Solution structure of a hydrophobic analogue of the winter flounder antifreeze protein Edvards Liepinsh 1 , Gottfried Otting 1 , Margaret M. Harding 2 , Leanne G. Ward 2 , Joel P. Mackay 3 and A. D. J. Haymet 4 1 Karolinska Institute, Tomtebodava ¨ gen, Stockholm, Sweden; 2 School of Chemistry, University of Sydney, NSW, Australia; 3 Department of Biochemistry, University of Sydney, NSW, Australia; 4 Department 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 pH 7.0 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 v 1 rotamers 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 ÔantifreezeÕ 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 M r , alanine rich, a helical structures. Within this class, HPLC6 (TTTT) [11], a 37-residue sequence containing three 11-residue repeats of ThrX 2 AsxX 7 is 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 3 10 -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  221] 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) Eur. J. Biochem. 269, 1259–1266 (2002) Ó FEBS 2002 hydrogen 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. MATERIALS AND METHODS 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% H 2 O/10% D 2 O at concentrations of 11 m M (pH 4.9) and 2 m M (pH 7.0). The 2-m M 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 10 °C 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 15 °C to support the resonance assignment and check for conformational differences. The in-phase lineshape of NOESY cross peaks was used to determine J HN,Ha coupling constants [26]. The COSY and TOCSY cross-peaks were visually inspected to determine the relative magnitude of the J Ha,Hb couplings of C b H 2 methylene groups. The ROESY spectrum at 15 °C (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 program XEASY was 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 ÆÆÆH distances <2.4 A ˚ and internuclear O ÆÆÆH-N angles < 35°.Plotsof the structure were prepared with MOLMOL [30]. Accession numbers The coordinates of the 20 energy-refined DYANA conformers 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 m M KH 2 PO 4 (pH 8.0) containing 50 m M KCl, to give initial loading concentrations of 1.0, 0.3 and 0.1 m M . Sample aliquots (200-lL) were loaded into 12-mm double- sector cells, and data were collected at 0 °CinanAn-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 v 2 values. Visualization of the plots of apparent molecular mass vs. concentration and W vs. concentration was carried out using the program OMMENU [32]. RESULTS 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 m M ,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 m M concentration (the concentration used for NMR structure determination), as no significant chemical shift changes were Table 1. Sequence alignment of TTTT and VVVV2KE. 1 2 13 24 35 TTTT D TASDAAAAAAL TAANAKAAAEL TAANAAAAAAA TAR VVVV2KE D VASDAKAAAEL VAANAKAAAEL VAANAKAAAEA VARCONH 2 1260 E. Liepinsh et al. (Eur. J. Biochem. 269) Ó FEBS 2002 observed in the concentration range 0.1–2 m M (measured at 5 °C and pH 3.55). 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 15 °C 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 H N resonance when the pH was lowered to pH 2 (data not shown) [33,34]. This hydrogen bond seems to be highly populated at neutral pH, where the H N resonance 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 ahelical 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 H a and Lys19 H N , 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 H N chemical shifts of Ala15 and Ala26 (0.011 p.p.m. per °C between 5 and )5 °C) and Asn16 and Asn27 (0.015 p.p.m. per °C) 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 H N showed 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 NH 2 group of Arg37 were too large to suggest any involvement in a stable hydrogen bond. Yet, the temperature coefficients of the two NH 2 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. Ó FEBS 2002 NMR structure of type I antifreeze protein (Eur. J. Biochem. 269) 1261 Although no restraints were used for this NH 2 group in the structure calculations of VVVV2KE, and Arg37 was largely disordered (Fig. 3B), most of the conformers formed the corresponding hydrogen bond between Arg37 NH 2 and Val35 O. The amino-acid side-chains of VVVV2KE assumed the same v 1 rotamer position in all 20 conformers, while different rotamers were found beyond the b carbons. Only the side chain of Ser4 populated all three staggered v 1 rotamers. 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 Ôb-conformerÕ, and the less bent conformer as the Ôs-conformerÕ. 