Local Water Dynamics around Antifreeze Protein ... - ACS Publications

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Local Water Dynamics around Antifreeze Protein Residues in the Presence of Osmolytes: The Importance of Hydroxyl and Disaccharide Groups Anand Narayanan Krishnamoorthy,* Christian Holm, and Jens Smiatek* Institut für Computerphysik, Universität Stuttgart, D-70569 Stuttgart, Germany ABSTRACT: Antifreeze proteins (AFP) and antifreeze glycoproteins (AFGP) are synthesized by various organisms to enable their cells to survive low temperature environments like in the polar regions. The presence of antifreeze proteins leads to a temperature difference between the melting and freezing point of the solution known as thermal hysteresis. It is nowadays common knowledge that the antifreeze activity of AFPs is mainly determined by a short-range effect which includes a direct binding to the ice phase. Recently, experimental findings also revealed a long-range effect which implies a significant retardation of the water dynamics to facilitate the ice-binding process specifically for AFGPs. The aim of this work is to examine the dynamics of water molecules around different antifreeze protein residues by using atomistic molecular dynamics simulations. A prototype of AFP from antarctic notothenioids with the main subunit alanine−alanine-threonine (AAT) and a mutant (polyalanine) together with the residues of an antifreeze glycoprotein (AFGP) were simulated and compared with respect to their influence on the local water shell. The analysis of the water hydrogen bond characteristics and the dipolar relaxation times reveals a strong retardation effect of the water dynamics around the AFGP prototype. Our numerical results reveal the significant importance of polar units like threonine and disaccharides for the direct binding of water molecules in terms of hydrogen bonds and a significant retardation of water dynamics. In addition, a considerable change of the hydration dynamics is additionally observed in the presence of osmolytes like urea and hydroxyectoine. Our findings indicate that this effect is even more pronounced in the presence of kosmotropic osmolytes. as well as their chemical composition,1 antifreeze glycoproteins (AFGP) are a separate class of antifreeze active proteins that are found in fishes like the antarctic notothenioids and in the northern cod.5−8 The chemical structure of AFGPs compared to AFPs consists of repeating alanine−alanine-threonine (AAT)x units where x typically ranges from 4 to 50 but with the difference of a covalently bonded disaccharide group to threonine. The general antifreeze mechanism behind AFGPs has been discussed in the framework of a short-range and a long-range effect.5−8 The short-range effect mainly includes the inhibition of large ice crystal growth by a direct adsorption of AFGPs and also of AFPs to an ice surface. Thereby a quasi-irreversible binding is achieved whose natural consequence is the appearance of thermal hysteresis.2,3,7,8 The occurrence of this adsorption process has been discussed as one of the most difficult recognition mechanisms in nature8 which has to be facilitated by a significant retardation of the local water dynamics known as the long-range effect.5−7 Indeed, terahertz (THz) spectroscopy results in combination with computer simulations have indicated a significant retardation of the water dynamics on a length scale up to 2 nm around typical

1. INTRODUCTION Antifreeze proteins (AFP) and antifreeze glycoproteins (AFGP) are special classes of proteins in biological cells that enable organisms to survive in cold environments.1,2 These macromolecules provide cell protection by depressing the freezing point of water in small volumes by reacting in a noncolligative manner.3 The corresponding effect has been studied in a pioneering work whose results reveal that the presence of AFP at the interface between solid ice and liquid water inhibits the thermodynamically favored growth of a macroscopic ice crystal.3 The resulting difference between the melting and the freezing point of the solution is known as thermal hysteresis. The corresponding temperature difference is used to classify AFPs with respect to their antifreeze activity. Antifreeze proteins in polar fishes exhibit a thermal hysteresis around 2 °C which implies a moderate AFP effect whereas the presence of hyperactive insect AFPs results in larger temperature differences up to 5 °C.4−8 The technological applicability of AFPs possess some potential with regard to their usage as additives to improve the quality of frozen food, as cryoprotective agents for organ and cell cryopreservation, and also as chemical adjuvants to cancer cryosurgery. Further development also includes the usage of AFPs to increase the tolerance of transgenic plants and animals to cold environments.1,9,10,15 In addition to ordinary AFPs which can be classified according to their structural motifs © 2014 American Chemical Society

