Local Ice Melting by an Antifreeze Protein - ACS Publications

Jun 2, 2012 - ABSTRACT: Antifreeze proteins, AFP, impede freezing of bodily fluids and ... Molecular Dynamics, we show that type I AFP can also induce...
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Local Ice Melting by an Antifreeze Protein Matteo Calvaresi,*,† Siegfried Höfinger,*,†,‡ and Francesco Zerbetto*,† †

Dipartimento di Chimica “G. Ciamician”, Università di Bologna, Via F. Selmi 2, 40126 Bologna, Italy Department of Physics, Michigan Technological University, 1400 Townsend Drive, 49931 Houghton, Michigan, United States

‡

S Supporting Information *

ABSTRACT: Antifreeze proteins, AFP, impede freezing of bodily fluids and damaging of cellular tissues by low temperatures. Adsorption-inhibition mechanisms have been developed to explain their functioning. Using in silico Molecular Dynamics, we show that type I AFP can also induce melting of the local ice surface. Simulations of antifreeze-positive and antifreeze-negative mutants show a clear correlation between melting induction and antifreeze activity. The presence of local melting adds a function to type I AFPs that is unique to these proteins. It may also explain some apparently conflicting experimental results where binding to ice appears both quasipermanent and reversible.

I. INTRODUCTION The interaction of proteins with solid surfaces is a fundamental phenomenon with implications for nanotechnology, biomaterials, and biotechnological processes.1 Biological macromolecules may control many aspects of crystal formation,2 such as size, shape, assembly, and growth.3 In Nature, control is exerted through specialized proteins that can recognize specific surfaces during crystal growth, induce oriented nucleation, or intercalate themselves in a regular manner into the crystal lattice itself. The specificity of a protein for a surface may originate from both chemical4 (for example, hydrogen bonding, polarity, and charge effects) and structural5 (size and morphology) recognition mechanisms. This complementarity may, in turn, result in regulation of crystal growth and morphology or in induction of crystal nucleation.6 There are many examples in Nature where materials formation is controlled through protein/inorganic system interaction, as in the biosynthesis of bone,7 dental structures,8 mollusc shells,9 and particles formed by singlecelled organisms.10−12 The translation of these concepts from Nature into strategies for a “bottom up” approach to the synthesis of materials has become one of the most important areas in nanobiotechnology.13,14 Peptides and proteins have been used as nucleators, growth modifiers, or enzymes in the synthesis of inorganics9,11,15−17 or to control the growth and morphology of, for example, gold18 and silver crystals.19 A fundamental understanding of the basic principles underlying protein−surface interactions is key to a successful application of these concepts from Nature in the fabrication of novel materials. Unfortunately, three-dimensional structures of proteins directly interacting with crystals are still largely unknown, and the complementarity between these macromolecules and the mineral surfaces can be deduced only by indirect experiments. One exception to the general lack of information in this area is given by antifreeze proteins, where Xray and NMR structures of some classes of AFPs are available © 2012 American Chemical Society

and many experimental and mutational data have been made publicly accessible. Antifreeze proteins are inhibitors of ice crystal growth. They may represent an ideal model for the investigation of the interaction between a protein and a surface (ice, in this case). Organisms living at ambient temperatures below 5 °C are called psychrophiles.20 They have colonized all permanently cold environments (around 85% of Earth), from alpine and Arctic soils, to high latitudes and ocean waters deeper than 1000 m, to Arctic ice, glaciers, and snowfields. To survive and proliferate at such low temperatures, they have to overcome the hostile environment inherent to the permanent coldness in which they live. In fact, cells of living organisms are usually irreversibly damaged during freezing, causing subsequent cell death. However, a number of organisms, including fishes, plants, insects, fungi, and bacteria, have been identified to survive at temperatures below freezing.21 To avoid freezing, these organisms produce antifreeze proteins. These extracellular proteins, peptides, or glycopeptides of various sizes and shapes decrease the freezing point of bodily water by binding to ice crystals during formation, thereby inhibiting crystal growth. Five different types of AFPs22 have been isolated and characterized (types I−IV, together with a glycosylated form known as antifreeze glycoprotein (AFGP)). The ability of AFPs to affect crystal growth dynamics has suggested their use in practical applications,23 such as improving the properties of frozen foods, cryopreservation of transplant organs and cells, cryosurgery, and aquaculture, among others. One of the most studied forms of AFPs is the type I AFP,24−27 a monomeric, 37-amino acid long, right-handed, αhelical protein found in arctic winter flounder (Pseudopleuronectes americanus). The primary sequence of this protein Received: March 7, 2012 Revised: May 2, 2012 Published: June 2, 2012 2046

