Induced Ice Melting by the Snow Flea Antifreeze Protein from

Article pubs.acs.org/JPCB

Induced Ice Melting by the Snow Flea Antifreeze Protein from Molecular Dynamics Simulations Guido Todde,† Christopher Whitman,‡,† Sven Hovmöller,† and Aatto Laaksonen*,†,¶,§ †

Department of Material and Environmental Chemistry, Arrhenius Laboratory, Stockholm University, 10691 Stockholm, Sweden Facoltá di Biologia e Farmacia, Cittadella Universitaria di Monserrato, S.S. 554 bivio Sestu, 09042 Monserrato (CA), Italy ¶ Science for Life Laboratory, 17121 Solna, Sweden § Stellenbosch Institute of Advanced Study (STIAS), Wallenberg Research Centre at Stellenbosch University, 7600 Stellenbosch, South Africa ‡

S Supporting Information *

ABSTRACT: Antifreeze proteins (AFP) allow different life forms, insects as well as fish and plants, to survive in subzero environments. AFPs prevent freezing of the physiological fluids. We have studied, through molecular dynamics simulations, the behavior of the small isoform of the AFP found in the snow flea (sfAFP), both in water and at the ice/water interface, of four different ice planes. In water at room temperature, the structure of the sfAFP is found to be slightly unstable. The loop between two polyproline II helices has large fluctuations as well as the C-terminus. Torsional angle analyses show a decrease of the polyproline II helix area in the Ramachandran plots. The protein structure instability, in any case, should not affect its antifreeze activity. At the ice/water interface the sfAFP triggers local melting of the ice surface. Bipyramidal, secondary prism, and prism ice planes melt in the presence of AFP at temperatures below the melting point of ice. Only the basal plane is found to be stable at the same temperatures, indicating an adsorption of the sfAFP on this ice plane as confirmed by experimental evidence.

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Knight et al. has been questioned in both theoretical20 and experimental21 studies, suggesting a reversible binding of AFPs onto ice. Three main hypotheses have been proposed to characterize the AFP−ice interaction. First it was thought that the protein forms hydrogen bonds with its polar groups to the ice oxygens creating a receptor−ligand interaction.17,22 Then new experiments and simulations did lead to the hypothesis that the hydrophobic portion of the protein accumulates at the ice−water interface.11,23−25 A third hypothesis was then suggested; it asserts that the protein accumulates in the ice− water interface, shaping the ice surface.26 In a recent study, Calvaresi et al. show by molecular dynamics (MD) simulations on the type I AFP that this protein can induce local melting of ice and complete melting of nanoscopic ice crystals. They concluded that this might explain some apparently conflicting experimental results where binding to ice appears to be both quasi-permanent and reversible.27 An understanding of the AFP activity and mechanism could have important applications in many different areas, ranging from food industry to storage of human organs for transplants. Recently a new AFP has been isolated from Canadian snow flea,28 and its atomic structure has been solved by Pentelute et al.15 Two isoforms of snow flea antifreeze proteins (sfAFP)

INTRODUCTION Antifreeze proteins (AFP) were discovered more than 40 years ago by DeVries and Wohlschlag1 in the sera of Antarctic fishes.1−4 They were reported to have an abnormally low freezing temperature of their blood serum.5−7 DeVries and Wohlschlag isolated a glycoprotein partially responsible for the freezing-point depression of the blood serum.1 A few years later, Duman and DeVries8 and Hew and Yip9 reported of nonglycoproteins having the same antifreeze activity. Since then many other AFPs were found in fishes,10,11 insects,12−15 and even plants16 that encounter freezing conditions in their habitat. This particular class of proteins can lower the freezing point of a solution without affecting the melting point or the osmotic pressure of the same solution. The resulting temperature difference between the melting and freezing point of the solution is called thermal hysteresis (TH). This is why AFPs are also called thermal hysteresis proteins (THP) or thermal hysteresis glycoproteins (THGP). It is so far accepted that AFPs exert their activity by binding to an ice crystal surface.17,18 Through an absorption−inhibition mechanism they create microcurvature19 of the ice surface, making it less favorable for the ice to grow due to the Gibbs− Thomson (or Kelvin) effect. This model suggests that a stable binding of AFP onto a specific ice surface plane17,18 is a crucial condition for the ice growth inhibition. The nature of the AFP−ice interaction has been widely debated. The irreversible binding character of AFPs onto specific ice facets proposed by © XXXX American Chemical Society