Received: July 15, 2014 Revised: September 9, 2014 Published: September 10, 2014 11613

dx.doi.org/10.1021/jp507062r | J. Phys. Chem. B 2014, 118, 11613−11621

The Journal of Physical Chemistry B

Article

bulk solution. We attribute this finding to the presence of disaccharide units. Furthermore, our results reveal that the antifreeze activity is additionally increased in the presence of hydroxyectoine and urea. The binding mechanisms of both osmolytes to AFP prototypes are investigated by the evaluation of the local Kirkwood−Buff integrals. We are able to elucidate different binding mechanisms between the osmolytes and the prototypes of AFP which allow us to understand their influence on the water dynamics. All our results are in good agreement to recent experimental findings. The paper is organized as follows. The theoretical background and the simulation details are presented in the next two sections. The results are shown and discussed in section 4. A short summary is given in section 5.

AFGPs.5−7,11,12 The success of atomistic molecular dynamics (MD) simulations to explain these experimental findings and to study the properties of local water shells has been also illustrated by previous studies.13,14,17,18 Moreover, further experimental observations with respect to mutation studies for AFPs proved that mainly the threonine residues are responsible for the strong affinity to ice crystals.16,19,20 The authors of some of these studies came to the conclusion that this effect is less pronounced if the threonine residues are replaced by alanines which significantly changes van der Waals as well as hydrophobic interactions. Due to these and other findings, the hydration properties of AFPs and AFGPs are still in the main focus of research.17,18 In addition to AFPs and AFGPs, small organic molecules, socalled osmolytes, also show an increased retardation of the water dynamics within their local hydration shell.20,21 In terms of their biological function for organisms, osmolytes are mainly involved in the maintenance of cell volume and cell humidity.21−25 The presence of these molecules allows microorganisms to resist extreme living conditions like drastic temperature variations and high salinity.23,24 Interestingly, most osmolytes are biologically inert and accumulate at high concentration in the cytoplasm without interfering with the overall cellular functions and can be separated in two different classes.21,26,27 Osmolyte molecules that maintain protein structures are called kosmotropes (order-maker). Typical kosmotropes are hydroxyectoine, betaine, and trehalose. In contrast, molecules like urea which induce protein destabilization are called chaotropes (disorder-maker).28−32 The corresponding structures of hydroxyectoine and urea are shown in Figure 1. Specifically the study of osmolyte interactions with

2. THEORETICAL BACKGROUND 2.1. Hydrogen Bond Analysis. The main focus of the present work is to investigate and to compare the water dynamics around prototypes of AFP, AFGP, and polyalanine. The analysis of this behavior mainly involves the study of the hydrogen bond characteristics. The geometrical criterion used to determine the existence of a hydrogen bond is given by r ≤ rHB = 0.35 nm and α ≤ αHB = 30° where rHB denotes the maximum distance between a donor and an acceptor pair for hydrogen bonds within the maximum angle of 30°. The lifetime of the hydrogen bond is calculated from the average autocorrelation function of the existence criterion C HB(t ) =

⟨si(t0)si(t )⟩ ⟨si 2(t0)⟩

(1) 36,37

with si(t) = (0,1) for a hydrogen bond i at time t. The hydrogen bond forward lifetime τHB can then be calculated by τHB =

∫0

∞

C HB(t ) dt

(2)

which allows us to yield an estimate for the average existence time.26,27 The inverse hydrogen bond forward lifetime, also known as rate constant kf is related to the activation free energy via

Figure 1. Structural formula of urea (left side, chaotrope) and hydroxyectoine (right side, kosmotrope) in its neutral form. A recent publication21 has indicated the pronounced stability of zwitterionic hydroxyectoine in aqueous solution.

kf =

proteins is an ongoing field of research. Several studies state that urea directly interacts with the protein backbone,30,31,33 whereas kosmotropes like hydroxyectoine tend to stabilize proteins in an indirect mechanism in which the osmolytes are preferentially excluded from the protein surface.21,26,27,30 Within the context of antifreeze activity, it has been recently indicated that AFPs show a significantly more pronounced thermal hysteresis effect in the presence of osmolytes.6,20 The underlying molecular mechanism is still under debate whereas further studies also show that the presence of cosolutes like polyhydroxyl and polycarboxyl groups in an AFP solution enhance the antifreeze activity considerably.34,35 In this paper, we present the results of atomistic MD simulations for prototype units of antifreeze proteins as well as residues of antifreeze glycoproteins in the presence and absence of osmolytes with regard to the local water dynamics. The properties of the local hydration shell are mainly investigated by the study of the hydrogen bond correlation function as well as the dipolar relaxation times. Our results indicate a significant increase of the hydrogen bond forward lifetimes and the dipolar relaxation times in the presence of AFGPs compared to the