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contains the repetitive motif Thr-X2-Asp(Asn)-X7,28 where X is usually an alanine (Ala). When viewed along the α-helical axis the protein exhibits three distinguishable faces: a hydrophobic face comprising Ala and the methyl groups of Thr residues, a hydrophilic face formed by Arg, Glu, Ser and Asn residues, and another hydrophilic face formed by Thr and Asp(Asn) residues. Selection of the AFP face that binds to ice was first thought to be mediated by H-bonds between the Thr and Asp(Asn) residues and oxygen atoms of the ice surface (lattice matching hypothesis).29 Later, the dipole moment of the AFP was proposed to determine the relative orientation of the protein with respect to the specific adsorption plane on ice (dipole matching hypothesis).25 More recently, the role of hydrogen bonds was revised by mutational analysis that demonstrated that Thr residues can be exchanged to Val (but not to Ser) without compromising antifreeze activity.27,30−32 An alternative model was proposed, based on a steric match between the AFP and the ice surface (receptor−ligand AFP binding hypothesis).22 All current mechanisms share the idea of initial AFP binding to ice with subsequent modulation of crystal growth (adsorption−inhibition mechanism).33 The process under investigation is difficult to study experimentally. It requires (a) a single-molecule method with subnanometer resolution (the diameter of a water molecule is about 0.3 nm), (b) hyperfast instrumental refocusing (to distinguish adsorbed AFPs from free forms), (c) subnanosecond resolution of the instrumental response, (d) the flexibility to cope with varying dimensions of the solid (an emerging ice crystal is a dynamically growing object), and (e) the versatility to operate at proper physiological conditions (solubility problems27 were reported in early studies on type I AFP). With such challenging experimental conditions, a “computational microscope”34 can be useful. Atomistic molecular simulations can explore the dynamics at the molecular level and address the effects of surface chemistry on the adsorption of proteins.35−40

two steps comprising (i) charge removal and (ii) vdW decoupling, where the latter was performed in two windows (λ = [0,0.8]; λ = [0.8,1]) using modified LJ-potentials to avoid the well-known end point singularity (option klambda = 6). Five point Gaussian integration has been used to evaluate ∫ λ=1 λ=0⟨(∂V)/(∂/λ)⟩dλ numerically. Individual sampling points were subjected to (a) minimization (500 steps), (b) 50 ps equilibration to 300 K, 1 bar, using a 12 Å cutoff and a time step of 1 fs, (c) 0.5 ns of production TI at identical conditions, as employed for equilibration, (d) graphical control for convergence of TI, and (e) numerical integration. A vacuum run is used for subtraction of internal degrees of freedom and solvated systems are modeled either by TIP3P water or by TIP4P/ice water. Resulting solvation free energies are summarized in Supporting Information, Table 1, together with results from previous theoretical work and experimental measurements. All in all, the difference between TIP3P and TIP4P/Ice turns out to be small (±1 kcal/mol) except for Gln and Tyr. In conclusion, solvation free energy calculations on amino acid side chain analogues prove that TIP4P/ice may be combined with AMBER99 parameters to result in a model chemistry of almost identical quality than the one provided by the standard TIP3P model. Simulation Protocol. An equilibration protocol consisting of seven individual steps was applied, resulting in an unconstrained well-tempered NPT ensemble at target conditions. Individual trajectories were recorded, always sampling 10 ns of MD simulation. Set Up of Structures and Geometries. AFP26 (PDB = 1WFA, 1WFB)/ice complex geometries were retrieved from the Sonnichsen lab (wild-type protein and Thr13Ser/Thr24Ser and Thr13Val/Thr24Val mutants).49 The ice slabs were rotated and translated until slab walls coincided with the xz, yz, and xz planes. A number of border forming ice waters in the plane needed to be translated about the box length until periodic boundary conditions were established in rectangular box coordinates. Final ice slab dimensions were 23.3 × 67.1 × 91.8 Å for the plane (i.e., 4320 ice slab forming waters). The thickness of the slab was 23.3 Å. The AFP was oriented in the yz plane, hence, crystal lattice periodicity was maintained in directions y and z. Solvent water molecules were added in direction x on top of the ice slab surface that had the AFP attached (5911 solvent waters). AMBER 99 force field parameters42 were used for the AFP. All ice slab forming water molecules as well as all solvent waters were described by the TIP4P/ice model46 (operating at 260 K). Initial Minimization and Equilibration. About 1000 steps of steepest descent minimization were performed with SANDER.41 All ice slab forming waters and the AFP were restrained to their original position using a force constant of 5 (kcal/mol) Å−2. The minimized structure (only cleared from severe sterical clashes) was subsequently considered for a 7 step equilibration protocol. Particle Mesh Ewald summation was used throughout (cut off radius of 10 Å for the direct space sum). Bonds involving H-atoms were constrained using the SHAKE algorithm50 and a time step of 2 fs was applied in all equilibration runs. Individual equilibration steps included the following: (i) 50 ps of heating to 165 K using restraints on ice and the AFP (force constant 1 (kcal/mol) Å−2) within an NVT ensemble and temperature coupling according to Berendsen.50 (ii) 50 ps of equilibration MD at 165 K to switch from NVT to NPT and adjust the simulation box. Restraints on ice and the AFP were applied (force constant 1 (kcal/mol) Å−2). Isotropic