Received: September 5, 2014 Revised: October 24, 2014

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dx.doi.org/10.1021/jp508992e | J. Phys. Chem. B XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry B

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were found28 having molecular weights of 6.5 and 15.7 kDa, respectively. A crude extract of fleas showed a thermal hysteresis of ∼6 °C.28 The sfAFP has been shown to belong to the hyperactive class of AFPs,29 having its activity one or two orders of magnitude higher than most AFPs found in fishes and plants. The sfAFP is very interesting also for its structure. So far no known homologue has been found among either AFPs or globular proteins. The small isoform of sfAFP modeled in this study is a glycine rich polypeptide composed of 81 residues (37 of which are glycines) arranged in six antiparallel polyproline type II helices. The protein also contains four cysteines that form two disulfide bonds between residues 1−28 and 13−43. The entire shape of the protein can be seen as a domino tile with two faces: one hydrophilic and one hydrophobic, giving the protein its amphipathic character. The hydrophobic face of sfAFP has been proposed as the ice binding site of the protein.15,30,31 Different AFPs have been shown to bind to a specific ice plane,17,26,32 while in the case of the largest sfAFP isoform, experiments show that it can bind to all surfaces of ice, including prism and basal plane.31 Furthermore, binding to the basal plane has been proposed as an explanation for the hyperactivity of certain AFPs, like sfAFP.29 MD simulations have been a useful tool in the investigation of this class of proteins.27,33−35 In this work we have studied, through MD simulations, the behavior of sfAFP both in water and at the ice/water interface at different temperatures. The former in order to investigate the dynamics of sfAFP and the stability of its structure; the latter to describe sfAFP interactions with four different ice crystal planes. We have also investigated the different orientations of the protein with respect to the ice surface. The hydrophobic face of sfAFP has been proposed as the ice binding site. Both protein faces, hydrophobic (proposed as binding site to ice15,30,31) and hydrophilic, have been placed against the ice surface.

Table 1. Box Dimensions and Water Molecule Content ice plane

box dimensions (Å)

prismatic secondary prismatic bipyramidal basal

65 61 60 69

× × × ×

66 71 66 67

× × × ×

60 58 75 57

water molecules 8640 8192 9600 8640

to move freely. In this way we obtained all four ice planes at the interface with water. The final configuration was then used as the starting structure for the ice/liquid water simulations. For all four ice planes a 10 ns MD was performed in the NPT ensemble storing the configurations every 1 ps. Six different temperatures were chosen for the trajectory production: 220, 225, 227, 229, 230, and 232 K. The temperatures range from the melting point of hexagonal ice (Ih) reported in literature for the TIP4P57 model to temperature 12 K below that point. sfAFP in Liquid Water. The starting configuration of the sfAFP was the crystal structure, solved by X-ray diffraction at 0.98 Å resolution, obtained from the Protein Data Bank,58 PDB ID 2PNE.15 The protein potential energy was minimized in vacuum for 1000 steps with a Steepest Descent algorithm. The obtained protein structure was placed in an empty cubic box and then filled with ∼10000 TIP4P water molecules. In order to neutralize the system one water molecule was substituted with one chloride ion. The water (only) was then energyminimized before a 10 ps MD was computed, keeping the protein position fixed. The protein was then allowed to move in the following energy minimization, while water was constrained. A last minimization of the entire system was then performed. At this point MD simulations in the NPT ensemble were started: 100 ns were produced at both 298 and 277 K. The trajectories were saved every 1 ps. sfAFP/Ice/Liquid Water. The structures obtained after the equilibration protocol followed for the ice/liquid water systems were used to insert the protein in the two phase water systems. The protein coordinates, obtained from the protein energy minimization in vacuum, were translated and rotated until the flat hydrophobic face of the protein was parallel to the ice plane. The procedure was repeated in order to place also the hydrophilic protein face parallel to the ice plane. For each protein face two different distances between the protein and the ice surface were used. The protein was first placed at a distance of 4−5 Å from the ice surface and then translated 2 Å toward the ice surface. The starting box dimensions are equal to those of the ice/liquid water systems. The empty volume needed to insert the protein was obtained removing all water molecules within 3 Å from the protein (∼450 molecules). The water potential energy was then minimized for 1000 steps with a Steepest Descent algorithm, keeping the protein fixed. Thereafter a short simulation of 50 ps was performed by imposing constraints on all protein bonds. At this point 20 ns MD simulations were carried out for all systems at four different temperatures 220, 225, 227, and 229 K. The trajectories were saved every 1 ps.

