Biomacromolecules 2000, 1, 268-274
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Structure-Function Relationship in the Antifreeze Activity of Synthetic Alanine-Lysine Antifreeze Polypeptides Andrzej Wierzbicki,*,† Charles A. Knight,‡ Travis J. Rutland,† Donald D. Muccio,§ Brandon S. Pybus,§ and C. Steven Sikes| Department of Chemistry, University of South Alabama, Mobile, Alabama 36688; National Center for Atmospheric Research, Boulder, Colorado 80397; Department of Chemistry, University of Alabama at Birmingham, Birmingham, Alabama 35294; and Department of Biological Sciences, University of South Alabama, Mobile, Alabama 36688 Received February 15, 2000; Revised Manuscript Received April 6, 2000
Recently antifreeze proteins (AFP) have been the subject of many structure-function relationship studies regarding their antifreeze activity. Attempts have been made to elucidate the structure-function relationship by various amino acid substitutions, but to our knowledge there has been no successful from first principles design of a polypeptide that would bind to designated ice planes along a specific direction. In this paper we show the results of our first attempt on an entirely de novo design of an alanine-lysine-rich antifreeze polypeptide. This 43 residue alanine-lysine peptide exhibits characteristic nonequilibrium freezing point depression and binds to the designated (21h0) planes of ice along the [122] vector. The structural and thermodynamic properties of this polypeptide were determined using circular dichroism spectroscopy and its nonequilibrium antifreeze properties were investigated using an ice-etching method and nanoliter osmometry. Introduction Some plants, insects, and fish living in cold environments are able to survive temperatures below the freezing point of water by producing antifreeze proteins (AFP) or antifreeze glycoproteins (AFGP).1-4 These macromolecules prevent potential damage from freezing in a noncolligative fashion by binding to specific planes of ice crystals. The adsorption results in the cessation of ice growth from melt and a nonequilibrium depression of the freezing point. This freezing point depression depends on AFP/AFGP concentration and is believed to operate by the Kelvin effect. Fish antifreeze proteins are categorized into R-helical, alanine-rich type I AFPs, cysteine rich globular type II AFPs, and small globular type III AFPs.4 Recently another type of fish antifreeze protein from the longhorn sculpin was reported, namely type IV.5 The most extensively studied are the type I AFPs which are generally alanine-rich, R-helical peptides of 36-44 residues (3300-5000 Da), in some cases exhibiting 11 residue long repetitive segments. A helical structure has been also attributed to antifreeze glycoproteins, which have disaccharides attached to the threonine side chains of their ALA ALA THR repeat units. Type II and type III AFPs on the other hand are nonhelical molecules without repetitive amino acid sequences. Type II AFPs are large (120+ residues, ca. 14 000 Da), cysteine-rich proteins composed of β-sheets, reverse turns and helices. Type III * To whom correspondence should be addressed. † University of South Alabama. ‡ National Center for Atmospheric Research. § University of Alabama at Birmingham. | University of South Alabama.
AFPs are of intermediate size, containing about 65 residues (6500 Da), and lack a predominant amino acid type. NMR studies of this protein from ocean pout (Macrozoarces americanus) have shown that it contains β-sheets, two antiparallel triple β-strands, and one antiparallel double β-strand. Ice-etching6 and molecular modeling studies7 of type III globular AFP were not able to identify the preferred directionality of binding of these proteins on their respective adsorption planes. Mutation studies8 and structural X-ray analysis9 revealed that the ice-binding motif of these proteins was expressed on the protein surface which was unusually flat, affording formation of a hydrogen-bonding network between the protein and flat ice surface. Type IV antifreeze protein consists of four amphipathic R helices of similar length which are folded into a four-helix bundle with an R-helix content of about 60% at 1 °C. This unusual, relatively large antifreeze protein (108 residues long, 12 299 Da) shows about 22% similarity to certain apolipoproteins, some of which are known to form helix bundle structures.10 Elucidation of structure-function relationship in the antifreeze activity of antifreeze proteins (AFPs) plays a fundamental role in de novo design of antifreeze polypeptides. During the past decade our understanding of this structure-function relationship has significantly increased and at the same time new intriguing questions have been posed. Following the ice-etching studies of Knight,6 explicitly identifying ice planes responsible for binding of several known AFPs, it became possible to model interactions between AFPs and ice and thus to account for the stereospecificity of interactions between the AFPs and ice. For example for type I APFs it has been proposed6 that 16.7 Å
10.1021/bm000004w CCC: $19.00 © 2000 American Chemical Society Published on Web 05/11/2000
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Figure 1. Sequences of the antifreeze polypeptides discussed in this paper: (1) shorthorn sculpin AFP; (2) 43-mer; (3) 32-mer; (4) 27-mer; (5) winter flounder AFP. Enclosed in frames termini areas for the winter flounder and the 43-mer indicate amino acids that are common to these polypeptides.
