Optimum Number of Anchored Clathrate Water and Its Instantaneous

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B: Biophysical Chemistry and Biomolecules

Optimum Number of Anchored Clathrate Water and Its Instantaneous Fluctuations Dictate Ice Plane Recognition Specificities of Insect Antifreeze Protein Sandipan Chakraborty, and Biman Jana J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b00548 • Publication Date (Web): 06 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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The Journal of Physical Chemistry

Optimum Number of Anchored Clathrate Water and its Instantaneous Fluctuations Dictate Ice Plane Recognition Specificities of Insect Antifreeze Protein

Sandipan Chakraborty, Biman Jana*

Department of Physical Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata-700032

*Corresponding author: Dr. Biman Jana: Department of Physical chemistry, IACS, Kolkata-700032, India. Phone: +91 33 2473 4971; Fax: +91 33 2473 2805; E-mail: [email protected]

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Abstract Ice recognition by antifreeze proteins (AFPs) is a subject of topical interest. Among several classes of AFPs, insect AFPs are hyperactive presumably due to their ability to adsorb on basal plane. However, the origin of the basal plane binding specificity is not clearly known. Present work aims to provide atomistic insight into the origin of basal plane recognition by an insect antifreeze protein. Free energy calculations reveal that the order of binding affinity of the AFP towards different ice planes is: basal plane > prism plane > pyramidal plane. Critical insight reveals that the observed plane specificity is strongly correlated with the number and their instantaneous fluctuations of clathrate water forming hydrogen bonds with both ice binding surface (IBS) of AFP and ice surface, thus anchoring AFP to the ice surface. On basal plane, anchored clathrate water array is highly stable due to exact match in the periodicity of oxygen atom repeat distances of the ice surface and the threonine repeat distances at the IBS. The stability of anchored clathrate water array progressively decreases upon prism and pyramidal plane adsorption due to mismatch between the threonine ladder and oxygen atom repeat distance. Further analysis reveals that hydration around the methyl side-chains of threonine residues becomes highly significant at low temperature which stabilizes the anchored clathrate water array and dual hydrogen bonding is a consequence of this stability. Structural insight gained from this study paves the way for rational designing of highly potent antifreeze-mimetic with potential industrial applications.

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Introduction Macromolecules that control ice growth draw immense research interests in recent years due to their potential application in biotechnological industries and regenerative medicines.1-3 Several synthetic polymers,1 biomimetic poly-vinyl alcohol (PVA)3-5 and polyampholytes6 have been shown to elicit ice growth inhibition activities. Recently, the cryopreservation of erythrocytes has been reported by using hydroxyl-ethyl starch and PVA.1,7 Cryopreservation of primary rat hepatocytes by ice growth inhibitory polymers also has been shown recently.2 Designs of these synthetic polymers are inspired from its natural counterparts, antifreeze proteins (AFPs). This special class of protein is evolved due to the natural selection of cold-adapting organisms to survive in sub-zero conditions. There are enormous structural diversities of AFPs present in different species. AFPs are commonly classified as fish AFPs (type I-IV), insect AFPs and antifreeze glycoproteins.8 Among several classes of AFPs, insect AFPs show the highest degree of ice growth inhibition and therefore termed as hyperactive.9 Ice recrystallization inhibition (IRI) and thermal hysteresis (TH) are the two common measurements of anti-freezing activities. However, their inter-relation remains highly controversial and strongly depends on measuring techniques.10 At low concentration, AFPs inhibit growth of large ice crystal from small ice nucleus which is known as IRI.11 On the other hand, at high concentration, AFPs inhibit ice growth by TH activity which is non-equilibrium depression of freezing point of ice without altering the melting point.8 TH activity qualitatively can be explained by the adsorption-inhibition model of ice recognition. According to this model, TH activity can be rationalized in terms of “Gibbs-Thomson effect”.8,12,13 Binding of APFs to a growing ice front induces curvature between two bound 3



AFPs. With further AFP binding, there is gradual decrease in the effective radius of the newly grown ice surface. When the radius falls below the critical limit, ice growth halts. However, the microscopic insights into the ice-recognition by natural antifreeze proteins or by synthetic polymers are highly controversial.14 Earlier concept of ice recognition by AFP was thought to be mediated by direct hydrogen bonding between ice and AFP.15-17 However, experimental mutational data invalidated the hypothesis. Zhang et al.18 demonstrated that the anti-freezing activity of type I AFP completely abolishes upon mutation of threonine at the IBS with another hydrogen bonding amino acid, serine. However, upon valine (devoid of hydrogen bonding sidechains) mutation, the AFP remains active. These results implicate the role of hydrophobicity during ice recognition. Although recently, using computer simulation it has been shown that biomimetic antifreeze polymer poly(vinyl alcohol) (PVA) binds to the prismatic faces driven by hydrogen bonding, facilitated by distance matching between the hydroxyl groups of PVA and ice surface.19 Thus it might be possible that mechanism of ice recognition of antifreeze polymer is distinctively different than anti-freeze protein. It is pertinent to mention that antifreeze polymers are structurally more related to antifreeze glycoprotein and also bear no resembles with fish or insect AFPs. Furthermore, crystal structures of type III and insect AFP reveal that the ice binding surface (IBS) is flat and hydrophobic compared to non-IBS planes.20-23 Therefore, lately a snugfit model was proposed where the IBS of AFP fits on the ice plane driven by strong steric complementarity and van der Walls interactions.20,22,23 However, the ice/water interface is not that rigid to provide surface complementarity, rather it is a slowly diffusive water layer that extends over 10-15 Å from ice surface.24,25 This observation seriously questions the validity of the snug-fit model. Unavailability of proper structure-function relationship of antifreeze activity impedes developments of highly potent novel antifreeze polymers with industrial application. 4