II. METHOD Parameters. All-atom molecular dynamics simulations were carried out with the AMBER41 suite of programs. AMBER 99 force field parameters42 were employed to describe the AFP and corresponding mutants. Successful application of such parameters was demonstrated in previous theoretical studies43−45 that also included AFP action and are further validated here. Water molecules were described by TIP4P/ice,46 a specifically developed water model for liquid/solid equilibria close to the freezing point of water. The recent development of a computational model that reproduces the dynamics and properties of melting ice46 prompted us to combine it with long-standing models that describe the structure and dynamics of proteins.47 To accept the resulting activity of type I AFPs, it is necessary to provide an independent verification (cross terms between TIP4P/ice−AMBER 99), which was obtained by calculating the free energy of solvation of a set of amino acid side chain analogues. In Supporting Information, Table 1, the results are summarized together with results from previous theoretical work and experimental measurements. The differences are small (±1 kcal/mol), except for Gln and Tyr, where they are less than 10%. The same set of 14 examples of amino acid side chain analogues reported in Chang et al.48 has been selected for solvation free energy calculations using AMBER (TI module in SANDER following instructions given at http:// ambermd.org/tutorial/shirts). Free energies were computed in 2047

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H-Bond Formation and Lifetimes. H-bond analysis was carried out for all AFPs (and mutants) exclusively focusing on H-bonds formed between solvent and the solute. AMBER’s ptraj module was used throughout. Distance cutoffs of 3.5 Å and angle cutoffs of 150° were employed to identify acceptable H-bonds, and residence frequencies had to be greater than 0.05 for a particular H-bond to be taken into account. Individual contributions were averaged if a particular residue turned out to be involved in more than one H-bond simultaneously. Both types of MD simulations, that is, AFPs (and mutants) in pure aqueous solution as well as AFPs (and mutants) attached to the ice slab and covered by liquid water were analyzed. Separate evaluations were carried out for (i) the ice binding face (IBF) and (ii) the fraction not forming the IBF. In addition to occupation numbers (counting the number of instances a certain H-bond was actually established in the set of available snapshots), H-bond lifetimes (focusing on the duration of uninterrupted occurrences of a certain H-bond) were analyzed. Principal Component Analysis (PCA). Principal component analysis (PCA) was carried out for all AFPs (and mutants). Individual snapshot structures were superimposed piecewise on the average structure (selecting the AFP) of each trajectory analyzed. A covariance matrix for positional fluctuations was computed (restricted to Cα atoms) and diagonalized using ptraj of AMBER.41 Eigenvectors were projected on the average structure computed from all available structural snapshots.