spacing of threonine residues, contained in the 11 residue motif TAAN(D)A7, might be responsible for winter flounder AFP recognition and binding along the [1h22] vector on the (201) plane of ice. This hypothesis was later supported by molecular modeling studies.11-13 Another type I APF from Alaskan plaice, of similar structure, conforms exactly to the same binding model. Wierzbicki et al.12 proposed that for the type I antifreeze protein from the shorthorn sculpin, which lacks the 16.7 Å long repetitive motif containing threonine residues, the stereospecific binding occurs along the [122] direction on (21h0) planes. This binding is based on the protein-crystal surface enantioselective recognition that utilizes both R-helical protein backbone matching to the (21h0) surface topography and matching of side chains of polar/charged residues with specific water molecule positions in the ice surface. The shorthorn sculpin AFP is a heterogeneous, alanine-rich peptide with predominantly R-helical secondary structure. The hydrophilic side of this amphipathic protein displays several different polar and charged groups, defining a binding surface that is not clearly repetitive (Figure 1). Unlike most type I AFPs, shorthorn sculpin AFP contains no repeats of the 11 residue sequence TAAN(D)A7. Repeat spacing of threonines has been implicated in winter flounder AFP binding based on structural complementarity and hydrogen-bonding capability with respect to ice. An alternative spacing however occurs along the hydrophilic surface of many shorthorn sculpin species AFPs. This spacing involves primarily charged residues such as lysines (K). In the protein used for that study K9 and K31 (Figure 1) were 33.8 Å (22 residues) apart whereas R12 and K23 were 16.9 Å (11 residues) apart. These distances are nearly exact multiples of the 16.7 Å spacing of the (21h0) ice surface along the [122] vector, indicating that these four basic residues instead of threonines may form the critical, specific hydrogen bonds for adsorption. So far, all analyzed type I, II, III, and IV antifreeze proteins contain amino acids that are equipped with groups capable of hydrogen bonding to the ice surface. Recently the importance of these hydrogen-bonding residues for antifreeze activity has been called into question by a series of new investigations.14-17 In these studies, using site-specific substitutions of hydrophilic residues (believed to be responsible for binding of AFPs to ice) by hydrophobic residues, the importance of hydrophobic interactions with ice has been postulated as the plausible leading mechanism of recognition and binding of type I AFPs to ice. In this paper, we will present the design of the alaninelysine nonequilibrium antifreeze polypeptide. We used the shorthorn sculpin AFP structure as a departure point for our de novo design of an antifreeze polypeptide. We reduced a
large variability of shorthorn sculpin amino acid composition and designed a polypeptide in which ice recognizing and binding motif would be entirely based on alanine-lysine residues and which would bind only to the designated (21h0) planes of ice along the [122] direction. These planes have been recognized earlier as planes for adsorption of shorthorn sculpin AFP.6,12 We anticipated that the presence of alanine residues would be conducive to the formation of an R-helical structure in water whereas the appropriately distributed lysine residues not only would participate in ice recognition and binding but also, due to their excellent water solubility, will provide a necessary solubility for the polypeptide. Since our modeling has shown12 that R12 is in the 16.9 Å register with K23 we postulated that when R12 is replaced by K12 residue, together with A7 they define a framework template that could be replicated through the molecule. Thus, KAAKA7 will form, assuming a perfectly helical structure, an 11 residue long repetitive motif that will match a 16.7 Å spacing of ice on the (21h0) plane along the [122] direction.18 Methods Peptide Synthesis and Purification. A 43 peptide long polypeptide was synthesized and HPLC purified, according to our sequence, by MacroMolecular Resources at Colorado State University, as were all remaining synthetic, nonrandom polypeptides discussed in this paper. Circular Dichroism (CD) Studies. CD spectra were recorded on an AVIV 62DS circular dichroism spectrometer. Spectra