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AFPs draw immense research attention recently due to its unusual hydration properties.26-35 Modulations of the dynamics of hydration water over long distances have been demonstrated in case of antifreeze glycoproteins.34,36 Even, presence of ice-like ordered water around the IBS has been reported for AFP.37,38 However, the relation between the observed unusual hydration patterns on the ice growth inhibition ability of AFP is still not clear. Nutt and Smith proposed that the preconfigured hydration water around the IBS of AFP help the protein to adsorb on the ice plane.29 P. L. Davies and his group observed an array of ordered water directly hydrogen bonded to the IBS, termed as anchored clathrate water.39,40 However the atomistic details of the process of AFP adsorption and the role of anchored clathrate water remain unclear since none of the study probed AFP on ice surface, explicitly. Recent microfluidic experiments demonstrated that AFPs indeed adsorb strongly on ice surface41 and the nature of binding is irreversible.41,42 Recently we have demonstrated direct role of the ordered water on ice recognition by both type I43 and insect AFP.44 According to our hypothesis, AFP adsorbs on the ice plane through water cage framed around the IBS, particularly around the methyl side-chains of threonine residues. Ice/water interface as well as surface of the ice plane complete the caging around the IBS.44 The complex undergoes dynamic crossover to a hydrogen bonded complex upon further growth of one layer of ice front. This model of ice plane recognition corroborates the experimentally observed time-dependent TH activity45 as well as irreversible nature of AFP binding to ice surface.46 Interestingly, it has been shown that type I AFP inhibits gas hydrate crystal growth through a similar water mediated adsorption mechanism. The proposed binding mode further supports our observed water cage mediated ice surface adsorption mechanism.47 Fluorescence microscopy reveals that spruce budworm AFP (sbwAFP) accumulated on both prism and basal planes.42 The affinity towards the basal plane is related to the observed hyperactivity of insect 5



AFPs.42 However, how this water mediated binding model rationalizes the experimentally observed binding specificities and hyperactivity is still unknown. Also the principal structural features of the IBS that stabilize the ordered water need to be explored in critical details to understand structure-function relation of antifreeze activity which can be used to design more potent antifreeze mimetic. Here, we have explored the mechanisms behind the observed ice plane binding specificities of an insect AFP and the origin of anchored clathrate water molecules which mediates binding of AFP on ice surface with the aid of equilibrium simulations, free energy calculations and other relevant analysis.

Methods All the molecular dynamics simulations and free energy calculations were carried out by GROMACS 4.5 packages48-51 using OPLS/AA52,53 force field and SPC/E54 water model. It is noteworthy that the melting temperature (Tm) of SPC/E water is 215 K which is lower than the Tm of four-point and five point water model.55 However, Bryk et al., also demonstrated that the basal and prism plane of ice interfaces can be appropriately mimicked by SPC/E water at the temperature 225 ± 5 K.56 Within this range, density, translational, orientational and dynamic order parameters smoothly change from crystal to liquid across the interface.56 Preparation of the systems 3-D co-ordinates of the insect AFP protein isoform 501, referred as sbwAFP, from Spruce budworm (Choristoneura fumiferana) were obtained from Protein Data Bank (PDB ID: 1m8n).57 There are six crystallographic water molecules that form dual hydrogen bonds with the threonine 6


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residues of the IBS, known as anchored clathrate water.39 These six water molecules were retained and all other hetero-atoms were not considered during the simulation. In the IBS of sbwAFP contains an array of threonine residues (residue number 7, 23, 39, 54, 69, 84 and 101). Apart from THR 7, all other six threonine residues form hydrogen bonds with the anchored clathrate water molecules. These six threonine residues, i.e., residue number 23, 39, 54, 69, 84 and 101 were mutated to serine residues to construct the serine mutant AFP. Serine mutant was derived from the wild-type protein using the mutagenesis toolkit implemented in Visual Molecular Dynamics (VMD) packages.58 Molecular docking of AFPs with different ice planes In this work we have considered three different ice planes, i.e., basal, prism and pyramidal planes. Initial AFP-ice docked complexes were obtained by a rigid body docking procedure using the PatchDock webserver.59,60 For the wild-type sbwAFP, molecular docking was carried out with all the three ice surfaces. For serine mutant, docking was performed with the basal plane only. In all the docking procedures, anchored clathrate water molecules bound to the AFP surface were considered. Lowest energy docked complexes were considered as an initial structure for binding free energy calculations. Calculations of potential of mean force (PMF) for AFPs adsorption on different ice planes Each of the docked AFP-ice complex was then equilibrated using equilibrium molecular dynamics simulation before potential of mean force calculations. In each case, the ice slab was aligned on X-Y plane and then solvated with water in a rectangular water box with periodic boundary condition. For wild type and serine mutant sbwAFP bound to basal plane, the size of

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the simulation box was 100 × 80 × 100 Å3 whereas in case of wild type AFP bound to prism and pyramidal plane, the box size was 100 × 80 × 120 Å3 and 100 × 100 × 120 Å3, respectively. The box was so chosen that the ice slab does not collide with its image from neighboring boxes in X and Y direction. Along the Z-direction, the box length is greater than double of the final pull distance between AFP and ice plane center of mass. All the water molecules (solvent water, ice plane and anchored clathrate water) were defined by SPC/E water model. Three Cl- ions were added in each system to make it charge neutral. In all the simulations, the ice slabs were kept frozen. Each system was then subjected to 500 steps of energy minimization using steepest descent algorithm. Each minimized complex was subjected to 1 ns position restrained dynamics where the ice slab was kept frozen and protein backbone atoms were restrained, but water molecules were allowed to move freely in NVT ensemble at 225 K. Then for each system a 3 ns NVT simulations was performed at 225 K where both proteins and water molecules were allowed to move freely except for the ice slab. Temperature was maintained by employing an external bath with a coupling constant of 0.1 ps using v-rescale algorithm and electrostatic interactions were calculated using particle mesh Ewald summation method. Umbrella sampling technique was used to calculate the PMF profiles of AFP binding to different ice planes. The center of mass distance along the Z-axis between AFP and ice slab was considered as the reaction co-ordinate and the AFP was pulled from the ice surface along the Zdirection with an interval of 0.1 nm using an umbrella potential with a force constant of 5000 kJ mol-1 nm-2. In each umbrella window, 1 ns equilibration simulation followed by 2 ns production run was performed in NVT ensemble. All the other simulation parameters were kept same as mentioned above. Weighted histogram analysis method (wham)61 was then used to construct the PMF profile. Sufficient overlaps among the all the windows were confirmed by histogram 8