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 v 1 rotamers 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 H N H a H b Others Asp1 – 4.18 2.85, 3.02 Val2 8.45 3.91 2.05 C c1 H 3 0.96, C c2 H 3 1.02 Ala3 8.24 4.24 1.43 Ser4 8.68 4.24 3.90, 4.00 Asp5 8.46 4.49 2.80, 2.67 Ala6 8.24 4.22 1.48 Lys7 8.06 4.13 1.89, 1.92 C c H 2 1.36, 1.49; C d H 2 1.70; C e H 2 2.96 Ala8 8.04 4.19 1.50 Ala9 8.15 4.17 1.48 Ala10 8.00 4.18 1.52 Glu11 8.30 4.07 2.16, 2.03 C c H 2 2.27, 2.50 Leu12 7.83 4.26 1.70, 1.84 H c 1.65; C d1 H 3 0.90, C d2 H 3 0.93 Val13 7.75 3.70 2.14 C c1 H 3 0.96, C c2 H 3 1.09 Ala14 7.91 4.22 1.48 Ala15 8.40 4.18 1.52 Asn16 8.62 4.55 2.80, 2.92 H d21 7.68, H d22 6.88 Ala17 8.20 4.24 1.50 Lys18 8.09 4.14 1.89, 1.92 C c H 2 1.37, 1.50; C d H 2 1.66; C e H 2 2.96 Ala19 7.99 4.20 1.49 Ala20 8.14 4.18 1.50 Ala21 7.98 4.18 1.52 Glu22 8.31 4.08 2.17, 2.03 C c H 2 2.26, 2.51 Leu23 7.82 4.26 1.70, 1.84 H c 1.65; C d1 H 3 0.89, C d2 H 3 0.93 Val24 7.76 3.70 2.14 C c1 H 3 0.96, C c2 H 3 1.09 Ala25 7.91 4.22 1.48 Ala26 8.43 4.18 1.53 Asn27 8.67 4.55 2.79, 2.94 H d21 7.69, H d22 6.87 Ala28 8.24 4.24 1.51 Lys29 8.12 4.14 1.91, 1.94 C c H 2 1.37, 1.49; C d H 2 1.67; C e H 2 2.95 Ala30 7.99 4.20 1.51 Ala31 8.13 4.18 1.47 Ala32 7.94 4.18 1.50 Glu33 8.20 4.08 2.09, 2.01 C c H 2 2.24, 2.46 Ala34 7.86 4.15 1.47 Val35 7.76 3.82 2.10 C c1 H 3 0.93, C c2 H 3 1.03 Ala36 7.93 4.18 1.43 Arg37 7.90 4.18 1.84, 1.87 C c H 2 1.65, 1.74; C d H 2 3.17,3.20; H e 7.24; NH 2 7.24, 7.27 1262 E. Liepinsh et al. (Eur. J. Biochem. 269) Ó FEBS 2002 chemical shift difference between the 1 H-NMR resonances of the C-terminal NH 2 group 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 NH 2 and 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 H N is 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 H N and Glu33 O are also close in the crystal structure of TTTT. Thesidechainv 1 angles 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 OH by a CH 3 group without affecting the position of the other CH 3 group. 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. DISCUSSION 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 Oat10°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 +3andi/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 H a and Lys18 H N , 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 drawnwithdottedlines.TheN-caphydrogenbondbetweenthe carboxyl group of Asp1 and the backbone amide of Ser4 is identified in bold. Ó FEBS 2002 NMR structure of type I antifreeze protein (Eur. J. Biochem. 269) 1263 In 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 Ôice-bindingÕ 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 +3andi,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 H N chemical 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 v 1 angles 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. Parameter Value Number of assigned NOE cross peaks 599 Number of nonredundant NOE upper-distance limits 414 Number of scalar coupling constants a 57 Number of dihedral-angle restraints 91 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 and C¢ (A ˚ ) b 0.53 ± 0.15 Rmsd to the mean for all heavy atoms (A ˚ ) b 0.88 ± 0.16 Ramachandran plot appearance c Most favoured regions (%) 99.7 Additionally allowed regions (%) 0.3 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]. 1264 E. Liepinsh et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Earlier 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 v 1 rotamers 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 NH 2 group 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- paredto10°C. 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]. CONCLUSIONS 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. ACKNOWLEDGEMENTS ThisresearchwassupportedinpartbyanAustralianResearchCouncil 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.). REFERENCES 1. DeVries, A.L. (1983) Antifreeze peptides and glycopeptides in cold-water fishes. Ann. Rev. Physiol. 45, 245–260. 2. Davies,P.L.&Hew,C.L.(1990)Biochemistryoffishantifreeze proteins. FASEB J. 4, 2460–2468. 3. Davies, P.L. & Sykes, B.D. (1997) Antifreeze proteins. Curr. Opin. Struct. Biol. 7, 828–834. 4. 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(2000) NMR experiments for the sign determination of homonuclear scalar and residual dipolar couplings. J. Biomol. NMR 16, 343–346. 41. Otting, G. (1997) NMR studies of water bound to biological molecules. Prog. NMR Spectrosc. 31, 259–285. 42. Laskowski, R.A.,Rullmannn, J.A., MacArthur, M.W., Kaptein, R. & Thornton, J.M. (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486. 1266 E. Liepinsh et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . chemical shift changes were Table 1. Sequence alignment of TTTT and VVVV2KE. 1 2 13 24 35 TTTT D TASDAAAAAAL TAANAKAAAEL TAANAAAAAAA TAR VVVV2KE D VASDAKAAAEL VAANAKAAAEL VAANAKAAAEA VARCONH 2 1260. 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. display and analysis of macromolecular structures. J. Mol. Graphics 14, 29–32. 31. Johnson, M.L., Correia, J.J., Yphantis, D .A. & Halvorson, H.R. (1981) Analysis of data from the analytical