were recorded on solutions containing 14-25 µg/ mL peptide in a stirred 1 cm cell. Data points were taken every 0.5 nm with a 1-3 s averaging time. Spectra were baseline corrected by subtracting buffer spectra obtained in an identical manner. Temperature was regulated using an AVIV single-cell thermoelectric device. The thermal unfolding curves for all peptides were measured in temperature mode at a single wavelength (225 nm) using a 1.5 nm bandwidth and a 10 s averaging time. A temperature probe was used to accurately measure the cell temperature (( 0.1 °C). Secondary structures were calculated using Prosec by AVIV based on the method in ref 19. Antifreeze Activity Measurements. Determination if antifreeze solutions act as true nonequilibrium antifreezes, by lowering the freezing point by the Kelvin effect,6 was done by observing the effect of a peptide solution on the growth morphology of ice crystals using a Clifton nanoliter osmometer (Clifton Technical Physics, Hartford, NY) in a cryomicroscope setup equipped with a video/printer. This setup includes a Clifton Nanoliter Osmometer mounted on a Nikon Optiphot 2 microscope with Sony CCD IRIS video
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Table 1. Comparison of the Helical Content in Water (2.2 °C) and TFE (-6˚C) and Thermodynamic Data Fit of the Thermal Unfolding in Water Using the Zipper Model peptide
helix % (H2O)
coil % (H2O)
helix % (TFE)
coil % (TFE)
Tm (°C)
∆H (kcal/(mol res))
∆S (cal/(mol K res))
27-mer 32-mer shorthorn sculpin 43-mer
50 46 64 59
50 54 37 41
69 72 75 63
31 28 25 37
27 5 32 35
-2.4 -2.4 -2.4 -2.4
-8 -8.6 -7.9 -7.8
camera, Sony Trinitron monitor PVM1343MD, and Sony Mavigraph color video printer UP-2000. This setup was interfaced with a Canon ES6000 8 mm video camcorder that allowed real time imaging. Image transfer and processing was done using ZIPSHOT (ArcSoft Inc, Fremont, CA), a PC-based attachment and software package for image capturing and processing. Antifreeze activity was determined as described previously20 using a 0.1 M solution of ammonium bicarbonate (pH ∼8) as a solvent. Ice Etching. We used 0.28 mg/mL of 43-mer solution to produce the etch pattern that enabled the adsorption plane and adsorption alignment to be deduced.6 Single-crystal, oriented ice hemispheres were grown from the solution using a coldfinger with a seed crystal frozen on, oriented with a prism plane normal to the axis of the coldfinger (parallel to the base of the hemisphere). The AFP molecules interacted with the growing surface where the orientation was correct for adsorption. The adsorption orientations were determined by slow evaporation-etching of the hemisphere surface in air, since the presence of nonvolatile adsorbed molecules produces a rough surface, like ground glass, while pure ice evaporates to a mirror-smooth surface.6 Results and Discussion Defining an antifreeze as a solute that prevents ice crystal growth, producing a nonequilibrium “freezing point” which differs from the melting point (hence the term freezing hysteresis), it is valuable to recognize several other distinct antifreeze effects: (1) alteration of the crystal growth habit, (2) inhibition of recrystallization, (3) adsorption onto ice, and (4) incorporation into growing crystals (engulfment of the adsorbed molecules by growing ice crystals). The simplest and most commonly accepted definition of nonequilibrium antifreezes that we will use in this paper is that they are water-soluble compounds that prevent ice crystal growth over a measurable range of supercooling. We used the shorthorn sculpin AFP structure as a departure point for our de novo design of an antifreeze polypeptide. Since our modeling has shown12 that R12 is in the 16.9 Å register with K23 we postulated that when R12 is replaced by K12 residue, together with A7 they define a framework template that could be replicated through the molecule. Thus, KAAKA7 will form, assuming a perfectly helical structure, an 11 residue long repetitive motif that will match a 16.7 Å spacing of ice on the (21h0) plane along the [122] direction.18 In the de novo designing of a nonequilibrium antifreeze polypeptide using the KAAKA7 motif as an ice-recognizing pattern, the assumption was made that the polypeptide will assume a nearly ideal R-helical structure in solution to match the 16.7 Å spacing along the [122] vector on the (21h0) plane.