⎛ ΔF† ⎞ kT 1 = B exp⎜ − ⎟ τHB h ⎝ kBT ⎠

(3)

where kBT is the thermal energy, h is the Planck constant, and ΔF† expresses the activation energy to break an arbitrarily chosen hydrogen bond.26,38 2.2. Kirkwood−Buff Integrals. The radial distribution function around molecules or particles can be expressed by gαβ (r ) =

ρβ (r ) ρβ , ∞

(4)

where ρβ(r) denotes the local density of a molecular species β at a distance r around molecule α where ρβ,∞ denotes the global density in the bulk phase.43 The cumulative particle number function is given by fαβ (d) = 4πρB

∫0

d

r 2gαβ (r ) dr

(5)

which gives the number of molecules of type β around α within a distance d. The Kirkwood−Buff integral is defined by 11614


The Journal of Physical Chemistry B r=R

Gαβ = lim Gαβ (R ) = lim 4π R →∞

R →∞

∫r=0

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and the force field for urea and hydroxyectoine have been described elsewhere.21 It has to be noted that we used the zwitterionic form of hydroxyectoine in our simulations due to the fact that recent quantum-chemical calculations indicated a stronger stability in an aqueous environment compared to the neutral form which is more stable under vacuum conditions.21 All molecular dynamics simulations were performed in explicit SPC/E water55 and under periodic boundary conditions. Several sets of simulations were performed to study the prototype antifreeze protein−water dynamics. Electrostatic interactions have been calculated by using the particle mesh Ewald method.56 The time step in all simulations was δt = 2 ps, and we applied the LINCS algorithm to constrain all bonds.57 The Berendsen thermostat58 was used in all simulations with a thermostat relaxation time of 0.1 ps and a temperature of 300 K. After energy minimization with the steepest descent method and a warm up phase of 2 ns, we conducted a production run of 20 ns for each simulated system. The influence of the mutation T → A has been studied by the simulation of (AAT)6 and (A)18 in a cubic simulation box of (5 × 5 × 5) nm3 which was filled with 4080, respectively 4069, water molecules. To analyze the influence of the disaccharide chains, a single AFGP prototype with (AAT)2 where two disaccharide chains were attached to the threonine residues was solvated with 1500 water molecules in a (3.617 × 3.617 × 3.617) nm3 cubic simulation box. For the study of osmolyte effects, we have simulated a (AAT)15 chain which was dissolved in a cubic box of (4.69 × 4.69 × 4.69) nm3 in which further 237 urea molecules (concentration c = 3.8 mol/L), respectively 168 hydroxyectoine molecules (c = 2.8 mol/L), were inserted. The results of these simulations have been compared to a single (AAT)15 chain in a pure aqueous environment with the same box dimensions. Although the box sizes differ in our simulations, it has to be mentioned that our analysis is not affected by this. It was additionally ensured that the monomer concentration does not exceed a critical concentration. We mostly focus on very local behavior like hydrogen bond properties such that known concentration dependent effects like macromolecular crowding59 and hydrodynamic screening60 or finite size effects for dynamic properties61 do not influence our results due to only small differences in the box length which do not considerably change the numerical values. The hydrogen bond density ρHB was calculated by the ratio of the number of hydrogen bonds to the total solvent accessible area σt, respectively hydrophilic solvent accessible surface area σ+ in agreement to a recent publication.26 All atoms whose partial charge exceeds q > |0.3| e have been considered as hydrophilic and the solvent accessible surface area was calculated by the algorithm proposed in ref 62.

r 2(gαβ (r ) − 1) dr (6)

where the relation is valid in the limit of an infinite distance R = ∞.39−42 In terms of a finite distance r, one typically estimates the values of the radial Kirkwood−Buff integral by Gαβ (r ) = 4π

∫0

r

r 2(gαβ (r ) − 1) dr

(7)

where the lower integration limit defines the molecular surface and the upper value is given at the point where the values for Gαβ(r) converge.41,42 Finally the radial preferential binding parameter is given by νβγ(r ) = ρβ , ∞(Gαβ (r ) − Gαγ (r ))

