The solution structure reveals an a helix bent in the same direction as the more bent conformer of the published crystal structure of TTTT, while the side chain v1rotamers of VVVV2KE are
Trang 1Solution structure of a hydrophobic analogue of the winter flounder antifreeze protein
Edvards Liepinsh1, Gottfried Otting1, Margaret M Harding2, Leanne G Ward2, Joel P Mackay3
and A D J Haymet4
1
Karolinska Institute, Tomtebodava¨gen, Stockholm, Sweden;2School of Chemistry, University of Sydney, NSW, Australia;
3
Department of Biochemistry, University of Sydney, NSW, Australia;4Department of Chemistry and Institute for Molecular Design, University of Houston, TX, USA
The solution structure of a synthetic mutant type I antifreeze
protein (AFP I) was determined in aqueous solution at
pH7.0 using nuclear magnetic resonance (NMR)
spectro-scopy The mutations comprised the replacement of the four
Thr residues by Val and the introduction of two additional
Lys-Glu salt bridges The antifreeze activity of this mutant
peptide, VVVV2KE, has been previously shown to be
sim-ilar to that of the wild type protein, HPLC6 (defined here as
TTTT) The solution structure reveals an a helix bent in the
same direction as the more bent conformer of the published
crystal structure of TTTT, while the side chain v1rotamers of
VVVV2KE are similar to those of the straighter conformer
in the crystal of TTTT The Val side chains of VVVV2KE
assume the same orientations as the Thr side chains of TTTT, confirming the conservative nature of this mutation The combined data suggest that AFP I undergoes an equi-librium between straight and bent helices in solution, com-bined with independent equilibria between different side chain rotamers for some of the amino acid residues The present study presents the first complete sequence-specific resonance assignments and the first complete solution structure determination by NMR of any AFP I protein Keywords: antifreeze; a helices; proteins; winter flounder; NMR spectroscopy
During the last two decades, at least four classes of
structurally diverse antifreeze or thermal hysteresis proteins
(type I–IV AFPs) have been isolated from the serum of cold
water fish (for reviews see [1–6]) These compounds have in
common the ability to lower the freezing point of blood
serum, thus allowing fish to survive in subzero ocean
temperatures While some progress has been made in the
structural characterization of these proteins [7–10], the exact
mechanism by which they are able to inhibit ice growth is
not fully understood
Most studies have focused on the type I antifreeze
proteins [5], which are structurally the simplest members of
the AFPs Fourteen type I proteins have been identified
from the right-eye flounders and sculpins [5], and these
proteins are characterized by being low Mr, alanine rich,
a helical structures Within this class, HPLC6 (TTTT) [11], a
37-residue sequence containing three 11-residue repeats of
ThrX2AsxX7is by far the most extensively studied protein
and is the only type I AFP for which a solid state structure
has been reported Single X-ray diffraction [8,12] showed
that in the solid state this protein is completely a helical in
conformation with the exception of the last unit, which
adopts a 310-helix conformation The protein has also been studied by NMR spectroscopy [13] but due to the high number of alanine residues in the sequence, which led to significant spectral overlap, full resonance assignments were not possible These studies confirmed the global helical conformation of the peptide and allowed the rotamer conformations of a number of residues to be determined, but clear evidence for the presence of helix-stabilizing interactions arising from the capping motifs observed in the crystal structure was not obtained More recently, meas-urements of chemical shifts and rotational correlation times
of TTTT in supercooled water [14] showed no evidence for any structural change in the protein at temperatures below the freezing point
Structure–activity studies on TTTT, summarized previ-ously [5], have identified the importance of the Thr residues
at positions 2, 13, 24 and 37 (highlighted in bold in the sequence in Table 1), plus surrounding residues, for ice growth inhibition activity While the Thr residues were assumed to be involved in hydrogen-bonding interactions with ice for many years [15–18], more recent mutations [19–23] have identified the hydrophobicity provided by the c-methyl group of Thr, in addition to hydrogen bonding involving other residues, as a key factor related to the ability
to inhibit ice growth However, a plausible model that explains the selective interaction of TTTT with the [2 0 22 1] plane [15] has not emerged (for a full description of the different ice interfaces, see [5]) Recent computational studies on the nature of the ice/water interface have allowed the first real simulations of the interaction of TTTT with the fluid interface to be carried out [24] These studies support experimental data on mutants [19–23] that have shown that
Correspondence to M M Harding, School of Chemistry, F11,
Uni-versity of Sydney, N.S.W 2006, Australia Fax: + 61 29351 6650,
Tel.: + 61 29351 2745, E-mail: harding@chem.usyd.edu.au
Abbreviations: AFP I, type I antifreeze protein; NMR, nuclear
magnetic resonance; TTTT, HPLC6 polypeptide; NOE, nuclear
Overhauser effect; AU, analytical ultracentrifugation.