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analysis. Same methodology was used to construct PMF profiles for all the cases. Validation of the methodologies, water models used to construct the PMF profile for ice adsorption by insect AFP is provided in our recent publication.44 The sbwAFP-ice complex corresponds to each PMF minimum was then subjected to 30 ns NVT equilibrium simulations at 225 K and 210 K for further characterization of ice adsorption and in all the simulations, ice slab was kept frozen in all the three dimensions. Free energy calculation using metadynamics: The interfacial water as collective variable Free energy simulations were performed using metadynamics methodology implemented in PLUMED 1.362 plugin for GROMACS 4.5. Major problem in deriving complete free energy landscape of biological macromolecules is its multi-dimensionality. Metadynamics is an effective procedure where free energy is expressed in terms of few collective variables (CVs). In this process the dynamics of the system is biased by a history-dependent repulsive potential constructed as a sum of Gaussian functions along the trajectory of the simulation. The sum of Gaussians is then used to reconstruct the free energy of the system. Gaussians are defined by hill height and sigma values.63,64 Sampling of the configurational space is enhanced by the rate of added Gaussians. 1-D free energy surfaces for wild-type AFP adsorption on basal, prism and pyramidal plane were constructed using interfacial water as collective variable (CV). The CV is defined using a switching function which has following generalized expression65:

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  r −r  1−  i 1 n0   r0 S wat CV = ∑  i  1 −  ri − r1   r   0

n    ri − r2   1 −     r0 m     ri − r2   1 −     r0

n       m       

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(1)

Here, ‫׀‬ri –r1‫ ׀‬are distance between any threonine oxygen at the IBS of wild-type sbwAFP and clathrate water oxygen. ‫׀‬ri –r2‫ ׀‬are distance between oxygen at the surface of respective ice plane in each case and clathrate water oxygen. Values of n=8, m=12 and r0= 2.5 Å were used in all the free energy calculations. We have observed that clathrate water molecules are involved in strong hydrogen bonding interactions with characteristic donor-acceptor distances that vary between 2.2 to 2.8 Å. Therefore, we have chosen r0= 2.5 Å and using this value the obtained numbers of interfacial water matches with the number of anchored clathrate water calculated with the dual hydrogen bonding criterion, i.e., water form dual hydrogen bonds with IBS and ice surface. Increasing the value of r0 increases the number of interfacial water molecules however, the qualitative differences of different ice plane adsorption by AFP remains similar. The structure of the AFP-ice complex corresponds to each PMF minimum was considered as a starting structure for metadynamics run. Simulations were carried out at 225 K in NVT ensemble and continued until the point of desorption of AFP from ice surface in each case. The Gaussian function of height 0.4 kJ/mol with the sigma value of 0.1 nm was added in every 2 ps. Equilibrium simulation of wild-type and serine mutant sbwAFP at different temperatures Both the wild-type and serine mutant sbwAFP were initially energy minimized in vacuo using the steepest descent algorithm and then solvated in a cubic box of SPC/E explicit water with periodic boundary condition such that protein atoms were at least 10 Å apart from the box edges. 10


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Both the systems were then made charge neutral by adding three Cl- ions. Each system was then subjected to 500 steps of steepest descent energy minimization followed by 5000 steps of energy minimization using conjugate gradient algorithm. Each minimized solvated AFP was equilibrated for 1 ns using position restrained dynamics in the isothermal-isobaric (NPT) ensemble where the protein was restrained using a force of 1000 kJ/mol.nm2 in all three dimensions while the rest of the systems were allowed to move freely. For each system, the position restrained equilibrations were performed in four different temperatures (210 K, 225 K, 250 K and 298 K) in NPT ensemble. Temperature was kept constant at desired value by employing an external bath with a coupling constant of 0.1 ps using v-rescale algorithm. The pressure was kept constant (1 bar) by employing isotropic Parrinello-Rahman barostat with the time-constant set to 2 ps. Electrostatic interactions were calculated using particle mesh Ewald summation method. For each system, the 50 ns production runs were carried out in the NPT ensemble at those four different temperatures. During the production run both protein and water molecules were allowed to move freely. The trajectories were stored at every 1 ps.

Result and Discussions Here, we have explored the molecular origin of the observed ice plane binding specificities of the insect AFP with the aid of free energy calculation and equilibrium simulations. We have performed four binding free energy estimations using PMF calculations: wild-type sbwAFP adsorption on basal, prism, pyramidal planes and serine mutant AFP adsorption on basal plane. Structures of the IBS of sbwAFP are shown in Figure 1A where the residues considered for the

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construction of serine mutant have been highlighted. Surface topologies of the three different ice planes considered in the study also have been shown in Figure 1B.

Figure 1: (A) Energy minimized structure of the sbwAFP is shown in cartoon representation. Anchored clathrate waters are shown in VDW mode whereas the perfectly aligned threonine residues, referred as THRs ladder, are shown in stick mode. Threonine residues at the ice binding surface that have been mutated to serine residues to construct the serine mutant sbwAFP have been highlighted. (B) Surface topologies of three different ice planes are shown. Ice water is shown in stick mode.

The primary objective for the construction of serine mutant AFP is to understand the structureactivity relation of hyperactive insect AFP. Notably, the IBS of the AFP is exclusively designed by perfectly oriented threonine residues, the THRs ladder, rather than serine residues (amino acid 12


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with similar hydrogen bonding groups, but devoid of the methyl group). Therefore, a legitimate question is that the hydroxyl groups are sufficient to stabilize the anchored clathrate water or methyl side chains of threonine residues are needed to form the stable array of anchored clathrate water? To explore the molecular origin of the stability of the anchored clathrate water, we have specifically designed the mutated sbwAFP. Binding free energy calculations of wild-type and mutant AFP with different ice planes: Potential of Mean Force (PMF) calculations Highest binding affinity has been observed when the wild-type AFP is adsorbed on the basal plane, evident from the PMF profiles (Figure 2). Also wild-type AFP shows considerable binding affinity with the prism plane however it is substantially lower than the basal plane adsorption. Wild-type AFP shows least binding affinity towards the pyramidal plane (Figure 2). Interestingly, serine mutant shows considerable affinity towards the basal plane. While the calculated binding affinity is lower compared to the adsorption of wild-type AFP on the basal and prism planes, however it is higher than the pyramidal plane adsorption by wild-type sbwAFP. It is noteworthy that we have generated the initial AFP-ice complexes in all the four cases using a rigid body docking procedure implemented in the PatchDock Server.

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Figure 2: PMF profiles for the adsorption of wild-type sbwAFP to different ice planes: the basal plane of ice (black), prism plane (red) and pyramidal plane (blue). Also the PMF profile for serine mutant sbwAFP adsorption to the basal plane is shown as green line. Error estimations were carried out from the bootstrap analysis. Closer insights into the minima of the PMF profiles are shown in the inset.