(8)

where γ represents water molecules, α stands for the protein surface and β denotes osmolyte molecules. The values of νβγ(r) can be used to elucidate the binding or exclusion behavior of osmolytes to the protein surface. In case of a negative value for the radial preferential binding parameter within a specific region, it can be shown that the osmolyte is preferentially excluded at this point in contrast to a positive value which implies a preferential binding behavior.26,27,41,42 2.3. Dipole Autocorrelation Function. A further important tool to study the properties of the solvent and its relaxation behavior is given by the analysis of the dipolar relaxation times.26,27,44−46 The autocorrelation time for the dipolar orientation vector μ⃗ between two water molecules has been shown to follow ⟨μ ⃗ (t )μ ⃗ (t0)⟩ ≈ exp( −t /τ )β

(9)

with the stretching exponent β. autocorrelation function is defined by

26,27,44−46

Cμ(t ) =

The corresponding

⟨μ ⃗ (t )μ ⃗ (t0)⟩ ⟨μ ⃗ 2 (t0)⟩

(10)

The average relaxation time ⟨τ⟩ is then given according to ⟨τ ⟩ =

∫0

∞

exp( −t /τ )β dt =

τ ⎛1⎞ Γ⎜ ⎟ β ⎝β⎠

(11)

which can be derived by the integration of eq 9, respectively eq 10.

3. SIMULATION DETAILS Atomistic molecular dynamics simulations were performed to study the water dynamics around prototypes of AFPs and AFGPs by using the software package GROMACS 4.5.5.47−49 The structural unit of the prototype antifreeze protein, respectively prototype antifreeze glycoprotein, is considered to be an (AAT)x chain with x = 4−50 which can be found in Dissostichus mawsoni5 where A denotes alanine and T stands for threonine. The hydroxyl side-chain of threonine can undergo O-linked glycosylation which in terms of the AFGPs results in the presence of a covalently bonded disaccharide group β-Dgalactosyl-(1 → 3)-α-N-acetyl-D-galactosamine.5 The structures of AFP and AFGP prototypes5,50,51 were created by using the PRODRG server.52 The so-obtained force fields were finally edited and reparameterized according to ref 53 with respect to the values of the GROMOS43A1 parameter set for alanine and threonine.54 We have also investigated a pure polyalanine chain which was created by using the same strategy. The structure

4. NUMERICAL RESULTS 4.1. Water Dynamics around the Prototype Protein Residues. Recent experimental studies5−7 validated that the significant retardation of water dynamics around AFGPs, also known as long-range effect, is mandatory to assist the direct binding of these proteins to ice phases. It has been discussed that this property of AFGPs is strongly related to the antifreeze activity.7,18 Moreover, it has been shown that the long-range effect perturbs the hydration shell over long distances up to 2 nm around the proteins.5 In order to understand the underlying effects and mechanisms of the long-range effect, we investigated the water dynamics around prototype antifreeze proteins in allatom MD simulations by the study of the hydrogen bond 11615