(Received 28 September 2001, revised 21 December 2001, accepted
7 January 2002)
Trang 2hydrogen bonds involving the hydroxyl groups of the four
Thr residues is not the primary reason for the interaction of
TTTT with the ice/water interfacial region
We have recently designed and synthesized analogues of
TTTT in which the relative size, hydrophobicity and
hydrogen bonding characteristics of the side chains at
positions 2, 13, 24 and 37 were systematically varied [21,22]
Four additional charged residues K7, E11, K29 and E33
(italicized in the VVVV2KE sequence shown in Table 1)
were incorporated into the sequence to improve solubility
and minimize aggregation The valine-substituted analogue
VVVV2KE showed similar behaviour to TTTT at low
concentrations [22] and showed conclusively that models for
the mechanism of ice growth inhibition that are dominated
by hydrogen bonding involving the Thr hydroxyls are
incorrect
This paper reports determination of the solution structure
of VVVV2KE The additional charged residues in this
sequence provided chemical shift dispersion in the
alanine-rich segments compared with TTTT and thus allowed the
first solution structure of a type I protein to be determined
Such experimental solution data are important in modelling
the interaction of these peptides with the ice/water interface,
in order to provide a mechanism for the selective interaction
of the peptide with the [2 0 22 1] ice plane, and hence to
allow the rational design of synthetic AFPs
M A T E R I A L S A N D M E T H O D S
Materials
VVVV2KE was obtained and purified as previously
described [20,22] Sample concentrations were determined
by amino-acid analysis NMR samples were prepared in
unbuffered 90% H2O/10% D2O at concentrations of
11 mM (pH4.9) and 2 mM (pH7.0) The 2-mM sample
was desalted by ultrafiltration
NMR spectroscopy, collection of conformational
restraints and structure calculation
NMR spectra were recorded on Bruker DMX-600 and
Varian Unity INOVA-800 NMR spectrometers The
NOESY spectrum used for collection of NOE distance
restraints was recorded at 10C on the 800-MHz NMR
spectrometer, using the 2-mM sample This spectrum was
recorded with a mixing time of 80 ms, using the 3-9-19
sequence for water suppression [25] In addition, NOESY,
ROESY, TOCSY and DQF-COSY spectra were recorded
using the 11-mM sample at temperatures between )8 C
and 15C to support the resonance assignment and check
for conformational differences The in-phase lineshape of
NOESY cross peaks was used to determine JHN,Hacoupling
constants [26] The COSY and TOCSY cross-peaks were
visually inspected to determine the relative magnitude of the
J couplings of CbH methylene groups The ROESY
spectrum at 15C (mixing time 50 ms) was used to identify spin-diffusion cross-peaks in the NOESY spectrum recor-ded with an 80-ms mixing time The programXEASYwas used for resonance assignments and peak integration [27] DYANA [28] and OPAL [29] were used for the structure calculations and energy minimization, respectively Stand-ard parameters were used for both programs The energy minimization was performed in a water shell of 6 A˚ Hydrogen bonds were identified by O Æ Æ Æ Hdistances
< 2.4 A˚ and internuclear O Æ Æ Æ H-N angles < 35 Plots of the structure were prepared withMOLMOL[30]
Accession numbers The coordinates of the 20 energy-refinedDYANAconformers
of VVVV2KE and the resonance assignments were depos-ited in the Protein Data Bank with the accession code 1K16 The NMR chemical shifts were deposited at the Bio-MagResBank (BMRB) under the accession code 5157
Analytical ultracentrifugation (AU) Sedimentation experiments were performed on a Beckman XL-A analytical ultracentrifuge VVVV2KE was dissolved
in 50 mMKH2PO4(pH8.0) containing 50 mMKCl, to give initial loading concentrations of 1.0, 0.3 and 0.1 mM Sample aliquots (200-lL) were loaded into 12-mm double-sector cells, and data were collected at 0C in an An-60ti rotor (45 000 and 54 000 r.p.m.) Data were acquired as absorbance vs radius scans (at 240 and 360 nm) at 0.001-cm intervals and as the sum of 10 scans Data were collected at 3-h intervals and compared to determine when the samples had reached chemical and sedimentation equilibrium After subtraction of the 360-nm scans, the data from all speeds and loading concentrations were fitted simultaneously to a number of models using the program NONLIN [31]; the quality of each fit was determined by inspection of residual plots and v2values Visualization of the plots of apparent molecular mass vs concentration andW vs concentration was carried out using the programOMMENU[32]
R E S U L T S
Analytical ultracentrifugation Figure 1 shows the results of AU experiments on VVVV2KE at three concentrations, including the fitted curves obtained using an ideal single species model The combined residuals of the fit are presented in the bottom panel of Fig 1 The derived molecular mass for the peptide shows that in the concentration range less than 1 mM, and under the conditions used for these measurements, the major species present in all cases is the monomeric peptide The peptide also appears to be monomeric at 2 mM concentration (the concentration used for NMR structure determination), as no significant chemical shift changes were
Table 1 Sequence alignment of TTTT and VVVV2KE.