All the initial AFP-ice complexes are hydrogen bonded complexes where the THRs ladder is directly hydrogen bonded to the respective ice surfaces. The characteristic center of mass (COM) distance between the THR ladder and ice surface oxygen along the Z-axis is 0.46 nm. However, 14


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the minima in PMF of wild-type AFP adsorbed on basal, prism and pyramidal plane appears ~0.56 nm and for serine mutant, the minimum appears ~ 0.50 nm (Figure 2, inset). Structure of the wild-type AFP-basal plane complex corresponds to the PMF minimum is shown in Figure 3AI. The bound state is not a hydrogen bonded complex rather an adsorbed complex has been observed. In this state the methyl groups of the THRs ladder orient toward the ice plane. The observed complex structure is similar to our earlier reported structure of the adsorbed sbwAFP on basal plane.44 Details of this structure and its implications on the anti-freezing activity have been discussed in critical details in our recent publication.44 An important observation is the role of anchored clathrate water molecules on the binding of AFP to the ice surface. In case of basal plane adsorption, the anchored clathrate water forms cooperative dual hydrogen bonds with both the THRs ladder and ice surface. In fact, all the anchored clathrate water molecules are engaged in highly tetrahedral hydrogen bonding network. Perfectly positioned hydrogen bond donors and acceptors on the THRs ladder and ice surface facilitate this tetrahedral hydrogen bond network formation (Figure 3AII). Anchored clathrate water forms dual hydrogen bonds with two adjacent threonine residues of the THRs ladder and a single hydrogen bond with the ice surface.

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Figure 3: Structures of the wild-type (A) and serine mutant (B) sbwAFP-ice complexes correspond to the PMF minima are shown. Side views of the bound state of the wild-type AFP (AI) and serine mutant (BI) on the basal plane are shown. Protein is rendered in brown cartoon representations and ice plane as light blue surface. THRs/SERs ladder is shown as cyan sticks whereas the anchored clathrate water molecules are represented in vdW representation. Ice surface waters that are in close proximity are rendered as red sticks and hydrogen bonding interactions are shown as red dotted lines. A closer look into the binding details of the THRs/SERs ladder of wild type sbwAFP (AII) and serine mutant (BII) on the basal plane in presence of anchored clathrate water are shown. Anchored clathrate water molecules are 16


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represented in vdW mode and ice surface and THRs ladder are shown in stick representation. Hydrogen bonds are shown as red dotted lines.

Interestingly, when the THRs ladder is mutated with the serine residues, the PMF minima becomes a hydrogen bonded minima where the SERs ladder directly forms hydrogen bonds with the ice surface (Figure 3BI). All the hydrogen bonding contacts between anchored clathrate water molecules and IBS of the AFP are lost. However, due to confinement these water molecules do not diffuse away rather form hydrogen bonds with the ice surface only (Figure 3BII). Thus the OH groups alone are not sufficient enough to provide the stability to the anchored clathrate water array rather methyl side chains of THRs ladder are needed to stabilize the array. Furthermore, we have analyzed the structures of AFP-ice complexes correspond to the PMF minima in case of AFP adsorbed on prism and pyramidal planes (Figure 4). In both the cases, the minima correspond to a state where AFP is adsorbed on the ice surface and the initial hydrogen bonded complex is not the PMF minimum (Figure 4 AI-AII & 4 BI-BII). The bound state is very similar to the AFP adsorbed state on the basal plane. In both the AFP adsorbed states, the methyl side-chains of THRs ladder orient towards the respective ice surface. Critical insight reveals that the observed differences of differential binding plane affinity are related with the stability of the anchored clathrate water array (Figure 4AII & 4BII).

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Figure 4: Structures of the wild-type sbwAFP-ice complexes correspond to the PMF minima for prism plane (AI) and pyramidal plane (BI) adsorption are shown. Protein and THRs ladder are rendered in cartoon and stick representations, respectively. Ice plane is shown as light blue surface, whereas the anchored clathrate water molecules are represented in vdW representation. Ice surface waters that are in close proximity of the IBS are rendered as red sticks and hydrogen bonding interactions are shown as red dotted lines. A closer look into the binding details of the THRs ladder at the IBS of wild type sbwAFP on prism plane (AII) and pyramidal plane (BII) are shown. Anchored clathrate water molecules are represented in vdW mode and ice surface and THRs ladder are shown in stick representation. Hydrogen bonds are shown as red dotted lines. 18


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Structure of the adsorbed AFP on prism plane corresponding to the PMF minimum reveals a considerable destabilization of the anchored clathrate water compared to the basal plane adsorption. Among the six anchored clathrate water molecules, three water molecules are involved in tetrahedral dual hydrogen bonding network with THRs ladder and ice surface. Two anchored clathrate water molecules shift towards the THRs ladder while the remaining water molecule at the edge of the THRs ladder diffuses away from the binding surface of the AFP. High destabilization of the co-ordinated tetrahedral water network of the anchored clathrate water molecules is also observed when the AFP adsorbed on the pyramidal plane. Only two of the anchored clathrate water molecules maintain the initial dual hydrogen bonded network with both the IBS and ice surface. Rest of the water molecules either shift towards the THRs ladder or ice surface. The anchored water at the edge of the IBS ultimately diffuses away from the binding surface of AFP. It is noteworthy that each ice surface has a unique surface topology (Figure 1B) and significantly different from each other. In case of basal plane, there is perfect match between the threonine repeat distance at the IBS and the oxygen atom repeat distance at the ice surface. Therefore the anchored clathrate water molecules find hydrogen bond donor and acceptor groups at appropriate position such that it can form highly tetrahedral dual hydrogen bonding network involving both the THRs ladder of the IBS and ice surface. However, such periodicity is disturbed in case of prism and pyramidal plane. The repeat distances of threonine in THRs ladder matches partially with the oxygen repeat distance of prism plane and the degree of this match is lowest in case of pyramidal plane. Therefore, the anchored water molecules do not find donor/acceptor in appropriate position to form a stable tetrahedral dual contact hydrogen bonding network involving both THRs ladder and ice surface. 19



Number and its fluctuations of anchored clathrate water dictate binding specificities: Evident from equilibrium simulation To critically analyze the clathrate dynamics when the AFP is adsorbed on different ice planes, we have performed equilibrium simulations. The structures correspond to the PMF minima when AFP adsorbed on basal, prism and pyramidal plane are subjected to equilibrium simulations. Evident from the crystal structure as well as from the free energy simulations, anchored clathrate water molecules are involved in a network of highly tetrahedral hydrogen bonding network where it forms at least two hydrogen bonding contacts with the IBS and one hydrogen bonding interaction with the ice surface. We have used distance criterion to define a hydrogen bond, i.e., if a donor-acceptor pair is within the distance of 3.5 Å then the tagged pair is considered to be involved in hydrogen bonding interactions. We have therefore analyzed the number of water molecules that form such dual contact tetrahedral hydrogen bonding interactions involving both the IBS and ice surface throughout the simulation timescale and results are shown in Figure 5.