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bonds with all proteins in comparison to water−water hydrogen bonds becomes even more obvious with regard to eq 3. Thus, the occurrence of larger τHB by log τHB ≈ ΔF†/kBT implies that longer hydrogen bonds life times are directly related to larger activation free energies that are needed to break the bonds. Hence, the values for τHB can be interpreted as good estimators for the energetic stability of hydrogen bonds. With respect to the results for the AFP compared to the polyalanine chain, the crucial influence of hydroxyl groups, as the main difference between alanine and threonine becomes obvious in terms of stronger hydrogen bonds in the presence of threonine. This finding has been recently introduced as the main reason for the ice-binding behavior of AFPs to ice crystals.20 Moreover, the importance of disaccharide groups becomes evident by the presence of increased forward hydrogen bond lifetime for AFGPs compared to that for AFPs. We can conclude that the presence of hydroxyl and disaccharide groups as the main differences between the proteins are essential for the observed effects. These results clearly illustrate the importance of threonine and polar disaccharide groups for the increase of the hydrogen bond lifetimes which is more or less a local effect in the first hydration shell. Interestingly, our results indicate that this behavior is attributed to the chemical unit of threonine instead of the presence of higher order secondary structure elements. Nevertheless our findings support the outcome of mutation experiments where it was found that the thermal hysteresis activity of AFP decreases when the threonine residues are replaced by the more apolar alanine residues.19 Therefore, we can conclude that the presence of threonine and disaccharide chains implies a strong influence on the direct binding of water molecules. In addition, we have also focused on the number of hydrogen bonds between the AFP and polyalanine chain to study the different hydration properties. The results are shown in Table 2. It can be clearly seen that the hydrogen bond densities ρ+HB are highest for AFGP followed by AFP which indicates a good solubility due to the presence of polar threonine and disaccharide groups. In general, the values for the hydrophilic as well as the total hydrogen bond density indicate a difference of ΔρHB ≈ 0.4 nm−2, respectively Δρ+HB ≈ 0.8 nm−2, between the AFP and the mutant, respectively ΔρHB ≈ 1.2 nm−2 and Δρ+HB ≈ 2.4 nm−2 between AFGP and the polyalanine chain. These results significantly illustrate the high solubility of AFGPs and therefore the high impact on the local surrounding aqueous water shell. The global influence on the water−water hydrogen bonds is shown in Figure 3. Here, we have calculated the hydrogen bond correlation function between water molecules in the presence of the proteins. It can be clearly seen that a significant strengthening of the water hydrogen bonds is achieved for AFGPs but also for polyalanine and AFPs compared to a pure aqueous solution. Hence, one can conclude that the hydrogen

characteristics and the dipolar relaxation times around the residues of typical AFPs and AFGPs. In the following we omit the designation prototype for the sake of readability. Furthermore, it has to be noted that all values have been determined by an averaging procedure over all corresponding molecules in the simulation box which means that any spatial dependence is absent. Figure 2 shows the results for the hydrogen bond correlation function CHB(t) according to eq 1 between water molecules and

Figure 2. Hydrogen bond lifetime correlation function CHB(t) for hydrogen bonds between water molecules and AFGP (blue line with squares), AFP (red line with circles), and the mutant of a polyalanine chain (gray line with diamonds) compared to water−water hydrogen bond life times (black line with triangles). The residue number for all protein molecules is x = 6 except for the AFGP where it is x = 2.

AFP, AFGP and the polyalanine chain. It can be clearly seen that all proteins lead to a significant increase of the corresponding hydrogen bond forward life times τHB compared to hydrogen bonds in an aqueous bulk solution in the absence of macromolecules. The exact values for τHB are shown in Table 1. Moreover, even for the slightly hydrophobic polyalanine Table 1. Hydrogen Bond Forward Life Times τHB According to the Results Shown in Figure 2 for Water Hydrogen Bonds with the Corresponding Molecules τHB (ps)

protein chain AFP and water AFGP and water polyalanine and water water and water

9.20 13.81 7.09 2.28

± ± ± ±

0.95 1.29 0.81 0.16

chain, we observe a significant increase of τHB. This finding can be related to the characteristics of apolar surfaces and the corresponding hydrophobic hydration concept.27,42 Thereby the presence of apolar surfaces leads to an increased water order and a strengthening of the hydrogen bond network around apolar solutes.27 The increased strength of hydrogen

Table 2. Number of Hydrogen Bonds nHB between Water and the Polyalanine Chain (Mutant), Respectively AFP and AFGP, Total and Hydrophilic Solvent Accessible Surface Area σt and σ+ and the Corresponding Total Hydrogen Bond Density ρHB and Hydrophilic Hydrogen Bond Density ρ+HB protein chain

nHB

σt (nm2)

ρHB (nm−2)

σ+ (nm2)

ρ+HB (nm−2)

mutant in water AFP in water AFGP in water

24.74 ± 3.96 31.21 ± 3.15 19.71 ± 2.53

12.10 ± 0.75 12.92 ± 0.74 6.09 ± 0.19

2.02 ± 0.45 2.45 ± 0.38 3.24 ± 0.52

4.55 ± 0.57 4.98 ± 0.46 2.53 ± 0.16

5.44 ± 1.55 6.27 ± 1.21 7.79 ± 1.49

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compared to a pure aqueous solution which is in agreement with the results shown in Figure 3. Hence, the presence of hydroxyl groups in threonine as can be seen by comparing the results for AFP and the polyalanine chain increases the strength of a direct water binding and can be considered as a local effect. It has to be noticed that all water molecules within the whole simulation box with a box length of minimally r ≈ 3.6 nm have been taken into account for the corresponding analysis. All these results let us conclude that the general presence of disaccharide groups as given in AFGP is essential for the occurrence of slower water dynamics whereas the hydroxyl groups of threonine are mainly involved in direct water binding mechanisms. An additional evidence for this observation is given by the corresponding water diffusion constants Dcm around the proteins as calculated by

Figure 3. Hydrogen bond autocorrelation function CHB(t) between water molecules in a pure aqueous solution (black line with triangles) and in the presence of AFP (red line with circles), AFGP (blue line with squares), and a polyalanine (gray line with diamonds).