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METHODS AND MODELS Force Field. All the molecular dynamics simulations were performed using Gromacs 4.5.4.36−39 In all simulated systems the protein was modeled with the OPLS-AA force field,40−46 while the TIP4P model47 was used for water molecules. All MD simulations were performed in NPT ensemble. The pressure was 1 atm and controlled by the Parrinello−Rahman barostat.48,49 The temperature was kept constant by the Nose-Hoover50,51 thermostat. The van der Waals interactions were computed within a cutoff distance of 10 Å, and the Coulombic interactions were treated with the PME52,53 method. All bonds involving hydrogen atoms were treated by the LINCS algorithm.54 Ice/Liquid Interface. The starting ice slabs were downloaded from the Sonnichsen lab.55 The starting ice slab was translated and replicated in order to create a simulation box large enough to subsequently accommodate the protein. The final dimension of the boxes depends on the simulated ice plane. For notation on the different ice planes see, for example, the review by Madura et al.56 The box dimensions and the number of water molecules are shown in Table 1. The potential energy of the initial configurations were minimized for 1000 steps with a Steepest Descent algorithm. Three short MD simulations (50 ps) were thereafter sequentially performed at increasing temperatures (100, 150, and 200 K) at NPT ensemble imposing the position restraints on all water oxygen atoms. The system was then simulated 50 ps at 300 K allowing the water molecules in one-half of the box

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RESULTS AND DISCUSSION sfAFP in Liquid Water. The flexibility of the protein along its sequence was evaluated at both simulated temperatures (298 and 277 K) by the root mean-squared fluctuations (RMSF) presented in Figure 1. The two RMSF profiles are similar to each other (correlation coefficient 0.7), but the simulation at 298 K shows fluctuation intensities that are on average double B



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large fluctuation of the C-terminus (Figure 1) at the same temperature. These data seem to confirm the prediction based on the model of the sfAFP structure made by Lin et al.30 In their study a break between loops 4 and 5 was predicted. The Ramachandran plot59 computed from the 100 ns MD simulations compared with the X-ray data (Figure 3) shows a clear decrease in the population of the PPII helix area (−75°, 150°), especially at 298 K. Residues that adopt PPII conformation in the crystal structure explore other areas of the Ramachandran plot in the MDs; β-strand (−150°, 150°) and α-helix (−60°/−45°) regions become populated. These two areas are mostly populated at 298 K. The Ramachandran plots obtained from the simulations of sfAFP at the four different ice/water interfaces (see Supporting Information Figures S4−7) instead clearly show that the protein structure is very stable. At the ice/water interface the PPII helix area is highly populated showing a structure very close to the crystallographic one. To better understand which residues were changing their backbone conformation we have divided the protein in Gly and no-Gly residues. The respective Ramachandran plots show (see Supporting Information Figures S1 and S2) that no-Gly residues populate both β-strand and αhelix areas. All the Gly residues, instead, are mainly found close to the PPII helix (−105°, 150°) area. Properties, like solvent accessible surface (SAS) and hydrogen bonds, shown in Table 2, also indicate a slight instability of the sfAFP structure at 298 K. The high number of hydrogen bonds formed by the protein with water at 298 K seems as a consequence of the wide protein surface exposed to the solvent. The values of SAS and HBs found at the ice/water interface for the protein are considerably smaller than those obtained from simulation at 298 K. The number of HBs between protein residues is on the contrary higher when the protein is at the interface. Comparing all these values it seems that when the protein is in bulk water at 298 K a portion of its internal hydrogen bonds are lost; at the same time the hydrophilic SAS grows, so water can now get in contact with hydrophilic residues and form new hydrogen bonds. In any case the protein structure does not change much after the simulation at 298 K, with the six PPII helices remaining close to each other keeping the protein compact. The minor structural changes