position scaling was used at default conditions. (iii) 50 ps of equilibration MD at 165 K to switch back from NPT to NVT and invoke temperature coupling according to Andersen,51 including restraints on ice and the AFP (force constant 1 (kcal/ mol) Å−2). (iv) 50 ps of equilibration MD at 165 K to release all restraints using NVT and an Andersen thermostat. (v) 50 ps of further heating from 165 to 260 K without restraints in the NVT ensemble (Andersen thermostat). (vi) 50 ps of equilibration MD restraints at 260 K to switch from NVT to NPT and adjust the simulation box (Andersen thermostat). Anisotropic (x-,y-,z-) pressure scaling was employed (NTP = 2 in SANDER). (vii) 400 ps of continued equilibration MD without restraints at 260 K in the NPT ensemble (Andersen thermostat, anisotropic pressure scaling). Production MDs. MD simulations were carried out for the series of equilibrated systems using SANDER.41 Simulation conditions were identical to the final equilibration step (vii). Overall sampling time for all considered systems was 10 ns. Snapshot structures were saved into individual trajectory files every 1000 time steps, that is, every 2 ps of molecular dynamics. Post Processing of Trajectories. Individual snapshot structures of all trajectories were centered around the initial 4357 residues (ice slab plus AFP) and periodic copies imaged back into the central box. The ice slab forming part of the initial snapshot structure was used as a reference frame to classify deviations of subsequent structural snapshots in the trajectory. Corresponding waters of the reference frame were identified for all waters in the snapshots and root-mean-square deviations (rmsd) were calculated for each of them. Mobility coefficients were computed with the help of these rmsd distances and values greater than 10 were reset to 10. Each residue was assigned its corresponding mobility coefficient in the range of 0 Å to 10 Å and colors were introduced for their structural representation (red: small RMSDs, blue: large RMSDs >10 Å, VMD52 visualization using Coloring Method Beta). A subset of representative structural snapshots in their color-coded form was assembled into a movie sequence describing the evolution of the system over the simulation time. The site-specific propensity for secondary structure formation was determined via the STRIDE program53,54 available within VMD.52 The STRIDE program consists of a knowledge-based algorithm that uses hydrogen-bond energy and statistically derived backbone torsional angle information to determine the secondary structure assignments in maximal agreement with crystallographers designations. Density Distribution Functions, g(r). Density distribution functions, g(r), the analogue of radial distribution functions for pure liquids except that a solute molecule is chosen as the reference point and the “solvent structure” around this solute molecule is analyzed for “occupation frequencies” of solvent atoms at various distances. AMBER’s ptraj facility has been used for all g(r) evaluations. Binning was done in 0.1 Å increments up to a maximum distance of 12 Å taking into account only “heavy atoms” for calculation of solute solvent distances (option “closest” in ptraj). Because the ice slab is composed of water molecules using TIP4P/ice parameters,46 the analysis can be carried out for simulations of AFPs (and mutants) in aqueous solution as well as for AFPs (and mutants) attached to ice and covered by liquid water. Two runs per species of AFP have been carried out taking into account either (i) the ice binding face (IBF) or (ii) the fraction not forming the IBF, respectively.

III. RESULTS MD Simulation of Ice. The {202̅1} plane of the ice crystal is the preferred attachment site for the type I AFP.33 A twophase system of ice, with the face {202̅1} exposed to liquid water was simulated at freezing point conditions. A snapshot of the water/ice system after 3 ns of MD simulation is shown in Figure 1a (top view in Supporting Information, Figure 1a). Colors represent the molecular mobility and range from red (little mobility) to blue (large molecular mobility). The figure

Figure 1. Molecular Dynamics simulation of the activity of Type I Antifreeze Protein. Color code: in red water molecules and AFP residues undergoing small movements, in blue water molecules and AFP residues undergoing large (>10 Å) displacement from the initial position. The {202̅1} plane of ice is the face in contact with liquid water. (a) Early (3 ns) and (b) later (10 ns) snapshots of the liquid/ solid system. The ice crystal structure is intact over the entire interval. (c) Early (3 ns) and (d) later (10 ns) snapshots of the liquid/AFP/ solid system. The AFP melts ice. 2048