2 ⟨( rcm ⃗ (t ) − rcm ⃗ (0)) ⟩ (12) t →∞ 6t where rc⃗ m denotes the center of mass position for a water molecule at different times t. The corresponding results are shown in Table 4. With regard to these findings, it can be also

Dcm = lim

bond network and also the water structure is significantly stabilized in the presence of the considered macromolecules. However, this effect is more pronounced for AFGPs which seems to be significantly related to the AFGP disaccharide groups due to the fact that the results for the polyalanine chain and the AFP are nearly identical. It becomes obvious that the presence of threonine compared to alanine does not have a pronounced influence on the water hydrogen bond dynamics. Finally, we have calculated the dipolar relaxation times of water molecules in the presence of the macromolecules. The corresponding results are shown in Figure 4 and Table 3. It can

Table 4. Water Diffusion Constant Dcm in the Presence of AFP, AFGP, and a Polyalanine Chain and in Pure Solution AFP AFGP polyalanine pure solution

Table 3. Mean Dipolar Relaxation Time ⟨τ⟩ for Water Molecules around AFP, AFGP, and a Polyalanine Chain Compared to a Pure Aqueous Solution system

dipolar relaxation time ⟨τ⟩ (ps) ∼4.8 ∼7.6 ∼4.7 ∼4.5

2.67 1.80 2.64 2.71

± ± ± ±

0.06 0.05 0.04 0.01

clearly stated that the long-range affect is explicitly attributed to the presence of the disaccharide groups in AFGPs as it becomes evident by the significantly lower diffusion constant compared to the other proteins. In terms of the dipolar relaxation times, the water−water forward hydrogen bond life times, and the diffusion constant, one can conclude that disaccharide groups strongly influence the water dynamics on a long length scale whereas the hydroxyl groups in AFPs mainly coordinate and bind water molecules in terms of a local effect. 4.2. Influence of Osmolytes on the Hydration Dynamics. As it has been discussed in the Introduction, the survival strategies of organisms also often rely on the presence of small osmolytes. Typical osmolytes in this context are urea as well as hydroxyectoine. Recent publications6,20 have pointed out the strong influence of osmolytes on the water dynamics in the presence of AFPs. In order to understand the mechanism as well as the interactions between osmolytes and AFPs, we have performed atomistic molecular dynamics simulations of hydroxyectoine and urea in the presence of a prototype AFP and a polyalanine chain where the repeat number of the structural unit for both is given by x = 15. We start the presentation of the results by the investigation of the corresponding water−protein hydrogen bond lifetimes in the presence as well as in absence of osmolytes as shown in Figure 5 and Table 5. It can be clearly seen that the presence of hydroxyectoine results in a strong increase of the water− protein hydrogen bond life times compared to the results shown in Figure 2. This effect is slightly less pronounced for urea. Hence, it can be concluded that the presence of chaotropic osmolytes in contrast to kosmotropic cosolutes only slightly increases the occurrence of the retardation effect around AFPs. We have also investigated the influence of

Figure 4. Dipole autocorrelation function Cμ(t) for water molecules in a pure aqueous solution (black line with triangles), an AFP solution (red line with circles), an AFGP solution (blue line with squares), and a polyalanine solution (gray line with diamonds).