From the earlier work on shorthorn sculpin AFP12 we deduced that at least 3-fold repeat of this binding motif, i.e., (KAAKA7)3, will be a necessary requirement for the polypeptide to exhibit the nonequilibrium antifreeze activity. Also, since the study on winter flounder antifreeze protein21 has emphasized the importance of C and N termini structure for maintaining the ideal R-helical structure of the polypeptide in solution, to prevent the loss of helicity of the (KAAKA7)3 polypeptide we added the C and N termini from the winter flounder AFP. Because the winter flounder AFP binds to entirely different sets of planes of ice, the hexagonal bipyramidal (201) planes, we would be able to exclude the possibility that the structure of the C and N termini will impact the binding, as would be the case if we were to use the shorthorn sculpin AFP C and N termini. Another reason for using the winter flounder termini is due to their high level of helical integrity, as shown by the X-ray data.21 Figure 1 shows (areas in frames) the common termini structure for the winter flounder AFP and the (KAAKA7)3 polypeptide with the C and N termini from the winter flounder AFP, which we will call hereafter the 43-mer. We tested the importance of C/N termini for maintaining the R-helix integrity by designing a 27-residue polypeptide (27-mer), also derived from the shorthorn sculpin AFP. All residues identified earlier as essential for the binding to (21h0), second-order prism planes of ice12 were retained but both C and N termini were removed entirely (Figure 1). This 27 residue polypeptide was inactive as a nonequilibrium antifreeze (∼10 mg/mL concentration). Another step in testing the antifreeze alanine-lysine based polypeptide involved synthesis of the 32-mer with no specifically designed C/N termini and with lysines in only four, out of the total of six, correct positions (Figure 1). The lysine residues were spaced to match the 16.7 Å periodicity of oxygen atoms on the (21h0) surface of ice. This biomimetic polypeptide, containing 25 alanine and 7 lysine residues, showed excellent solubility and also binding to (21h0) prism faces as demonstrated in etching studies (see the Discussion below); however, it did not exhibit a nonequilibrium freezing point depression. We have employed circular dichroism (CD) spectroscopy to determine the importance of the C/N termini by measuring the helicity content of the polypeptides. The far-UV CD spectrum of each peptide was measured in 91% trifluoroethanol (helix-inducing solvent) at -6 °C. Each spectrum showed the characteristic profile (negative bands at 208 and 225 nm; positive band at 190 nm) of peptides with high R-helical content. Each peptide had a calculated helical content of between 63 and 75% (Table 1) with the remaining fraction unordered structure. No other secondary structural components (β-sheet or β-turns) were predicted. To judge the helix stability, each peptide in deionized H2O was heated
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Figure 2. (a) CD spectra for the 43-mer in deionized water measured as a function of temperature. The spectral changes were completely reversible when the temperature was lowered to 2.2 °C. (b) Thermal unfolding curves for the 43-mer measured at 225 nm between 1 and 86 °C.