Trang 3observed in the concentration range 0.1–2 mM(measured at
5C and pH3.55)
NMR spectroscopy and structure calculation
Significant spectral overlap prevented the resolution of all
cross peaks In particular, the chemical shifts of residues
Glu11 and Ala14 were practically indistinguishable from
those of Glu22 and Ala25 (Table 2) Yet, the appearance of
the 800-MHz NOESY spectrum shows that, at least for the
amide protons, the degeneracy is not complete (Fig 2A)
Sequential connectivities between amide protons can be
traced from Ala3 to Ala36 without interruption (Fig 2A),
indicating a helical conformation The spectral overlap is
more severe for the resonances of the aliphatic protons
Nevertheless, many of the nuclear Overhauser effects
(NOEs) characteristic of a helical secondary structure could
be resolved (Fig 2B)
In the case of strongly overlapping cross peaks, as
observed for example for the homologous repeats Glu11–
Ala14 and Glu22–Ala25, upper distance limit restraints
were derived using the assumption that corresponding
NOEs from the different segments contributed equally to
the overlapping cross peak intensity Similarly, the same
dihedral angle restraints were used for homologous repeats,
when the corresponding COSY cross peaks overlapped, but
their assignment was otherwise unambiguous The use of
identical restraints for homologous, spectrally unresolved
peptide segments was motivated by the observation of
similar NOEs and coupling constants, when cross peaks
between homologous repeats could be resolved
NOEs with the terminal amino-acid residues were very
weak, presumably due to increased mobility Therefore, the
set of upper distance restraints of residues 2 and 37 was
supplemented by restraints obtained from the ROESY spectrum recorded at 15C and a much higher sample concentration Furthermore, a hydrogen bond between the carboxyl group of Asp1 and the amide proton of Ser4 was indicated by the observation of a large high-field shift of this
HNresonance when the pHwas lowered to pH2 (data not shown) [33,34] This hydrogen bond seems to be highly populated at neutral pH, where the HNresonance of Ser4 is the most low-field shifted amide (Fig 2)
Solution structure of VVVV2KE The solution structure of VVVV2KE, represented by the ensemble of 20 energy-minimized DYANA conformers, consists of a bent a helix spanning the entire length of the peptide The most pronounced bend seems to occur near Lys18 While all residues were engaged in proper a helical backbone hydrogen bonds in the conformer closest to the average structure and in the conformer with the smallest residual violations, the hydrogen bond between Lys18 and Ala14 was broken in eight of the 20 NMR conformers In four of these, the carbonyl oxygen of Ala14 was hydrogen bonded to the amide proton of Ala17 instead A straight helix around Lys18 was obtained in test calculations, and a short distance restraint was artificially introduced between Ala14 Haand Lys19 HN, at the expense of an increased number of distance-restraint violations in the resulting conformers As the corresponding i/i + 4 NOE was, however, absent (Fig 2B), it was not used in the final calculations
Temperature coefficients measured for the amide-proton chemical shifts between 5 and )5 C did not show any irregularities for these lysine residues Twofold to threefold larger values than average were, however, observed for the