AFP AFGP polyalanine pure solution

diffusion constant Dcm [10−5 cm2/s]

system

be clearly seen that the presence of the polyalanine chain and the AFP only slightly changes the dipolar relaxation times 11617



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Table 6. Mean Relaxation Time ⟨τ⟩ for Water Molecules around an AFP in Presence and in Absence of Osmolytes protein chain

relaxation time ⟨τ⟩ (ps)

AFP in water AFP in aqueous urea AFP in aqueous hydroxyectoine

∼4.9 ∼7.3 ∼80.1

induce a strong influence on the water dynamics which can be interpreted as a long-range effect around AFPs. Furthermore, it can be also remarked that the monomer concentration does not significantly influence the long-range water dynamics. This becomes explicitly obvious by comparing the results for the (AAT)15 chain in Table 6 and the (AAT)6 chain in Table 3 in pure water solution. For the understanding of the underlying binding mechanism of the osmolytes, we first study the accumulation behavior around the AFP via the cumulative number distribution function as shown in eq 5. The normalized results according to f(r)/f(rmax) where rmax = 2 nm are shown in Figure 7. Our

Figure 5. Correlation function CHB(t) for hydrogen bonds between water molecules and AFPs in a pure aqueous solution (red line with circles), a urea solution (blue line with squares), and in the presence of a hydroxyectoine solution (black line with diamonds). The repeat number for all protein molecules was x = 15.

Table 5. Hydrogen Bond Forward Life Times τHB for Hydrogen Bonds between Water and the AFP in Different Solutions protein chain

τHB (ps)

AFP in water AFP in aqueous urea AFP in aqueous hydroxyectoine

11.19 ± 1.11 15.87 ± 1.54 30.50 ± 3.04

osmolytes on the dipolar relaxation times of the aqueous solution. A change of this property should directly indicate a long-range effect, due to the fact that the dielectric relaxation behavior was calculated over all solvent molecules within the simulation box. The dipole autocorrelation function is shown in Figure 6 and the corresponding values for the mean relaxation Figure 7. Cumulative number of osmolyte molecules around the surface of the AFP. The red line denotes the results for urea, whereas the black line indicates the results for hydroxyectoine.

findings reveal that more urea molecules accumulate at the molecular surface of AFPs compared to hydroxyectoine. This clearly emphasizes the point that urea is favorably attracted to nearly all protein surfaces, which has been also validated by a recent publication.27 In contrast, the results for hydroxyectoine indicate an exclusion behavior from macromolecular surfaces which is a common principle for kosmotropic osmolytes.31 In order to systematically study the osmolyte accumulation behavior, we have evaluated the local preferential binding parameter according to eq 8 in Figure 8. It can be clearly seen that a preferential exclusion behavior is dominating for hydroxyectoine within a distance of 0.45 nm to the molecular surface whereas an overall preferential binding behavior can be detected for the interaction of prototype AFP with urea on distances larger than 0.2 nm. Hence, the observed characteristics are in good agreement to previous results and reflect the expected binding characteristics of the considered osmolytes.27,29−31,33 With regard to the above-discussed results, it becomes clear that the overall influence on the water dynamics is more pronounced for AFPs in the presence of kosmotropic osmolytes. It can be assumed that the strong zwitterionic properties of hydroxyectoine with the corresponding highly polar hydroxyl and carboxyl groups are mainly

Figure 6. Dipole autocorrelation function for water molecules in the presence of AFPs in a pure aqueous solution (red line with circles), an AFP within urea solution (blue line with squares), and in the presence of a hydroxyectoine solution (black line with diamonds).

times are presented in Table 6. We can deduce from these findings that the presence of hydroxyectoine strongly increases the mean dipolar relaxation time. The corresponding values for urea compared to the pure AFP solution are less pronounced. Hence, the molecular role of hydroxyectoine as a kosmotropic agent in terms of a strong water-structure maker becomes evident. Therefore, we conclude that kosmotropic osmolytes 11618