from 2.2 to 80 °C, and the spectra were recorded. In each case the spectral characteristics of a helical peptide changed to that of an unfolded peptide with increasing temperature. A clear isoelliptical point occurred at 200 nm in each case. For the 43-mer, shorthorn sculpin AFP, and the 27-mer the majority of the helical content remained at 20 °C while the spectrum of the 32-mer at 20 °C indicated that it was mainly unfolded at this temperature. Figure 2a shows the CD spectra for 43-mer (data for other polypeptides not shown) measured as a function of temperature. The spectral changes were completely reversible when the temperature was lowered to 2.2 °C. Thermal unfolding measurements were conducted for all polypeptides discussed in this study. Figure 2b shows the thermal unfolding curves for the 43-mer (data for other peptides not shown) measured at 225 nm between 1 and 86 °C. Thermal unfolding curves were fit to the zipper model,22,23 for the unfolding of small peptides, that assumes two equilibrium constants for the folding process. One constant measures the equilibrium for the addition of a helical residue to a polypeptide chain already in a helical conformation; another constant measures the equilibrium of adding a helical residue to a residue in a nonhelical segment of the polypeptide. The helix and coil lines are the limiting curves predicted from the fit of the data to the helix-to-coil model. The results of CD measurement shown in Table 1 indicate that the R-helical content in water of 27-mer was only 50% and for the 32-mer only 46%, compared to 64% for the shorthorn sculpin AFP. Loss of antifreeze activity (27-mer) and a very low helical content (32-mer) clearly indicated that in design-
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ing a nonequilibrium antifreeze one must consider that the solution structure may be far from the intended one and therefore a polypeptide may be inactive even if it has a appropriately designed ice-binding motif. Our earlier molecular dynamics studies of the shorthorn sculpin AFP12 indicated that due to water-polypeptide interactions the helicity loss may occur at the ends of the alanine-lysine polypeptide. Our CD studies of shorthorn sculpin antifreeze protein have revealed approximately 64% helical content (Table 1). Simple calculations, based on the idealized R-helical structure of the protein, showed that this will provide a rigid helical region equal to at least 33.4 Å. This is a necessary requirement for maintaining the binding residues in the rigid conformation that is needed for recognition and binding on (21h0) plane of ice along the [122] direction. Determined from the CD analysis, the 59% helicity content of the 43-mer and its Tm of 35 °C are very close to the values for shorthorn sculpin AFP (Table 1). Comparison of the 32mer and the 43-mer shows the dramatic impact of the termini on polypeptide helicity and thermal unfolding. Adding the C and N termini significantly improved the helicity by 13% and Tm by 30 °C, when compared to the 32-mer. These two structures have similar alanine-to-lysine ratio however the 32-mer is lacking the (KAAKA7) repetitive binding motif and it has one lysine more and two alanines less than the (KAAKA7)3 segment of the 43-mer. Changes introduced in the key lysine positions of 32-mer and its reduced helicity appear to have resulted in the loss of its antifreeze activity. The antifreeze activity of the four polypeptides was assessed first by inspecting their effect on the morphology of ice grown from the melt. When the morphological changes were seen, the freezing point depression was determined as a function of concentration. This nonequilibrium freezing point depression is defined as a difference between the melting and the freezing points.20 As indicated before, the 27-mer and the 32-mer were inactive at the concentrations used for the study (∼10 mg/mL), showing no ice morphology modifications. However the ice crystals grown in the presence of the 43-mer exhibited a characteristic ice inhibition morphology (Figure 3a), very similar to that of the shorthorn sculpin AFP (Figure 3b), of a hexagonal trapezohedron with its two hexagonal pyramids rotated exactly 30° with respect to each other. This ice crystal morphology could be directly linked to the adsorption planes of the 43-mer, which will be discussed below. The 43-mer polypeptide was able to entirely stop the growth of ice within its nonequilibrium freezing point depression range. However, when the concentration dependent freezing point was reached, ice crystals would grow rapidly in the form of needles elongated along the c axis of ice. Figure 3c shows the growth of ice at the concentration of 250 mg/mL of 43-mer when the freezing point of the solution has been reached. We have observed a similar pattern of growth in the presence of the natural type I antifreezes from fishes. It is interesting to note that ice morphology very similar to that of Figure 3b was obtained using a random (1:1) lysine/ alanine copolymer24 (Figure 3d). In contrast to the 43-mer,
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Figure 3. (a) Morphology of ice crystals grown from 100 mg/mL solution of the 43-mer within the thermal hysteresis, i.e., before the nonequilibrium freezing point was reached (magnification 400×). (b) Morphology of ice crystals grown from a 0.25 mg/mL solution of the shorthorn sculpin AFP analogue (magnification 400×). (c) Morphology of ice crystals grown from a 250 mg/mL solution of the 43-mer when the nonequilibrium freezing point is reached. Ice crystals grow in the form of needles along the c axis of ice (magnification 400×). (d) Morphology of ice crystals grown from a 160 mg/mL solution of (1:1) lysine/alanine random copolymer (magnification 400×).