HNchemical shifts of Ala15 and Ala26 (0.011 p.p.m perC between 5 and)5 C) and Asn16 and Asn27 (0.015 p.p.m perC) which might reflect conformational irregularities at these locations of the a helix While local flexibility would necessarily affect the amplitude and precise direction of the helical bend calculated from NOE data, a bend of the helix seems to be a genuine feature of VVVV2KE The hydrogen bond between the side chain of Asp1 and the backbone amide of Ser4 results in the presence of an N-cap (Fig 3B) When this hydrogen bond was removed from the list of restraints, it was found only in a minority of the conformers The presence of this hydrogen bond was, however, strongly supported by the chemical shift changes observed in the pH titration and it was consequently included as a restraint The chemical shift of Ser4 HNshowed the largest temperature coefficient of all amide protons (0.017 p.p.m per C between 5 and)5 C), suggesting that this hydrogen bond
is particularly short or is readily broken at higher temper-atures In contrast, the experimental evidence for the presence of a well-defined C-cap, as in the crystal structure
of TTTT [8], was less clear Any NOEs involving the terminal residue Arg37 were weak, probably due to increased mobility, and the temperature coefficients of the chemical shifts of the C-terminal NH2group of Arg37 were too large to suggest any involvement in a stable hydrogen bond Yet, the temperature coefficients of the two NH2 protons were significantly different and smaller for the high-field shifted proton, which in the crystal structure of TTTT hydrogen bonds to the carbonyl oxygen of Thr35 [8]
Fig 1 Analytical ultracentifugation data for VVVV2KE at
concentra-tions of 1 m M (diamonds), 0.3 m M (squares) and 0.1 m M (circles) Top
panel shows fits of data to an ideal-single species model and bottom
panel shows residuals derived from this fit.
Trang 4Although no restraints were used for this NH2group in the
structure calculations of VVVV2KE, and Arg37 was largely
disordered (Fig 3B), most of the conformers formed the
corresponding hydrogen bond between Arg37 NH2 and
Val35 O
The amino-acid side-chains of VVVV2KE assumed the
same v1rotamer position in all 20 conformers, while different
rotamers were found beyond the b carbons Only the side
chain of Ser4 populated all three staggered v1rotamers
Comparison between the structures of VVVV2KE
and TTTT
The crystal structure of TTTT contains two conformers in
the unit cell that differ widely in their helical bend (Fig 4)
[8] In the following, we refer to the more bent conformer as
the b-conformer, and the less bent conformer as the
s-conformer Interestingly, the b- and s-conformers are bent in opposite directions The overall bend observed in the NMR structure of VVVV2KE is in the same direction as in the b-conformer, placing residues 2, 13, 24 and 37, that are putatively involved in ice-binding, on the concave surface The two conformers of TTTT also differ by the side chain
v1rotamers of several residues, namely Asp1, Leu12, Lys18, Leu23 and Thr35 Both conformers display the backbone hydrogen bonds expected for an a helix spanning all residues, and include elaborate terminal cap structures As with the NMR structure of VVVV2KE, the N-terminal cap structure of TTTT includes a hydrogen bond between the side chain carboxyl group of Asp1 and the backbone amide
of Ser4 The C-terminal cap structure, however, makes use
of the Arg37 side chain to form a hydrogen bond to the backbone carbonyl oxygen of Ala33 [12] No evidence of this could be obtained in solution Interestingly, the
Table 2 1 H-NMR chemical shifts of VVVV2KE at 10 °C, pH 7.0 The chemical shifts were referenced to the water signal at 4.994 p.p.m The estimated error is ± 0.01 p.p.m The chemical shift values of stereospecifically assigned protons are in italics, where the first number is the shift of the proton with the lower branch number, e.g the b 1 proton.