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Figure 5:

(A) Number distribution of anchored clathrate water molecules that form dual

contacts with the IBS and a single contact with the ice surface during simulation time scale for wild-type sbwAFP adsorbed on basal (black), prism (red) and pyramidal (blue) is shown. (B) Average number of anchored clathrate water molecules and its fluctuations are shown when wild-type AFP adsorbed on different ice planes.

Evident from Figure 5A, three water molecules occupy anchored clathrate water positions when AFP adsorbed on the basal plane. However, in case of prism plane adsorption, the distribution possesses two peaks, the major peak represents adsorbed complex with two anchored clathrate water and the second one corresponds to four water molecules occupy the anchored clathrate positions. Interestingly, during pyramidal plane adsorption by the AFP, there are one-two anchored clathrate water molecules. However, the distribution is narrow in case of AFP adsorption on basal plane compared to the other two planes adsorption which implicates reduction in number fluctuations and hence greater stability of anchored clathrate array. We have further characterized the average number fluctuations of anchored clathrate water array () in all the three cases using the given equations and results are shown Figure 5B.

χ =

1  (| N clathrate − N clathrate |)   ∑ n  N clathrate 

(2)

has been calculated by averaging the instantaneous fluctuations (χi) over the entire simulation timescale. Nclathrate is the number of water molecules that forms two contacts with the THRs ladder and at least a single contact with the ice surface at the ith step and is the 21



average number of such clathrate water. Evident from the figure, least fluctuations have been observed in case of AFP adsorption on basal plane. Interestingly, when AFP adsorbed on the prism plane, although there is sufficient number of anchored clathrate water molecules however their fluctuation are quite high. This observation can be rationalized in terms of the degree of mismatch between the hydroxyl groups of THR ladder periodicity at the IBS and Oxygen atom repeat distances of ice surface. When AFP is adsorbed on pyramidal plane, the average number of anchored clathrate water is lowest and associated with high fluctuations indicating highest degree of destabilization of the anchored clathrate water array (Figure 5B). Considering both the number and their instantaneous fluctuations of anchored clathrate water rationalize the binding specificities of insect AFP obtained from PMF calculations, i.e., Basal plane > Prism plane > Pyramidal plane. However, results obtained from the equilibrium simulations are limited due to time-scale of the simulation thus free energy calculations are needed to reinforce the conclusion obtained from equilibrium simulations. We have performed free energy calculations using metadyamics to construct the 1-D free energy surface using water-bridge as an order parameter (Figure 6). Water bridges are contacts involving both the IBS and ice surface and mediated by water molecules.

22


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Figure 6: 1-D free energy surface obtained by considering the number of water-bridges as order parameter using metadynamics simulation. Wild-type sbwAFP adsorbed on basal plane: black, prism plane: red and pyramidal plane: blue. In case of basal plane adsorption, there is a single minimum corresponding to the number of water-bridge of 6.5 which is the highest in comparison to prism and pyramidal plane adsorption. Thus the number of water-bridge formation during the basal plane adsorption is highest and therefore the adsorbed complex is highly stabilized, in accordance with the PMF profile. Interestingly, the thermodynamic minima for prism and pyramidal plane adsorption of sbwAFP appear at the water-bridge CV value of 5.1 and 4.2, respectively. The free energy calculation further reinforces the observation obtained from equilibrium simulations and in line with the order of binding affinity of sbwAFP towards different planes obtained from the PMF calculations. All these results signify that the observed binding specificity of AFP is primarily

23



dictated by the cooperative dual hydrogen bond formation by anchored clathrate water molecules with both the IBS and ice surface. Functional transitions are possible when the AFP is adsorbed on basal or prism plane Drori et al.,45,66 recently showed that the thermal hysteresis (TH) activity of insect AFP increases with time. We have recently probed the origin of the time-dependent TH activity for the sbwAFP adsorbed on basal plane using the F4 order parameter. F4 is a highly sensitive order parameter which can distinguish between ice and clathrate water.67-69 F4 order is defined as the H-O-O-H torsion angle between two adjacent water molecules involved in hydrogen bonding network. Here, two hydrogen atoms are those which are the furthest away from the Oxygen of the other water molecule. Details of the calculations are discussed in Fidler et.al.,67 Value of F4 is positive for clathrate, negative for ice and almost zero for liquid water. At the point of initial contact to the ice plane in presence of an ice/water interface (Condition has been mimicked by simulating at 225 K, 10 K above the melting point of SPC/E water), AFP adsorption is mediated through the clathrate water shell around the IBS. However, in the TH regime when there is further growth of ice surface (Condition has been mimicked by simulating the adsorption at 210 K, 5 K below the melting point of SPC/E water) the complex undergoes dynamic crossover to a hydrogen bonded complex and hydration water around the IBS becomes ice like. Therefore, the anchored chathrate water molecules which form dual hydrogen bonding interactions with both the IBS and the ice surface now become part of the newly grown ice surface. As a result, the insect AFP is now directly hydrogen bonded with the newly grown ice surface. This mode of binding supports the experimentally observed irreversible binding mode.41,46 Details of these functional transitions are discussed in our recent paper.44 24


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Figure 7: Analysis of F4 order parameter distribution of water around the THRs ladder of the IBS of sbwAFP-ice adsorbed complexes from equilibrium simulation at 225 K (black) and 210 K (red) when AFP is adsorbed on basal plane (A), prism plane (B) and pyramidal plane (C).