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shows that the crystal lattice is maintained very well with a modest amount of perturbation at the liquid−solid interface.55 This trend continues throughout the entire simulation, here set at 10 ns for reasons that become clear in the next paragraph, Figure 1b (top view in Supporting Information, Figure 1b and Movie 1ab). In this time frame, the simulation protocol finds the ice slab in solution stable. MD Simulation of Ice/AFP. Next, the complex of AFP adsorbed onto the {202̅1} plane of ice was studied, in the same conditions as above. The simulation box contains 4320 water molecules in the ice slab and 5911 water molecules in the liquid phase, which makes for a concentration of AFP of 29 mg/mL or 8.8 mmol/L. This concentration falls into the “saturation domain”56 and should provoke a thermal hysteresis (TH) of ≈1.2 K.57 Notice that the original report of Duman et al.58 documented a thermal hysteresis of 0.6 K for the same AFP concentration. A snapshot, after 3 ns, is shown in Figure 1c. The N-terminus is on the left (top view in Supporting Information, Figure 1c). No significant differences are observed when compared to the water/ice system (compare Figure 1c to 1a). However, after ∼10 ns, Figure 1d (top view in Supporting Information, Figure 1d), the entire lattice structure of the ice slab is destroyed and the AFP finds itself swimming in liquid water that had formerly constituted the ice slab. The AFP has melted the ice slab (Supporting Information, Movie 1cd). The melting process starts beneath the C-terminus of the AFP and extends steadily toward the remaining intact fraction of the ice slab until all lattice structure is lost. The effect of induced melting is reproducible and confirmed in a repeated MD run with rigorously changed initial conditions (three additional cycles of 50 ps cooling/heating phases (ΔT = 95 K) followed by 450 ps of equilibration prior to production MD). MD Simulation of Ice/AFP Mutants. Experimental mutation of the two central Thr residues, Thr13 and Thr24, to either Ser or Val residues resulted in a virtually inactive Seranalogue, but a Val analogue with antifreeze-activity largely maintained (85% thermal hysteresis of wt-AFP)27,30−32 MD simulations for the two mutants were carried out under identical conditions as above. The 3 and 10 ns snapshots of MD simulations of the mutant AFP Thr13Ser/Thr24Ser are shown in Figure 2a and b (top views in Supporting Information, Figures 2a,b). No significant melting is observed (Supporting Information, Figure 11). The mutations trigger a conformational change in the middle of the α-helix (Supporting Information, Movie 2ab). In contrast, the antifreeze-positive mutant, Thr13Val/Thr24Val, still induces melting as shown in Figure 2c,d (top views in Supporting Information, Figures 2c,d and 11). Melting starts below the N-terminus of the mutant AFP Thr13Val/Thr24Val and proceeds to a lesser degree if compared to the wild-type (also see Supporting Information, Movie 2cd). The mutational studies in silico correlate with the experimental observations27,30−32 and confirm the discovery of this work that antifreeze activity can be linked to the local induction of melting of ice. Range of Functioning of the AFPs or the Melting Gap. The MD simulations were repeated at temperatures slightly above and below the working temperature of the previous results. For an increase of 5°, the ice slab melts very quickly, in 6.5 ns, regardless of the presence of an AFP (compare Supporting Information, Movies 3ab and 3cd with 1ab and 1cd). For a decrease of 10°, the AFP ceases to function (see Supporting Information, Figures 3e,f and Supporting Informa-

Figure 2. Molecular dynamics simulation of the activity of two mutants of Type I antifreeze protein. Color-code as in Figure 1. The {202̅1} plane of ice is the contact face with liquid water. (a) Early (3 ns) and (b) later (10 ns) snapshots of the double mutant AFP Thr13Ser/Thr24Ser known to forfeit antifreeze activity. No ice melting is observed. Snapshots of (c) 3 ns and (d) 10 ns of mutant AFP Thr13Val/Thr24Val known to preserve antifreeze activity. Considerable melting is observed.