Residue
Chemical shift
NH 2 7.24, 7.27
Trang 5chemical shift difference between the1H-NMR resonances
of the C-terminal NH2group increased by about 0.1 p.p.m
as the temperature was lowered to)2 C (data not shown),
suggesting that a hydrogen bond between Arg37 NH2and
the carbonyl oxygen of residue 35 may be significantly
populated at low temperatures, in agreement with the
crystal structure of TTTT [8] In contrast to the crystal
structure of TTTT, where Arg37 HNis hydrogen bonded to
Ala34 O, this amide proton consistently formed a hydrogen
bond with Glu33 O in the NMR structure of VVVV2KE
This difference, however, is hardly significant, as Arg37 HN
and Glu33 O are also close in the crystal structure of TTTT
The side chain v1angles observed in the NMR structure
of VVVV2KE are very similar to those observed in the X-ray conformers of TTTT (Table 3) In particular, the side-chain orientations of the valine residues in VVVV2KE are equivalent to those of the Thr residues in TTTT; i.e the ThrfiVal mutation effectively resulted in the replacement
of the OHby a CH3group without affecting the position of the other CH3group Five residues have different rotamer positions in the two TTTT conformers Except for Asp1, the rotamers of these residues in VVVV2KE are similar to those
of the s-conformer of TTTT (Table 4) There is thus no simple correlation between helix bend and side-chain conformation
D I S C U S S I O N
The molecular mechanism whereby TTTT and other type I proteins are able to inhibit ice growth via accumulation at the specific [2 0 22 1] plane remains a continued subject of discussion in the literature [5,6,24,35–37] The first molecu-lar dynamics simulation of a complete ice/TTTT/water system, that does not restrict ice lattice positions, and includes long-range electrostatic interactions, has been reported very recently [24] This study has allowed a comparison of the hydrogen bonding between the protein in water and the protein in the ice/water interfacial region
A
B
Fig 2 Selected spectral regions from the NOESY spectrum of
VVVV2KE in 90% H 2 O/10% D 2 O at 10 °C, pH 7.0 The spectrum
was recorded at a 1 H-NMR frequency of 800 MHz, using a mixing
time of 80 ms Cross peaks are labelled with the residue numbers of the
amino acids involved The first/second number refers to the residue in
the d 1 /d 2 frequency dimension, respectively (A) Cross peaks between
backbone amide protons (B) Cross peaks between a-protons in the d 1
dimension and amide protons in the d 2 dimension i/i + 3 and i/i + 4
NOEs are identified, where i is the residue number in the amino acid
sequence A circle marks the predicted location of the NOE cross peak
between Ala14 Haand Lys18 HN, which could not be detected even at
much lower plot levels.
20
29
37
11
1
37 29 20 11
1
Fig 3 Stereo views of the solution structure of VVVV2KE (A) Super-position of the 20 conformers representing the NMR structure of VVVV2KE (left panel) and single conformer closest to the average structure (right panel) The line drawings include all heavy atoms a-Carbon positions are identified by spheres, and the location of approximately every tenth residue is labeled by its number in the amino acid sequence (B) Stereo views of the N-cap (left panel) and C-cap (right panel) in the NMR structure of VVVV2KE The backbone atoms of the first five and last six residues, respectively, were super-imposed for minimum r.m.s.d Only bonds with backbone atoms and backbone carbonyl atoms are displayed, except for the side chain of Asp1 The N- and C-terminal ends are identified and hydrogen bonds drawn with dotted lines The N-cap hydrogen bond between the carboxyl group of Asp1 and the backbone amide of Ser4 is identified in bold.
Trang 6In parallel, recent experimental data on mutants that
incorporate systematic changes in both hydrophobicity
and hydrogen bonding characteristics have assisted in
defining the characteristics of the residues that are crucial
for activity and has led to new proposals for the ice-binding
face of the protein [22,36,37] Further molecular dynamics
studies are required to explain these new experimental
results with mutants and to explain why TTTT recognizes
and accumulates at the {2 0 22 1} planes of ice 1h the usual
form of hexagonal ice at 1 atm
The starting point for almost all simulations to date
[16,18,24,35,38,39] has been the X-ray coordinates of TTTT
[12] The protein is assumed to adopt a very similar
geometry in solution, and NMR studies on TTTT are
consistent with an a helical geometry [13] Simulations of
VVVV2KE with the ice/water interface should provide
significant insight into the mechanism of ice-growth inhibi-tion, as this is the first example of an active mutant that lacks hydrogen bonding side chains at positions 2, 12, 24 and 35 While CD data are consistent with an a helical structure [22], and substitution of ThrfiVal would not be predicted to significantly alter the helical conformation, it is important to confirm that the side chain conformations are unaltered and that the absence of hydrogen bonding residues at the C- and N-terminus does not affect the capping network and overall conformation of the peptide The structure determination of the VVVV2KE mutant of AFP I in solution was made possible by the increased chemical shift dispersion afforded by the two additional Lys/Glu salt bridges in this sequence compared to the wild type peptide (TTTT) There was still substantial resonance overlap, but it mostly affected the peptide repeats for which very similar conformations were suggested by the similarity
in chemical shifts With this assumption, the entire structure could be determined from experimental restraints