Figure 1. Root mean squared fluctuation of Cα atoms. The red segments on top of the plot represent the position of the six PPII helices along the protein sequence, while the gaps between segments represent the loops.

the intensities obtained at 277 K. Two distinct peaks emerge from both profiles. They are located at the loops between PPII 2 and 3 and PPII 4 and 5. The latter is clearly the most flexible portion of the protein at 298 K, excluding the C-terminus. In Figure 2 is shown the Cα−Cα matrix distance between residue pairs. Comparing the distance matrix from the 298 K MD simulation and the corresponding matrix calculated from the X-ray coordinates, the main difference is a greater distance between the protein segments 25−30 and 55−59. In the 277 K MD simulation this distance is closer to the crystallographic one. The two segments correspond to the position, along the protein sequence, of the two highest peaks in the RMSF profile. In the crystal structure these loops are held mutually by four hydrogen bonds (HB). Three of them are formed between backbone atoms: Cys28-Gly55 (HB1), Gly27-Gly55 (HB2), and Gly30-Val59 (HB3); one is formed between the side chains of residues Asp29 and Thr57 (HB4). In the 298 K MD simulation, HB3 and HB4 are immediately broken and never reformed, while HB1 and HB2, after being broken at the beginning of the MD simulation, reform for just a few nanoseconds during the MD simulation. At 277 K, instead, HB1 and HB2 are repeatedly broken and reformed. HB3 and HB4 break in the first 10 ns and never reform also at 277 K. Moreover, in Figure 2 (298 K) the C-terminus is found to move away from the protein body. This is consistent with the

Figure 2. Cα−Cα inter-residue distance matrix. C



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Figure 3. Ramachandran plot. Left: X-ray structure, open squares represent Gly residues, solid triangles all other residues; Middle: data from the 298 K MD; Right: data from the 277 K MD. The color scale on the extreme right of the image refers to MDs data.

Table 2. Hydrogen Bond Count and Solvent Accessible Surface of sfAFP in Water at 277 and 298 K and at the Different Ice/Water Interfacesa property HB (sfAFP−water) HB (sfAFP−sfAFP) SAS total (nm2) SAS hydrophobic (nm2) SAS hydrophilic (nm2)

ice/water interface 145 53 41.1 22.9 18.2

(6) (1) (0.5) (0.4) (0.4)

277 K 154 45 42.4 22.8 19.6

(8) (3) (0.8) (0.5) (0.6)

298 K 159 40 44.2 23.4 20.8

(9) (4) (1.2) (0.8) (0.8)

a

Averages from simulations described in section sfAFP/Ice/Liquid Water The standard deviation is given in parentheses.

observed at the room temperature MD should not drastically affect the protein antifreeze activity. Ice/Liquid Interface. In order to find the melting temperature (Tm) of ice for our models, simulations of ice and liquid water in mutual contact were performed at six different temperatures 220, 225, 227, 229, 230, and 232 K in NPT ensemble. The Tm was estimated adopting the same criterion previously used by Fernandez et al.60 The melting or freezing of the system is evaluated by the net drift of the variation of total energy and by the formation or loss of ice- like structure. For all four planes we found a Tm of 228 K. In the graphs presented in Figure 4, the 227 K line always shows a negative drift, indicating a decrease of the total energy; in other words ice is growing. At 229 K instead, a positive drift indicates melting of the ice. However, in none of the simulations performed at 229 K is ice found to completely melt within 10 ns. The complete melting, within 10 ns of NPT simulations, takes place above 229 K for all simulated ice planes. The value of Tm found for the TIP4P water model applied on our systems (228 K) is very close to the 229 K estimated for the TIP4P model by Fernandez et al.60 using the same method. These results make our model of the ice/water systems suitable for the study of the influence of AFPs on ice. sfAFP/Ice/Liquid Water. At this point sfAFP was inserted in the same ice/water boxes as described in the previous section. The protein was placed with either its hydrophilic or hydrophobic face parallel to the ice surface at two different distances from the ice: A = 2−3 Å and B = 4−5 Å. The systems were simulated at four different temperatures (220, 225, 227, and 229 K), close or below the melting point of the TIP4P

Figure 4. Evolution of the total energy for the four ice planes at different temperatures.