however, ice crystals grown in the presence of this polymer continued to grow slowly, each time the temperature was lowered, never exhibiting the growth pattern seen in Figure 3c and thus never showing a true nonequilibrium freezing point depression. We also observed that increasing the lysine/ alanine ratio in these random copolymers to (2:1)25 and (3: 1)26 resulted in the entire loss of this morphology modification, with reversion to the uninhibited ice growth pattern. Freezing point hysteresis for the 43-mer polypeptide is shown in Figure 4. During the measurements, particular care
was taken to ensure that the temperature was lowered very slowly (approximately 5 mOsm/15 s) and thus to ensure that the growth of ice crystal was not occurring within the thermal hysteresis range for each concentration used in this study. Considering that the shorthorn sculpin AFP reaches its maximum of freezing point depression of approximately 1 °C at 16 mg/mL,27 the 43-mer is approximately 32 times less active on a per weight basis than the shorthorn sculpin AFP. No appreciable freezing point depression was observed for the 43-mer below 50 mg/mL.
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Figure 4. Nonequilibrium freezing point depression of the 43-mer vs concentration. Note that there was no nonequilibrium antifreeze activity observed below 50 mg/mL.
Determination of the adsorption planes for the 43-mer was done using the ice-etching technique,6,12 but without scraping the hemisphere surface before the evaporation. The scraping is used to ensure that the etching reveals only antifreeze molecules that have been engulfed within the crystal during its growth, and the 43-mer at the concentration used (0.28 mg/mL) did not produce a detectable etch in this way. Without scraping, however, the etch pattern was clear, and matched that of the shorthorn sculpin antifreeze,12 indicating binding on the secondary prism planes (21h0) aligned along [122]. The etch pattern exhibited by the 43-mer can be seen in Figure 5a, which shows the etched surface of the hemisphere after its flat side was frozen to a dull black metal plate that prevents reflections off the far side of the ice for the photography. Two large, distinct etched regions are visible at the right and left sides of the photo. The orientation of the tangent plane to the surface at the centers of the etched regions reveals the “adsorption plane” and the elongation direction of the etched regions on curved surface are normal to the 43-mer alignment direction on the adsorption plane.6 Rotating the ice hemisphere 30° about the c-axis, about a N-S line in Figure 5a, places the adsorption plane (21h0) normal to the direction of the view. Viewed at this angle, the elongation of the etched regions can be measured. Now if the circular outline is a compass face, with north at the top, the elongation of the etched regions is toward the ESE and WNW as it already appears in Figure 5a. The eastern azimuth would be 118° if the AFP orientation were ideal along [122] as reported in ref 6. We interpret the etching shown in Figure 5a as revealing adsorption on the ice, but adsorption such that the antifreeze molecules are pushed ahead with the growing interface and not engulfed in the crystal. Figure 5b shows the etching pattern of 5 mg/mL of random (1:1) alanine-lysine copolymer. Interestingly it binds to the same secondary prism plane (21h0), and the angle of the etch pattern elongation is similar to the angle determined for the shorthorn sculpin AFP and the 43-mer. This most likely indicates that the binding may occur along the vector [122] in this plane. It has to be stressed, however, that unlike the shorthorn sculpin AFP or the 43-mer, this random (1:1) polypeptide does not exhibit the nonequilibrium freezing point depression.