When AFP adsorbs on basal plane this dynamic transition from clathrate-like water to ice like water is clearly evident from Figure 7A upon changing the temperature from 225 K (black) to 210 K (red). This dynamic transition is also evident when AFP adsorbs on prism plane (Figure 7B). However the degree of magnitude of this dynamic transition is less compared to basal plane adsorption. Notably, the dynamical transition from clathrate-like water to ice-like water is absent when AFP adsorbs on the pyramidal plane (Figure 7C). Due to mismatch between the hydroxyl side-chain periodicity of THRs ladder and ice surface, the anchored clathrate array is highly destabilized when AFP adsorbs on pyramidal plane. This significantly lower the number of clathrate water around the IBS which results in low binding affinity as well as hinders the functional transition of the clathrate mediated adsorbed complex to hydrogen bonded complex. 25



Both hydrogen bonding and hydrophobic groups are needed to stabilize the anchored clathrate water array Interesting observation from the PMF calculation is that in case of serine mutant, the minimum is shifted to lower IBS-ice surface distance and the contact minima is a hydrogen bonded state. Therefore, the serine mutant AFP is more close to the ice surface which is also evident from the equilibrium simulation (Figure 8A). Throughout the simulation timescale, the center of mass distance between the protein and ice plane is lower in case of the serine mutant compared to the wild-type AFP. Thus the effective volume in between the IBS and ice surface is significantly lower in case of serine mutant. Therefore the anchored clathrate water molecules are trapped within the confined space and unable to diffuse away. They are marginally stable due to the confinement compared to the pyramidal plane adsorption however they are not involved in the dual hydrogen bonding network as observed in case of wild type AFP adsorbed on the basal plane.

26


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Figure 8: (A) Center of mass distance between AFP and ICE plane along the z-axis obtained from the equilibrium simulation of wild-type (black) and serine mutant (green) sbwAFP adsorbed on basal plane. (B) Normalized distribution of hydrogen bonds of the two AFP-ice complexes obtained from equilibrium simulation. AFP-anchored clathrate water hydrogen bonds: black; ice-anchored clathrate hydrogen bonds: red; AFP-ice surface hydrogen bonds: green.

Analysis of the hydrogen bond number distribution for wild-type and serine mutant AFP adsorbed on the basal plane obtained from equilibrium simulation is shown in Figure 8B. When wild-type AFP is adsorbed on the basal plane, the number of hydrogen bonds between the THRs ladder and ice surface are very low, as the adsorption is mediated through anchored clathrate water. On the other hand, anchored clathrate water molecules are forming 5-7 and 8-9 hydrogen bonds with the ice surface and THRs ladder, respectively (considering the all possible donoracceptor pairs). However, the hydrogen bonding pattern markedly changes in case of serinemutant. The serine mutant AFP binds directly to the basal ice plane and the number of hydrogen bonding interactions between the serine ladder and the ice surface are ~ 4-5. Therefore, the confined anchored clathrate water losses hydrogen bonding interactions with the IBS and the numbers essentially reduce to 2. The confined water molecules reorient themselves more towards the ice surface such that the hydrogen bonding interactions are maximized, evident from the increase in number of hydrogen bonding contacts between anchored clathrate water and ice surface in case of serine mutant in comparison to the wild-type AFP (Figure 8B). Thus serine mutation destabilizes the anchored clathrate water array. Therefore, the hydrogen bonding groups solely can’t stabilize the anchored clathrate water, rather methyl side-chains of 27



threonine residues are needed to stabilize the anchored clathrate array. To gain an atomistic insight into the process, we have performed equilibrium simulations of the serine mutant sbwAFP at four different temperatures (210, 225, 250 and 298 K) and compared with the wildtype AFP. Structure of the water shell around the IBS of wild type and serine mutant sbwAFP has been inferred in terms of the O-O-O angle distribution of water in the first layer of solvation shell around the Cγ of THRs ladder (wild-type) and Cβ of serine residues at the IBS of serine mutant and results are shown in Figure 9. The angle distribution is associated with two characteristic peaks: first one is ~ 100-108° represents tetrahedral water network whereas second one is ~ 60° is related to the interstitial water.70,71 Evident from Figure 9AI, there is a peak shift towards higher angle value in case of wild type compared to the serine mutant AFP at 210 K which implicates that the water network around the IBS is more tetrahedral in case of wild-type. At 225 K, there is a noticeable increase in the population of tetrahedral water around the IBS in case of wild-type in comparison to the serine mutant. More importantly, the principal peak in the angle distribution for serine mutant appears ~ 103° whereas in case of wild-type AFP it appears ~ 106° (Figure 9AII). Thus the hydration water around serine is less tetrahedrally ordered. At 250 K, the population of tetrahedral water around the IBS is nearly same for wild type and serine mutant AFP however the peak maximum shifts towards higher values in case of wild-type compared to the serine mutant (Figure 9AIII). Thus the hydration water around the serine mutant is less tetrahedral. However, the angle distribution of hydration water around the wild-type and serine mutant AFP becomes similar at 298 K (data not shown). Considering the fact that the SPC/E water has a melting point ~ 215 K, the 210-225 K can be considered as the working temperature for AFPs. In

28


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the low temperature regions, the hydration water around the threonine residues are more ordered than serine residues.

Figure 9: O-O-O angle distribution among water molecules within 5.5 Å of Cγ atoms of THRs ladder (wild-type, black) and Cβ atoms of serine ladder (green) of serine mutant at 210 K (AI), 225 K (AII) and 250 K (AIII).

We then characterized the effect of increased ordering on the hydrogen bonding ability of the THRs/SERs ladder. We have calculated the hydrogen bond time-correlation function, C(t), of water-IBS hydrogen bonding interactions for wild-type and serine mutant AFP at different temperatures. The function has a generalized form72:

C (t ) =

< h(t )h(0) > < h(0) 2 >

(3)

29



Where h(t) is 1 when there is hydrogen bonding between IBS and hydration water and it becomes 0 when all the hydrogen bonding interactions between donor-acceptor pair is completely broken. From the correlation function the intermittent hydrogen bond life-times have been calculated using the method of Luzar and Chandler72 and results are listed in Table 1. Table 1: Intermittent hydrogen bond lifetimes of the hydrogen bonding interactions between the THRs/SERs ladder and the hydration water of wild-type and serine mutant sbwAFP calculated at different temperatures are listed.

Temperature

IBS-Water hydrogen bond lifetime (ps) THRs

SERs

210 K

20.13

15.96

225 K

15.05

13.93

250 K

10.51

8.28

298 K

5.97

4.24

Evident from the table that in all the four temperatures (210, 225, 250 and 298 K), serine-water hydrogen bond lifetime is shorter than threonine-water hydrogen bond lifetime. Methyl group plays major role in ordering the hydration water.73 Slow dynamics of water around the threonine residues facilitates strong hydrogen bond formation ability in comparison to serine residues which is the origin of anchored clathrate water. A schematic representation of the phenomenon has been shown in Figure 10, based on the structure of the wild-type and serine mutant AFP obtained after 30 ns simulation. 30


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Figure 10: Structure of the hydration layer around the THRs ladder in case of wild-type sbwAFP (left side) and SERs ladder for the serine mutant (right side) obtained after 30 ns simulation performed at 210 K. Threonine/serine residues are shown as cyan sticks whereas the ladder is rendered in surface mode. Anchored clathrate water molecules are shown in vdW, water molecules are shown in stick mode and hydrogen bonding interactions are displayed as red dotted line.