tion, Movie 3ef). This simulation and a similar one carried out 20° below yielded de novo formation of ice (see Supporting Information, Figure 4). A simple model can explain the lowering of the temperature of onset of ice formation. Crystals form at 0 °C. In the absence of AFP, they grow with a kinetic rate, kgrowth. In the presence of AFP, the crystals melt as kmelt [AFP]n, where n is a generic kinetic exponent that can be equal to one or even very different from one if additional effects, such as Kelvin effect, are considered. Both kgrowth and kmelt are a function of temperature, with the former endowed by a more steep change with temperature. As long as kmelt [AFP]n > kmelt, ice crystals form and melt. Decreasing the temperature, kgrowth increases more than kmelt. When kgrowth becomes sufficiently large, ice starts to build up. Extending the Simulation of the Nonfunctional SERMutation. If induced melting can be “switched off” by lowering the simulation temperature then the nonfunctional SER mutant may be regarded a modestly working form at simply too low temperature. Directly testing this hypothesis, however, is compromised by the immediate onset of trivial melting (see the plus 5° results just mentioned). We, therefore, repeated the MD simulation of the nonfunctional SER mutant but extended the simulation time to 25 ns. Only after ∼22 ns, the SER mutant induced ice melting. A movie (recorded at identical frame rate to all the previous examples, supp_movie_2AB_extend.mpg) is provided as Supporting Information. The conclusion is that the experimentally determined 90% reduction of activity observed for the SER mutant is of kinetic origin. Structural Observations. The evolving secondary structural elements are summarized in Figure 3a−c. Both antifreezefunctional forms develop end-to-end domains in α-helical conformation with terminal turns unfolded (Figure 3a,b). In contrast, the antifreeze-negative mutant exhibits a midpoint interruption that separates two subdomains in α-helical conformation (Figure 3c). The linker is mainly structureless. Superposition of the average structures of the wild-type AFP and the nonfunctional mutant proves that the region of the turn 2049

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is the only structural difference between active and inactive forms of AFP (Figure 3d). From a biological point of view AFPs should intervene as early as possible, that is when the first nuclei of ice start to grow.59 This may well be when the size of the crystals is nanoscopic, and ordered crystal cores emerge while crystallographic planes begin to develop.60 Some surface reconstruction61 and a thin region of disordered surface water may appear at this stage.55 The present scenario is easily linked to a number of previously discussed models of AFP action and also to the presence of the Kelvin effect, which has been advocated to explain AFPs functioning.33 The Kelvin effect results from the fact that surfaces with different curvatures have different vapor pressures. Concave surfaces have a lower vapor pressure and convex surfaces have a higher vapor pressure. If the vapor pressure is lower, molecules desorb from the surface/ crystal, and ice disappears. If the vapor pressure is higher, molecules stick to the surface/crystal and ice grows. It is accepted that AFPs attachment to the surface and ice growth around them produces concave surfaces with lower vapor pressure and diminished rate of ice growth. Microscopic Activity of the AFP. Three additional simulations in liquid water of the wild-type and the two mutant AFPs allowed comparison with the simulations of the proteins adsorbed to the ice surface. Results are summarized in Figures 6−8 of the Supporting Information. No major differences appear (i) for the radial distributions of water/ice around the AFP faces, (ii) for the hydrogen bonding, and (iii) for the hydrogen bond lifetimes. These findings are in agreement with refs 62−64, where the major conclusion was that H-bonds do not explain the AFP effect. In view of these results, the identification of the helical interruption in the 90%inactive T13S T24S AFP mutant becomes crucial. The key

Figure 3. Evolution in time of the secondary structure of active and inactive forms of AFP: magenta, α-helix; dark blue, 3−10-helix; cyan, turn; white, coil. (a) AFP (wild-type, wt) remains in predominantly αhelical conformation with terminal turns unfolded. (b) The antifreezepositive mutant Thr13Val/Thr24Val exhibits largely wt-like secondary structure. (c) The antifreeze-negative mutant Thr13Ser/Thr24Ser develops an α-helix defect at the center. (d) Superposition of wt (blue) and Thr13Ser/Thr24Ser (red) structures with the indication of the sites of mutation and the developing turn/coil.