As the NMR structure of VVVV2KE is based on short-range restraints, the overall bend of the helix crucially depends on the calibration used for translating the NOE cross peaks into upper distance restraints Therefore, the bend could in principle be an artifact of the automatic calibration routine used in the DYANA calculations The largely different cross-peak intensities observed for different i,i + 3 and i,i + 4 NOEs (Fig 2B) suggest, however, that the a helix is indeed not as uniform and ideal as might be expected for an isolated helix Furthermore, the HNchemical shifts and their temperature coefficients suggest that the VVVV2KE structure is bent in the same direction as the more strongly bent b-conformer in the TTTT crystal structure [8] Superficially the bend seems to be strongest near Lys18 in both VVVV2KE and TTTT As VVVV2KE contains two additional Lys-Glu salt bridges, bends near the additional lysines would also be expected Indeed, the backbone hydrogen bond between Lys29 and Ala25 is formed in only half of the 20 NMR conformers of VVVV2KE, but the resulting bend does not affect the overall structure as much as that near Lys18, because Lys29
is close to the C-terminal end of the peptide The same is true for Lys7 near the N-terminal end, although this residue forms correct backbone hydrogen bonds to Ala3 in all but four of the NMR conformers
While the overall bend in the b-conformer of TTTT is accompanied by changes in the v1angles of several residues, the side-chain conformations in the NMR structure of VVVV2KE are more similar to those of the s-conformer These data can be reconciled by a model where helix bending is facile, proceeding independently of side chain conformations AFP I peptides in solution would thus be involved in an equilibrium between straight and bent helices and, independently, equilibria between different side chain conformations Notably, the conformational spread among the NMR conformers is merely a measure of the precision with which the restraints define the structure, i.e the conformers are not meant to sample the entire conforma-tional space accessible to the peptide Instead, the NMR structure attempts to reflect the most highly populated conformations, although the use of NOE distance restraints entails a bias towards conformers with shorter internuclear distances This bias is also likely to exaggerate the overall helix bend in the NMR structure of VVVV2KE
Fig 4 Stereo views of the crystal structure conformers of TTTT The
two different conformers found in the unit cell of the crystal structure
(PDB accession code 1WFA [8]) are displayed in a line drawing
rep-resentation as in Fig 3A, using a similar orientation and residue
labeling While neither conformer presents a perfectly straight helix,
the conformer in the right panel is more strongly bent than the
con-former in the left panel Throughout the present text, the left and right
conformers are referred to as s- and b-conformer, respectively.
Table 3 Structural statistics for the NMR conformers of VVVV2KE.
Number of nonredundant NOE
upper-distance limits
414
Intra-protein AMBER energy (kcalÆmol)1) )1651 ± 42
Sum of residual NOE-restraint violations (A˚) 4.6 ± 0.2
Maximum dihedral-angle restraint violations () 1.6 ± 0.3
Rmsd to the mean for N, C a
Rmsd to the mean for all heavy atoms (A˚)b 0.88 ± 0.16
Ramachandran plot appearance c
Generously allowed and disallowed regions (%) 0.0
a 33 3 J(H N,Ha ), 24 3 J( Ha,Hb ) b For all residues c From PROCHECK - NMR
[42].
Trang 7Earlier NMR studies of TTTT in aqueous solution
showed that the side chains of many of those residues, which
are presumably involved in ice-binding, can populate
multiple v1rotamers in solution, although the most highly
populated rotamers coincided with those found in the
crystal structure [13] A similar situation probably holds for
VVVV2KE, where different side chain rotamers may be
populated to some degree despite the unique rotamers for
most amino-acid side chains (Table 4) Except for some
evidence for increased hydrogen bonding by the C-terminal
NH2group at lower temperatures (see above), there was no
clear indication for more rigid or better-defined backbone or
side-chain conformations at subzero temperatures
com-pared to 10C In principle, the questions of helix bend and
conformational variation could be addressed more
accu-rately by measuring the residual dipolar couplings A
significant set of residual dipolar coupling data would,
however, require isotopically enriched peptide to overcome
problems of signal overlap and to measure the signs of the
dipolar couplings [40]
C O N C L U S I O N S
The solution structure of VVVV2KE provides an improved
basis for simulations of possible ice-binding modes
Furthermore, the availability of sequence-specific resonance
assignments paves the way for a site-specific study of water–
peptide interactions at subzero temperatures by the use of
intermolecular water-peptide NOEs [41] Such a study,
which can be performed in solution, seems particularly
interesting in view of the fact that the interaction of water
with the putative ice-binding surface of TTTT in the single
crystal is severely hindered by intermolecular contacts
between different peptide molecules in the crystal lattice [8]
Note added in proof: the amide chemical shift changes
and helix bend in VVVV2KE are supported by a recent
publication [Cicrpicki, T & Otlewski, J (2001) Amide proton temperature coefficients as hydrogen bond indica-tors in proteins J Biomol NMR 21, 249–261], which has shown that the temperature coefficients of the amide chemical shifts are particularly large on the concave face
of curved helices
A C K N O W L E D G E M E N T S
This research was supported in part by an Australian Research Council Grant (A D J H and M M H.), University of Sydney Sesqui Research and Development Grant (M M H), Welch Grant (A D J H.) and the Swedish Research Council (E L and G O.).