Figure 5. (a) Etch pattern exhibited by the 43-mer. The etched surface of the hemisphere is shown after its flat side was frozen to a dull black metal plate that prevents reflections off the far side of the ice for the photography. Two large, distinct etched regions are visible at the right and left sides of the photo. The orientation of the tangent plane to the surface at the centers of the etched regions reveals the (21 h 0) adsorption plane and the elongation direction of the etched regions on curved surface are normal to the 43-mer [122] alignment direction on the adsorption plane.6,12 (b) Etch pattern for a 5 mg/mL solution of (1:1) lysine/alanine random copolymer. The c axis orientation is the same as in part a. Two narrow etched regions are visible at the right and left sides of the photo. The orientation of the tangent plane to the surface at the centers of the etched regions reveals the (21 h 0) adsorption plane, the same as in part a, and the angle of the etch pattern elongation is similar to the angle determined for the shorthorn sculpin AFP and the 43-mer. This most likely indicates that the binding may occur along the vector [122] in this plane.
A recent paper by Zhang and Laursen28 presented another attempt on the design of lysine/alanine antifreeze polypeptides. Their approach was inspired by the winter flounder antifreeze protein,28 whose 11 residue TAAN(D)A7 icebinding motif was replaced by the KAAKA7 motif, derived earlier18 by us from the shorthorn sculpin AFP structurefunction relationship. According to the authors this 34 residue
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long polypeptide, although derived from the winter flounder sequence, “must bind either to another (higher order) crystallographic plane or by a different mechanism” than the binding plane and the binding mechanism of the winter flounder or shorthorn sculpin AFPs. It was shown earlier6 that although both winter flounder and shorthorn sculpin antifreeze proteins bind along the same 〈122〉 vector of ice Ih, their binding planes are different. We proposed12 that the 〈122〉 binding direction results from the 16.9 Å long 11 residue repeat of R helices, common for both of these AFPs (Figure 1). The AFP R helix can be depicted as a series of four lowpitch right-handed spirals of β carbons of side chains protruding from its backbone, such that every 11th residue falls approximately along a straight line, 16.9 Å apart.12 This periodicity almost exactly matches the length of the 〈122〉 vector. Moreover we have shown that the binding along this vector allows for ideal accommodation of hydrophobic side chains within the ice surface corrugation.12 We suggested that binding to different ice planes observed for these APFs results from the different nature of ice-binding residues for the winter flounder and shorthorn sculpin AFPs. Binding of winter flounder AFP to (201) planes of ice is facilitated via the short side-chain threonine residues that can bind only to the tops of the steps, spaced every 16.7 Å, across this surface. On the other hand the shorthorn sculpin AFP ice-binding surface is equipped with the long side-chain lysine residues that can be easily accommodated within the “a axis channels” propagating at the 60 degree angle to the normal to the (21h0) surface.12 Binding of the 43-mer, discussed in this paper, that occurs along the [122] vector on the (21h0) surface conforms to exactly the same principle. From Figure 1, one can easily see that the winter flounder and shorthorn sculpin AFPs are following the same design principle, both utilizing “equivalent” ice recognizing/binding patterns, TAAN(D)A7 and KAAKA7, respectively. This is even more obvious when the 43-mer polypeptide, whose structure was modeled after shorthorn sculpin AFP, is compared to the winter flounder sequence (Figure 1). We propose that the 34-residue polypeptide, discussed in ref 28, will bind along the 〈122〉 vector since it contains an 11 residue binding motif which is essentially R helical. Moreover even although its design is inspired by the winter flounder AFP, due to the above-mentioned “equivalence” between the shorthorn sculpin and winter flounder AFPs structures, and because the 34-mer contains lysine residues along its binding side it makes it essentially the same as the 43-mer. Accordingly we propose that the polypeptide of Zhang and Laursen28 binds to the shorthorn sculpin AFP second-order prism (21h0) planes along the [122] direction in a manner analogous to the binding mechanism of the 43mer. In conclusion, we have de novo designed the 43-mer lysine-alanine-rich polypeptide. The structure of this polypeptide was deduced from the structure-function relationship
Wierzbicki et al.
