
Analysis of the side-chain torsional angles in 25-28 permitted
calculation of the distance between C1 of the carbohydrate and
CR of the glycosylated amino acid (Figure 6B) in each analogue.
Monomers 26-28 have similar CR-C1 distances of 5.59, 6.15,
and 5.81 Å, respectively. However, this distance is significantly
longer in 25 (8.03 Å). It was initially anticipated that the increase
in the number of carbons in the side chain would result in a
proportional increase in the CR-C1 distance. However, our
results show that analogue 26, a potent inhibitor of ice
recrystallization, deviates significantly from this trend. We
believe this is due to the fact that the side-chain conformation
permits formation of a hydrophobic pocket, so the side chain is
“folded back” on itself. A representative “snapshot” of this
configuration in 26 is shown in Figure 8.
The free-energy profiles for the various torsional angles of
the carbohydrate ring were examined using the weighted
histogram analysis method (WHAM).51 While the lowest-energy
conformation of 26-28 was a skew-boat (1S3), 25 was calculated
4
to have a C1 chair conformation (Figure 6B). The importance
of skew-boat conformations with respect to carbohydrate
isomerization has been studied in a series of recent papers.52,53
In many carbohydrates, the skew-boat conformation has been
found to be up to 4 kcal mol-1 less stable than the more stable
chair conformation.53 It is highly likely that the lack of an
(51) Kumar, S.; Rosenberg, J. M.; Bouzida, D.; Swendsen, R. H.; Kollman,
P. A. J. Comput. Chem. 1995, 16, 1339–1350.
(52) (a) Biarnes, X.; Ardevol, A.; Planas, A.; Rovira, C.; Laio, A.;
Parrinello, M. J. Am. Chem. Soc. 2007, 129, 10686–10693. (b) Ionescu,
A. R.; Berces, A.; Zgierski, M. Z.; Whitfield, D. M.; Nukada, T. J.
Phys. Chem. A 2005, 109, 8096–8105.
(49) Galema, S. A.; Høiland, H. J. Phys. Chem. 1991, 95, 5321–5326.
(50) Dashnau, J. L.; Sharp, K. A.; Vanderkooi, J. M. J. Phys. Chem. B
2005, 109, 24152–24159.
(53) Kra¨utler, V.; Mu¨ller, M.; Hu¨nenberger, P. H. Carbohydr. Res. 2007,
342, 2097–2124.
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Solution Conformation of C-Linked AFGP Analogues
A R T I C L E S
co-workers,54 who reported that upon cold-induced peptide
denaturation, peptides are able to maintain their hydration state.
This is relevant to our work, as our IRI assays were performed
at subzero temperatures. As such, any contribution of carbo-
hydrate hydration to hydration of the peptide at temperatures
above 0 °C should be consistent at temperatures below 0 °C.
Conclusion
This paper has described the synthesis of several C-linked
AFGP analogues bearing amide-containing side chains having
varying lengths between the carbohydrate moiety and polypep-
tide backbone. Analogue 5 (containing a three-carbon side chain)
is a potent inhibitor of ice recrystallization, while analogues 4,
6, and 7 (containing four-, two-, and one-carbon side chains,
respectively) possess no IRI activity. Analysis of solution
conformation by circular dichroism spectroscopy failed to
indicate any significant conformation changes that might account
for the observed difference in activity. Consequently, the
formation of intramolecular hydrogen bonds between the
polypeptide backbone and the side chain was investigated, but
these studies failed to confirm the presence of strong intramo-
lecular hydrogen bonds on the NMR time scale. However,
detailed molecular dynamics simulations performed in water
indicated that monomer 26 (a model for C-linked AFGP
analogue 5) in solution adopts a unique conformation in which
the carbohydrate moiety does not extend away from the
polypeptide backbone into the surrounding solution; instead, the
side chain is folded back upon itself, forming a hydrophobic
“pocket” between the carbohydrate and the backbone. This
appears to be a highly favorable carbohydrate orientation for
interaction with the quasi-liquid layer of the ice lattice, resulting
in potent IRI activity. The other C-linked AFGP analogues
examined in this study did not adopt such conformations in
solution and failed to exhibit IRI activity. We further speculate
that formation of this hydrophobic pocket may influence the
hydration shell of the carbohydrate (and subsequently of the
whole glycopeptide), and this may also contribute to the ob-
served IRI activity. This work further illustrates the importance
of hydration and its influence in biological systems.
Figure 8. Snapshots from unrestrained MD simulations of minimum-energy
conformations of 25-28. The IRI-potent analogue 26 is highlighted by the
red box.
electronegative anomeric oxygen in these C-linked carbohydrate
derivatives may cause the skew-boat and chair conformations
to be closer in energy. If the energy difference is small enough,
as seen in our simulations, the two conformations may readily
interconvert. Hence, it is not clear whether the observed increase
in IRI activity for 26 is also attributed to a subtle difference in
the conformation of the carbohydrate residue.
The overall lowest-energy conformations of the various
analogues, which take into account the contributions from the
various torsion angles, CR-C1 distances, and inter/intramo-
lecular hydrophobic/hydrophilic interactions, are shown in
Figure 8. From this figure, it is evident that there is a drastic
conformational change between the IRI-active analogue 26 and
the IRI-inactive analogues 25, 27, and 28. Analogues 25, 27,
and 28 have an extended alkyl side chain, which subsequently
leads to a carbohydrate that has all of its faces freely exposed
to the bulk solvent. This may be due to (1) the increased
geometric constraints imposed in the shorter analogues 27 and
28, which prevent the carbohydrate from folding back on its
own backbone, and (2) the increased flexibility of the longer
analogue 25. In contrast, 26 contains a folded side chain that
positions the more hydrophilic face of the carbohydrate toward
the peptide backbone and away from the bulk solvent. We
believe this is important, as it drastically alters the hydration
shell of the overall glycoconjugate and hence of the peptide.
While the conformation of the carbohydrate moiety may also
influence hydration, it is likely that carbohydrate stereochem-
istry9 and exposure to bulk solvent are the predominant factors.
The influence of carbohydrate stereochemistry on peptide
hydration is unknown at this point. However, the importance
of peptide hydration was recently examined by Davidovic and
Acknowledgment. The authors acknowledge the Natural Sci-
ences and Engineering Research Council (NSERC), Canadian
Institutes of Health Research (CIHR), Canadian Foundation for
Innovation (CFI), Ontario Research Fund, and IBM Canada for
computing resources for financial support as well as Dr. Glenn
Facey for assistance with the VT NMR experiments. T.K.W. holds
a Tier 2 Canada Research Chair (CRC) in Catalysis Modeling and
Computational Chemistry.
Supporting Information Available: Procedures and charac-
terization data for all new compounds, structural assignments,
MD simulation data, and complete ref 27a (as SI ref 4). This
material is available free of charge via the Internet at http://
pubs.acs.org.
JA904169A
(54) Davidovic, M.; Mattea, C.; Qvist, J.; Halle, B. J. Am. Chem. Soc.
2009, 131, 1025–1036.
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