31



Interestingly, in case of wild-type sbwAFP after 30 ns of equilibrium simulation five water molecules from the hydration shell occupy the anchored clathrate water position, although we have started the simulation without considering the crystallographically resolved anchored clathrate water. However, upon mutation of the threonine residues with serine residues, at the end of the 30 ns of equilibrium simulation only two water molecules occupy the anchored clathrate water position. Among them one is actually retained as the anchored clathrate water due its ability to form strong hydrogen bonding interaction with THR 7 of the serine mutant. Most of the other anchored clathrate water molecules as observed in case of wild-type sbwAFP do not appear during the simulation timescale for the serine mutant.

Conclusions Present work aims to explore the molecular basis of basal plane affinity by a hyperactive insect antifreeze protein (sbwAFP isoform 501). Free energy calculations reveal that the order of binding affinity of the insect AFP towards different ice planes is as follows: basal plane > prism plane > pyramidal plane. Detailed analysis reveals that the observed plane specificity of insect AFP is primarily associated with the stability of anchored clathrate water molecules which essentially anchor the AFP to the ice surface by forming dual contact hydrogen bonds with both the IBS of AFP and ice surface. In basal plane, the proper match between the periodicity of oxygen atom repeat distances of ice surface and the side-chain hydroxyl groups of the threonine repeat distances at the IBS stabilizes the anchored clathrate water array. Since, anchor clathrate water molecules find hydrogen bond donors and acceptors at appropriate position on both the IBS and ice surface to form tetrahedral dual contact hydrogen bonding network. Notably, each 32


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ice plane possesses unique surface topology due to different oxygen atom repeat distances. The mismatch between the threonine repeat distances at the IBS and the oxygen atom repeat distance is higher in case of prism plane adsorption and the mismatch is highest in case of pyramidal plane adsorption. Therefore, highest destabilization of anchored clathrate water has been observed in case of pyramidal plane adsorption which leads to the lowest affinity of AFP towards the pyramidal plane. Interestingly, although the formation of dual contact hydrogen bond is the characteristic of anchored clathrate water but the hydrogen bonding groups alone can’t stabilize the anchored clathrate water. Upon mutation of threonine residues of the THRs ladder to serine residues significantly destabilizes the anchored clathrate water molecule. In fact, the serine mutant AFP forms direct hydrogen bonds to the ice surface and the dual hydrogen bonding characteristics of the anchored clathrate water are completely lost. Equilibrium simulations reveal that methyl side-chains of threonine residues play major role in stabilizing the anchored clathrate water. At low temperature, hydration around the methyl side-chains of threonine become highly significant and there is increased ordering of hydration water around the methyl groups. These ordered water molecules are less dynamic and therefore can form strong hydrogen bond with the hydroxyl group of threonine which is the origin of anchored clathrate water. The study provides for the first time atomistic resolution insight into the experimentally observed basal plane binding affinity of insect antifreeze protein. Structurefunction correlation obtained from the study laid the foundation of rational designing of highly potent antifreeze-mimetic with immense application possibilities.

Conflict of interest Authors declare no financial conflict of interest. 33



Acknowledgment This research is funded by the Department of Science and Technology SERB grant EMR/2016/001333. Authors also gratefully acknowledge the central supercomputing facility (CRAY) at Indian Association for the Cultivation of Science, Kolkata. SC acknowledges all the lab mates for stimulating discussions.

References (1)

Deller, R. C.; Vatish, M.; Mitchell, D. A.; Gibson, M. I. Glycerol-free

cryopreservation of red blood cells enabled by ice-recrystallization-inhibiting polymers. ACS Biomat. Sci. Eng. 2015, 1, 789-794. (2)

Deller, R. C.; Pessin, J. E.; Vatish, M.; Mitchell, D. A.; Gibson, M. I. Enhanced

non-vitreous cryopreservation of immortalized and primary cells by ice-growth inhibiting polymers. Biomat. Sci. 2016, 4, 1079-1084. (3)

Congdon, T. R.; Notman, R.; Gibson, M. I. Influence of block copolymerization

on the antifreeze protein mimetic ice recrystallization inhibition activity of poly(vinyl alcohol). Biomacromolecules 2016, 17, 3033-3039. (4)

Congdon, T.; Notman, R.; Gibson, M. I. Antifreeze (Glyco)protein mimetic

behavior of poly(vinyl alcohol): detailed structure ice recrystallization inhibition activity study. Biomacromolecules 2013, 14, 1578-1586.

34


Page 34 of 44

Page 35 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(5)

Olijve, L. L. C.; Hendrix, M. M. R. M.; Voets, I. K. Influence of polymer chain

architecture of poly(vinyl alcohol) on the inhibition of ice recrystallization. Macromol. Chem. Phys. 2016, 217, 951-958. (6)

Rajan, R.; Hayashi, F.; Nagashima, T.; Matsumura, K. Toward a molecular

understanding of the mechanism of cryopreservation by polyampholytes: cell membrane interactions and hydrophobicity. Biomacromolecules 2016, 17, 1882-1893. (7)

Mitchell, D. E.; Lovett, J. R.; Armes, S. P.; Gibson, M. I. Combining biomimetic

block copolymer worms with an ice-inhibiting polymer for the solvent-free cryopreservation of red blood cells. Angew. Chem. Int. Ed. 2016, 55, 2801-2804. (8)

Jia, Z.; Davies, P. L. Antifreeze proteins: an unusual receptor–ligand interaction.

Trends Biochem. Sci. 2002, 27, 101-106. (9)

Scotter, A. J.; Marshall, C. B.; Graham, L. A.; Gilbert, J. A.; Garnham, C. P.;

Davies, P. L. The basis for hyperactivity of antifreeze proteins. Cryobiology 2006, 53, 229-239. (10)

Olijve, L. L. C.; Meister, K.; DeVries, A. L.; Duman, J. G.; Guo, S.; Bakker, H.