of its antifreeze activity. We have characterized the structure of this polypeptide as predominantly R helical. We have determined that this polypeptide exhibits nonequilibrium freezing point depression, and we have the determined dependence of its antifreeze activity upon concentration. Binding to the (21h0) secondary prism planes of ice along the [122] direction, as expected by virtue of polypeptide design, was confirmed using an ice-etching technique. Although the observed antifreeze activity of the polypeptide was lower than that of the shorthorn sculpin AFP, we hope that this study will be helpful in future improvements in the design of antifreeze polypeptides. Acknowledgment. This work has been funded by the National Science Foundation Grant MCB-9723271. References and Notes (1) Davies, P. L.; Hew, C. L. FASEB J. 1990, 4, 2460. (2) Hew, C. L.; Yang, D. S. C. Eur. J. Biochem. 1992, 203, 33. (3) Duman, J. G.; Wu, D. W.; Olsen, T. M.; Urrutia, M.; Tursman, D. In AdVances in Low-Temperature Biology; JAI Press Ltd.: London, 1993; p 131. (4) Yeh, Y.; Feeney, R. E. Chem. ReV. 1996, 96, 601. (5) Deng, G.; Andrews, D. W.; Laursen, R. A. FEBS Lett. 1997, 7402, 17. (6) Knight, C. A.; Cheng, C. C.; DeVries, A. L. Biophys. J. 1991, 59, 409. (7) Madura, J. D.; Taylor, M. S.; Wierzbicki, A.; Harrington, J. P.; So¨nnischen, F. D. THEOCHEM 1996, 388, 65. (8) Chao, H.; So¨nnischen, F. D.; DeLuca, C. I.; Sykes, B. D.; Davies, P. L. Protein Sci. 1994, 3, 1760. (9) Jia, Z.; DeLuca, C. I.; Chao, H.; Davies, P. L. Nature 1996, 384, 285. (10) Wilson, D.; Wardell, M. R.; Weisgarber, K. H.; Mahley, R. W.; Agard, D. A. Science 1991, 252, 1817. (11) Madura, J. D.; Wierzbicki, A.; Harrington, J. P.; Maughon, R. H.; Raymond, J. A.; Sikes, C. S. J. Am. Chem. Soc. 1994, 116, 417. (12) Wierzbicki, A.; Taylor, M. S.; Knight, C. A.; Madura, J. D.; Harrington, J. P.; Sikes, C. S. Biophys. J. 1996, 71, 8. (13) Wen, D.; Laursen, R. A. Biophys. J. 1992, 63, 1659. (14) Chao, H.; Houston, M. E.; Hodges, R. S.; Kay, C. M.; Sykes, B. D.; Loewen, M. C.; Davies, P. L.; So¨nnischen, F. D. Biochemistry 1997, 36, 14652. (15) Haymet, A. D. J.; Ward, L. J.; Harding, M. M.; Knight, C. A. FEBS Lett. 1998, 430, 301. (16) Zhang, W.; Laursen, R. A. J. Biol. Chem. 1998, 273, 34806. (17) Haymet, A. D. J.; Ward, L. J.; Harding, M. M. J. Am. Chem. Soc. 1999, 121, 941. (18) We presented a preliminary account of the 43-mer antifreeze peptide based on the (KAAKA7)3 sequence at the 216th National Meeting of the American Chemical Society National Meeting, Boston, MA, August 1998; AGFD Division, paper 86. (19) Chang, T. C.; Wu, C. C.; Yang, J. T. Anal. Biochem. 1978, 91, 13. (20) Chakrabartty, A.; Hew, C. L. Eur. J. Biochem. 1991, 202, 1057. (21) Sicheri, F.; Yang, D. S. C. Nature 1995, 375, 427. (22) Zimm, B. H.; Bragg, J. K. J. Chem. Phys. 1959, 31, 526. (23) Cantor, C.; Schimmel, P. R. Biophysical Chemistry Pt. III: The behaVior of biological macromolecules; W. H. Freeman and Co.: San Francisco, CA, 1980. (24) Poly (Lys,Ala) 1:1, Sigma P-4024, MW(vis): 41 600, MW (LALLAS): 50 000. (25) Poly (Lys,Ala) 2:1, Sigma P-1276, MW(vis): 49 300, MW (LALLAS): 55 300. (26) Poly (Lys,Ala) 3:1, Sigma P-1151, MW(vis): 35 000 MW, (LALLAS): 31 000. (27) Davies P. L.; Hew C. L. FASEB J. 1990, 4, 2460. (28) Zhang, W.; Laursen L. A. FEBS Lett. 1999, 455, 372.
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