Figure 4. Type 1 AFP/ice interaction (a−c) and melting point elevation (d−f): AFPs in red, the ice front motion is indicated by the yellow arrows, location of the AFPs is identified by green arrows, water in baby blue, supercooled water in blue, ice lattice in cyan. (a) A regular growing ice front at the solid/liquid interface in freezing conditions; (b) a magnified view of (a) with the addition of two AFPs that recognized their specific binding surface (i.e., the {202̅1} plane) and have induced melting with formation of a small local pool (basin) of supercooled water. The influx of adjacent, denser liquid water forces the AFPs to remain local (also see Supporting Information, Figure 5); (c) ice continues to grow between the AFPs and curved surfaces emerge leading to the depression of the freezing point by Kelvin effect;33 (d) starting of reverse (inward) motion of the ice front in melting conditions in the presence of AFPs; (e) the AFPs actively melt ice and remain located at the bottom of the basin because of the density difference of melted and surrounding water; (f) cusps are formed, leading to the melting point elevation by Kelvin effect.33. 2050

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experimental results where binding to ice appears both quasipermanent66 and reversible.65

interactions in the molecular mechanism that leads to melting are those established through the presence of the helical element. A principal component analysis, PCA, of the dynamics showed that the movements of wild-type AFP and of the functional mutant are dominated by collective motion of the helix as a whole. In the inactive mutant, PCA finds the motion separated into the two α-helical subdomains in which the helix is broken. An operational AFP therefore needs a rigid helical element specifically adsorbed on the ice crystallite. Additionally, because at the atomistic level the AFP affects the structure of neither water nor ice, but the motion of the entire helix affects ice, and the conclusion is that the AFP destabilizes ice (the corresponding translational or diffusive motion of the AFP in liquid water cannot affect liquid water stability). Freezing Point Depression upon Induced Local Melting. Below freezing temperature, the ice front grows steadily (Figure 4a). AFPs, in red, identify and bind to the surface,66 triggering local melting (Figure 4b). The molten domain has the density of ice, that is, ∼9% lower than that of the surrounding liquid water (anomaly of water). The affected volume must (i) contract (see Supporting Information, Figure 5, which reports the contraction of the simulation cell during the melting), and (ii) equilibrate with adjacent liquid water that pours into it. Ba et al. report that AFPs become detached from the ice surface at the equilibrium melting temperature65 and Pertaya et al. find that AFPs remain attached to the ice surface for hours at temperatures within the hysteresis gap.66 Melting can induce detachment, while contraction of the supercooled water region together with the influx of denser water can prevent the AFPs from drifting away into the liquid domain and AFPs remain local. Ice can now grow a convex surface between two, or more, AFPs, which function as nucleation sites for the formation of curved surfaces on the ice front, or, in other words, defects for the steady growth of the ice front. Once the water/ice interface is curved, the usual arguments of the Kelvin effect apply.33 This mechanism, based on local melting, can explain the discrepancy observed in different experiments that reported both quasi-permanent attachment66 and reversible binding65 to the ice surface. Superheating (or Melting Point Elevation)67,68 upon Induced Local Melting. Above the freezing temperature, the curved ice-front moves backward toward the crystal interior. The AFPs are accommodated in the defects (Figure 4d). The AFPs actively melt ice (Figure 4e) and remain located at the bottom of the basin because of the density difference of melted and surrounding water. The active melting forms basins separated by cusps (Figure 4f). Once the cusps are formed, the usual arguments of the Kelvin-effect apply33 and superheating occurs.67 In this model, superheating above the melting point is not due to concave regions between AFPs, rather it is brought about by the concave melted regions created by the AFPs themselves. A feature that may be amenable to experimental verification.

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ASSOCIATED CONTENT

S Supporting Information *

(1) Molecular dynamics simulation of the winter flounder AFP(wt), top down view; (2) Molecular dynamics simulation of antifreeze-positive and antifreeze-negative mutants of winter flounder AFP, top down view; (3) Molecular dynamics simulation of the AFP/ice interface at slightly in/decreased simulation temperature; (4) De novo formation of ice at the AFP/ice interface; (5) Volume contraction upon melting; (6) Density distribution functions analysis, g(r); (7) H-bond formation analysis; (8) H-bond life times analysis; (9) Principal component analysis of global AFP motion; (10) Secondary structure in free solution; (11) Time evolution of ice slab forming waters; (12) AFP atomic fluctuations. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; siegfried.hoefinger@ unibo.it; [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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IV. CONCLUSIONS Molecular dynamics simulations show that a type I AFP can melt nanoscopic volumes of ice. The experimentally nonfunctioning mutant is found “kinetically less” melting in the simulations, while the functioning mutant quickly melt ice. The activity probed by the simulations takes place in a restricted temperature range around that of ice formation. The presence of local melting adds a function to type I AFPs that is unique to these proteins. It may also explain some apparently conflicting 2051

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