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binding of water molecules to the proteins. Thus, it can be assumed that the presence of threonine and disaccharide groups are of main importance for a strong water coordination. In addition to the hydrogen bond life times, we also studied the dipolar relaxation times of water molecules in the presence of AFP, AFGP, and polyalanine prototypes. It can be concluded from the corresponding results that the disaccharide groups significantly influence the water dynamics on a large length scale. It is also interesting to notice that the dipolar relaxation times in the presence of a polyalanine chain and an AFP prototype do not significantly change compared to a pure aqueous solution. Hence, the influence of the hydroxyl groups as present in threonine can be mainly attributed to a hydrogenbond mediated local water binding mechanism whereas the bulk water dynamics around AFPs are only slightly influenced. These findings are further evidenced by the values for the water diffusion constants around the protein prototypes. We also investigated the influence of osmolytes on the water dynamics in the presence of AFP residues. Our findings clearly indicate a significant increase of the hydrogen bond and dipolar relaxation times in the presence of hydroxyectoine. It is worth to mention that this effect is drastically more pronounced for kosmotropic osmolytes like hydroxyectoine which are preferentially excluded from the AFP prototype surface. In terms of the binding characteristics to the AFP, we have indicated a preferential exclusion as well as a preferential binding mechanism for hydroxyectoine, respectively urea. Thus, it can be assumed that the addition of kosmotropic osmolytes to the solution strongly decreases the dynamical behavior of the bulk water molecules. In contrast, chaotropic osmolytes which directly bind to the protein have a weaker influence on the hydration dynamics due to their vanishing presence in the bulk water phase for low concentrations. Our study clearly emphasizes the significant influence of AFGPs on the local water dynamics as well as the importance of disaccharide groups for the water retardation effect. It becomes evident that alanine and threonine are mainly responsible for the occurrence of a local effect due to strong water−protein hydrogen bonds, whereas the disaccharide groups in AFGPs strongly contribute to the long-range effect which results in a retardation of the bulk water dynamics. The influence of kosmotropic osmolytes further supports this behavior. These results may imply the combined usage of AFP, AFGP, and kosmotropic osmolytes to avoid freezing in industrial applications. Furthermore, it is clearly shown that the chemical structure instead of higher order secondary structure elements has a significant influence on the surrounding water dynamics. With regard to the occurrence of AFPs and osmolytes in nature, it can be barely negotiated that evolution always optimizes its molecular machinery to allow organisms to survive under different harsh conditions.

Figure 8. Local preferential binding parameter for the osmolytes and their binding characteristics to the surface of the AFP. The red line denotes the results for urea, whereas the black line represents the results for hydroxyectoine.

responsible for the strong retardation of the water dynamics. With respect to the typical position for hydroxyectoine, which is preferentially excluded from the AFP surface, the significantly higher impact for hydroxyectoine on the water dynamics due to its accumulation in the second solvation shell becomes reasonable. Urea, which is preferentially accumulated at the AFP surface, does not have such a strong influence on the global water behavior due to its vanishing presence in the aqueous bulk phase. With regard to these results, we can conclude that the observed strong retardation of water dynamics for AFPs in the presence of hydroxyectoine can be mainly attributed to the influence of the osmolytes. These findings may also explain the outcome of recent experiments.6,20

5. SUMMARY AND CONCLUSION The aim to understand the properties of antifreeze proteins and their interaction with the local aqueous environment has stimulated several experimental and theoretical studies over the last decades. A main goal has been the understanding of the mechanism behind the freeze resistance in the presence of AFGPs and AFPs. A consensus was achieved in which it was stated that antifreeze glycoproteins inhibit the growth of large ice crystals by the presence of a long-range and a short-range effect. The short-range effect mainly describes the mechanism of direct binding of ice to AFPs, respectively AFGPs. The difficult recognition of ice phases is facilitated by a long-range effect for which recent experimental studies5 have indicated a pronounced retardation of the water dynamics on large distances around AFGPs. We conducted atomistic molecular dynamics simulations to understand the molecular origins of this effect. We therefore focused on the chemical subunits of most antifreeze proteins and antifreeze glycoproteins which are given by (AAT) residue sequences to study the influence on the water dynamics in the presence of threonine residues and disaccharide groups. Our results clearly reveal that the life times of hydrogen bonds strongly depend on the presence of polar units, e.g., hydroxyl and disaccharide groups in the considered proteins. Thus, a clear increase of the water−protein hydrogen bond life times was observed following the order, polyalanine < prototype AFP subunit < prototype AFGP subunit. Our findings therefore imply that mainly hydroxyl groups are responsible for the

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Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest. 11619



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ACKNOWLEDGMENTS The authors thank Martina Havenith, Dominik Horinek, Joachim Dzubiella, Andreas Heuer, Hans-Joachim Galla, Rakesh Kumar Harishchandra, Julian Michalowsky, Martin Vögele, and Maria Fyta for fruitful discussions and useful hints. A.N.K. acknowledges funding from the Erasmus Mundus program “MathMods”. This work has been funded by the Deutsche Forschungsgemeinschaft through the Stuttgart Center of Excellence “SimTech” and the Sonderforschungsbereich 716.

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