model. A total of 64 simulations were then performed for 20 ns each. In Figure 5 are presented three snapshots from the simulation of sfAFP at the secondary prism ice plane at 229 K. After 4.0 ns the ice slab has clearly started to melt. After 10.5 ns the protein is completely surrounded by liquid water; the ice slab has entirely melted. The complete melting of ice was also observed for all the other MDs performed at 229 K. In Table 3 D



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Figure 5. Snapshots from the MD at 229 K of sfAFP (in purple) in the two water phases. (a) After 0.0 ns; (b) after 4.0 ns; and (c) after 10.5 ns. The view is from the basal plane, while the secondary prismatic ice plane (in cyan) is in contact with liquid water (in red).

simulations, the type I AFP found in the Arctic winter flounder.1 The mechanism they propose to explain the induced ice melting is the following: AFP binds to the ice surface triggering local melting of the ice slab; the molten portion of the slab has the density of ice, and this volume contracts and equilibrates with the adjacent liquid water. The melting process induces detachment of the protein from the ice slab, while the contraction of the supercooled water region together with the inflow of water of higher density can hinder the protein from moving toward the liquid domain. Hence, ice can grow a convex surface between one or more AFPs, where the proteins work as nucleation sites for the growth of the ice front. When the interface between ice and water becomes curved, the Gibbs−Thomson effect takes place, preventing further growth of ice. They claim that this mechanism could explain previous controversial experimental results where the protein was found either quasi-permanently61 or reversibly21 attached to the ice surface. Experimental evidence of the induced ice melting were recently presented by Ba et al.62 They studied the bulk melting of frozen AFP solutions by NMR microimaging experiment. This mechanism agrees with the one proposed by Wierzbicki et al. They abandon the idea of a receptor−ligand interaction63 between the AFP and the ice surface, because this would not take into account the interface between water and ice. Previous studies64−67 have shown that the ice/water interface has properties that lie between those of water and ice, and it has been estimated to be 10−20 Å thick. As proposed first by Haymet et al.24 and then reported also by other studies56,68 the AFPs accumulate at the ice/water interface. In all our MDs it is always possible to see at least a partial melting of the ice surface in contact with the protein, creating a concave curvature on the ice front. According to the mechanism described above, the local melting suggests that the protein is adsorbed at the ice/water interface. The local melting of the ice is triggered by the protein independently of the ice−protein distance. This is consistent with the interpretation of the interaction between ice and protein given by Haymet et al.24 The entire ice melting observed for the bipyramidal and secondary prism planes (at 229 and 227 K) might indicate a reversible adsorption of the protein onto these planes. Instead, the partial melting observed at the prism plane and the stability of the basal plane (both planes at 227 K) might indicate a more permanent adsorption of the protein at the ice/ water interface of these two planes. This interpretation of our results fits with the experimental findings of Scotter et al. Their crystal morphology study29 shows that the sfAFP binds to the basal plane and that this might be an explanation for the hyperactivity of sfAFPs. The larger isoform of sfAFP (15.7 kDa) instead was found attached to all ice planes,31 with the

Table 3. Ice Slab Evolution during the sfAFP/Ice/Water Simulations at Different Temperaturesa

T (K)

prism plane

secondary prism plane

bipyramidal plane

basal plane

A

A

B

A

B

A

B

G G 15.5 15.0

G S 16.5 10.5

G G M 8.0

G G 17.5 8.5

G S S 10.0

G S S 12.0

G S 14.0 12.5

G S M 10.0

G G 15.0 12.0

G G M 14.0

G S M 10.0

G G S 11.0

B

Hydrophobic Protein Face 220 K G G 225 K S G 227 K M M 229 K 12.0 10.5 Hydrophilic Protein Face 220 K G G 225 K G S 227 K M M 229 K 10.0 8.5