R E F E R E N C E S
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2 Davies, P.L & H ew, C.L (1990) Biochemistry of fish antifreeze proteins FASEB J 4, 2460–2468.
3 Davies, P.L & Sykes, B.D (1997) Antifreeze proteins Curr Opin Struct Biol 7, 828–834.
4 Yeh, Y & Feeney, R.E (1996) Antifreeze proteins – structures and mechanisms of function Chem Rev 96, 601–617.
5 Harding, M.M., Ward, L.G & Haymet, A.D.J (1999) Type I
antifreeze proteins – structure–activity studies and mechanisms of ice growth inhibition Eur J Biochem 264, 653–665.
6 Madura, J.D., Baran, K & Wierzbicki, A (2000) Molecular recognition and binding of thermal hysteresis proteins to ice.
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7 So¨nnichsen, F.D., Davies, P.L & Sykes, B.D (1998) NMR structural studies on antifreeze proteins Biochem Cell Biol 76, 284–293.
8 Sicheri, F & Yang, D.S.C (1996) Structure determination of a lone a-helical antifreeze protein from winter flounder Acta Crystallogr 52, 486–498.
9 So¨nnichsen, F.D., Deluca, C.I., Davies, P.L & Sykes, B.D (1996) Refined solution structure of type III antifreeze protein-hydro-phobic groups may be involved in the energetics of the protein–ice interaction Structure 4, 1325–1337.
10 Gronwald, W., Loewen, M.C., Lix, B., Daugulis, A.J., So¨nnich-sen, F.D., Davies, P.L & Sykes, B.D (1998) The solution struc-ture of type II antifreeze protein reveals a new member of the lectin family Biochemistry 37, 4712–4721.
11 Fourney, R.M., Joshi, S.B., Kao, M.H & Hew, C.L (1984) Heterogeneity of antifreeze polypeptides from the Newfoundland winter flounder, Pseudopleuronectes americanus Can J Zool 62, 28–33.
12 Sicheri, F & Yang, D.S.C (1995) Ice-binding structure and mechanism of an antifreeze protein from winter flounder Nature
375, 427–431.
13 Gronwald, W., Chao, H., Reddy, D.V., Davies, P.L., Sykes, B.D.
& So¨nnichsen, F.D (1996) NMR characterization of side chain flexibility and backbone structure in the type I antifreeze protein at near freezing temperatures Biochemistry 35, 16698– 16704.
14 Graether, S.P., Slupsky, C.M., Davies, P.L & Sykes, B.D (2001) Structure of type I antifreeze protein and mutants in supercooled water Biophys J 81, 1677–1683.
15 Knight, C.A., Cheng, C.-H.C & DeVries, A.L (1991) Adsorption
of a-helical antifreeze peptides on specific ice crystal surface planes Biophys J 59, 409–418.
16 Chou, K.C (1992) Energy-optimised structure of antifreeze pro-tein and its binding mechanism J Mol Biol 223, 509–517.
17 Wen, D & Laursen, R.A (1992) A model for binding of an antifreeze polypeptide to ice Biophys J 63, 1659–1662.
Table 4 Side chain rotamers in the NMR structure of VVVV2KE and
the X-ray structure of TTTT Only those residues are listed that are
none-alanine in both sequences The v 1 -angles listed are those of the
nearest staggered rotamer All angles are given in degrees.
)60
a
Where different side chain rotamers were observed in the two
crystal structure conformers, the first value pertains to the straight
conformer and the second to the bent conformer b Thr in TTTT.
c
Predominant rotamer All three rotamers are populated.
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