Columns “A” and “B” refer to the protein−ice distance, 2−3 and 4−5 Å, respectively. G = ice grows; S = ice is stable; M = ice melts. Times expressed in ns refer to the complete melting of the ice. a

is presented, in a simplified way, the ice evolution for all systems containing the sfAFP. The evolution of all systems was evaluated from the total energy variation (see Supporting Information Figure S3). At 229 K the ice always melts within 8−15 ns. At 227 K the ice slab is also found to melt, with the exception of the basal plane. When the protein is close to the basal plane, the ice slab is found stable at 227 K. If instead, the protein is close to one of the other ice planes, the ice slab melts entirely or partially. The ice melting is at this temperature (227 K) induced by the sfAFP, which changes the drift of the total energy. At 227 K, without sfAFP in the system, the total energy was always found to decrease (see Figure 4), indicating growth of the ice. Instead, when the protein is in contact or close to the ice slab, the total energy of the system if found to increase for all of the ice plane, except for the basal plane. In this last case, the total energy profile is found quite stable and flat. In summary, at 227 K the ice melting is induced (or speeded up at 229 K) by the sfAFP regardless which one of its faces is close to ice or of the distance from the ice slab. Concerning the different ice planes, only the basal plane does not melt at 227 K in the presence of sfAFP. The prism plane seems to behave somewhere in between the basal and the other two ice planes in the presence of sfAFP. In none of the simulations at 227 K does the ice slab (prism plane) melt entirely, but the total energy profile clearly indicates that the ice is melting. At lower temperatures (220, 225 K) the ice is always found either stable or growing for all simulated systems. The ice melting induced by an AFP has been already reported by Calvaresi et al.27 They have investigated, using MD E



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Notes

largest number of proteins attached onto the basal plane. Considering the experimental findings and our MD simulations, we propose that perhaps the hyperactivity of sfAFP is due not only to the interaction with the basal plane but also to the interactions with other ice planes. The adsorption onto the secondary prism, bipyramidal, and prism planes probably takes place at relatively high concentration of the protein. This would also explain the sigmoidal shape of the activity plot ([sfAFP] vs TH) of sfAFP.29

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by The Swedish Science Council. The simulations were performed on resources provided by the Swedish National Infrastructure for Computing (SNIC) at PDC, HPC2N, and NSC. The authors wish to thank Dr. Robert L. Campbell for corrections to the accepted manuscript.

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CONCLUSIONS The small isoform of sfAFP was studied both in water and at the ice/water interface, applying four different ice planes. In water, at 298 K, the structure of the sfAFP is found to be slightly unstable. Large fluctuations are observed between PPII helices 4 and 5. The hydrogen bonds formed between them in the crystal structure are broken during the MD, and the mutual PPII helices distance is found to increase. The Ramachandran plot shows clear decrease of the population of the PPII helix area. At the same time, α-helix and β-strand areas became more populated. The variation of other properties, like solvent accessible surface and number of hydrogen bonds, also points toward a slight instability of the sfAFP structure at room temperature. Anyway, the protein structure does not unfold, so its antifreeze activity should not be affected. The MD simulations of the sfAFP at the ice/water interface show that sfAFP can trigger local melting of the ice slab. At temperatures within the hysteresis range of the protein, the ice slab is found to melt either partially or entirely within 20 ns of simulation. The only ice plane found stable at the lowest simulated temperature within the hysteresis range (227 K) is the basal plane. The local melting of ice has been proposed as possible explanation for the controversial experimental results which show both quasi-permanent and reversible binding of the AFPs onto the ice surface. The local melting takes place independently of the protein−ice distance and of the temperature. Hence, the nature of the interaction between the AFPs and the ice surface is unlikely to be receptor−ligand type. The nature of the AFPs−ice interaction emerging from our and other previous theoretical studies24,27,33,56,68 consists in the adsorption of AFPs at the ice/water interface. In this picture of the interaction, the stability of the basal plane observed at 227 K is found consistent with the experimental finding of sfAFP adsorbed onto the basal plane.29 Furthermore, our simulations, interpreted in the light of the adsorption mechanism proposed by Calvaresi et al., indicate an explanation of the hyperactivity of sfAFP. The adsorption onto prism, bipyramidal, and secondary prism ice plane, taking place at relatively high protein concentration, could explain the sigmoidal shape of the activity plot of sfAFP.

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ASSOCIATED CONTENT

S Supporting Information *

Ramachandran plots of X-ray, 277 and 298 K MDs for Gly residues (1) and no-Gly residues (2); the energy plot of all sfAFP/ice/water systems (3); and the Ramachandran plot of all sfAFP/ice/water systems (4−7). This material is available free of charge via the Internet at http://pubs.acs.org/.

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REFERENCES

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