J.; Voets, I. K. Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins. Proc. Natl. Acad. Sci. USA 2016, 113, 3740-3745. (11)

Knight, C. A.; Duman, J. G. Inhibition of recrystallization of ice by insect thermal

hysteresis proteins: A possible cryoprotective role. Cryobiology 1986, 23, 256-262. (12)

Raymond, J. A.; DeVries, A. L. Adsorption inhibition as a mechanism of freezing

resistance in polar fishes. Proc. Natl. Acad. Sci. 1977, 74, 2589-2593. (13)

Davies, P. L.; Baardsnes, J.; Kuiper, M. J.; Walker, V. K. Structure and function

of antifreeze proteins. Philos. Trans. R. Soc. London B: Biol. Sci. 2002, 357, 927-935.

35



(14)

Voets, I. K. From ice-binding proteins to bio-inspired antifreeze materials. Soft

Mat. 2017, 13, 4808-4823. (15)

Knight, C. A.; Cheng, C. C.; DeVries, A. L. Adsorption of alpha-helical

antifreeze peptides on specific ice crystal surface planes. Biophys. J. 1991, 59, 409-418. (16)

Yang, D. S. C.; Sax, M.; Chakrabartty, A.; Hew, C. L. Crystal structure of an

antifreeze polypeptide and its mechanistic implications. Nature 1988, 333, 232-237. (17)

Wen, D.; Laursen, R. A. A model for binding of an antifreeze polypeptide to ice.

Biophys. J. 1992, 63, 1659-1662. (18)

Zhang, W.; Laursen, R. A. Structure-function relationships in a type I antifreeze

polypeptide: the role of threonine methyl and hydroxyl groups in antifreeze activity. J. Biol. Chem. 1998, 273, 34806-34812. (19)

Naullage, P. M.; Lupi, L.; Molinero, V. Molecular recognition of ice by fully

flexible molecules. J. Phy. Chem. C 2017, 121, 26949-26957. (20)

Yang, D. S. C.; Hon, W.-C.; Bubanko, S.; Xue, Y.; Seetharaman, J.; Hew, C. L.;

Sicheri, F. Identification of the Ice-Binding Surface on a Type III Antifreeze Protein with a “Flatness Function” Algorithm. Biophys. J. 1998, 74, 2142-2151. (21)

Sönnichsen, F. D.; DeLuca, C. I.; Davies, P. L.; Sykes, B. D. Refined solution

structure of type III antifreeze protein: hydrophobic groups may be involved in the energetics of the protein–ice interaction. Structure 1996, 4, 1325-1337. (22)

Baardsnes, J.; Davies, P. L. Contribution of hydrophobic residues to ice binding

by fish type III antifreeze protein. Biochim. Biophys. Acta (BBA) - Proteins Proteomics 2002, 1601, 49-54.

36


Page 36 of 44

Page 37 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(23)

Hakim, A.; Nguyen, J. B.; Basu, K.; Zhu, D. F.; Thakral, D.; Davies, P. L.; Isaacs,

F. J.; Modis, Y.; Meng, W. Crystal structure of an insect antifreeze protein and its implications for ice binding. J. Biol. Chem. 2013, 288, 12295-12304. (24)

Karim, O. A.; Haymet, A. D. J. The ice/water interface: a molecular dynamics

simulation study. J. Chem. Phys. 1988, 89, 6889-6896. (25)

Hayward, J. A.; Haymet, A. D. J. The ice/water interface: molecular dynamics

simulations of the basal, prism, {202̄1}, and {21̄1̄0} interfaces of ice Ih. J. Chem. Phys. 2001, 114, 3713-3726. (26)

Yang, C.; Sharp, K. A. Hydrophobic tendency of polar group hydration as a major

force in type I antifreeze protein recognition. Proteins: Struct. Func. Bioinform. 2005, 59, 266274. (27)

Sharp, K. A. The remarkable hydration of the antifreeze protein Maxi: A

computational study. J. Chem. Phys. 2014, 141, 22D510. (28)

Pooja, R.; Parbati, B. Shape dependence of the radial distribution function of

hydration water around proteins. J. Phys. Cond. Matter 2014, 26, 335102. (29)

Nutt, D. R.; Smith, J. C. Dual Function of the Hydration layer around an

antifreeze protein revealed by atomistic molecular dynamics simulations. J. Am. Chem. Soc. 2008, 130, 13066-13073. (30)

Midya, U. S.; Bandyopadhyay, S. Hydration behavior at the ice-binding surface of

the tenebrio molitor antifreeze protein. J. Phys. Chem. B 2014, 118, 4743-4752. (31)

Groot, C. C. M.; Meister, K.; DeVries, A. L.; Bakker, H. J. Dynamics of the

hydration water of antifreeze glycoproteins. J. Phys. Chem. Lett. 2016, 7, 4836-4840.

37



(32)

Meister, K.; Ebbinghaus, S.; Xu, Y.; Duman, J. G.; DeVries, A.; Gruebele, M.;

Leitner, D. M.; Havenith, M. Long-range protein–water dynamics in hyperactive insect antifreeze proteins. Proc. Natl. Acad. Sci. USA 2013, 110, 1617-1622. (33)

Ebbinghaus, S.; Meister, K.; Prigozhin, Maxim B.; DeVries, Arthur L.; Havenith,

M.; Dzubiella, J.; Gruebele, M. Functional importance of short-range binding and long-range solvent interactions in helical antifreeze peptides. Biophys. J. 2012, 103, L20-L22. (34)

Ebbinghaus, S.; Meister, K.; Born, B.; DeVries, A. L.; Gruebele, M.; Havenith,

M. Antifreeze glycoprotein activity correlates with long-range protein−water dynamics. J. Am. Chem. Soc. 2010, 132, 12210-12211. (35)

Pandey, H. D.; Leitner, D. M. Thermodynamics of hydration water around an

antifreeze protein: a molecular simulation study. J. Phys. Chem. B 2017, 121, 9498-9507. (36)

Mallajosyula, S. S.; Vanommeslaeghe, K.; MacKerell, A. D. Perturbation of long-

range water dynamics as the mechanism for the antifreeze activity of antifreeze glycoprotein. J. Phys. Chem. B 2014, 118, 11696-11706. (37)

Meister, K.; Strazdaite, S.; DeVries, A. L.; Lotze, S.; Olijve, L. L. C.; Voets, I.

K.; Bakker, H. J. Observation of ice-like water layers at an aqueous protein surface. Proc. Natl. Acad. Sci. USA 2014, 111, 17732-17736. (38)

Smolin, N.; Daggett, V. Formation of ice-like water structure on the surface of an

antifreeze protein. J. Phys. Chem. B 2008, 112, 6193-6202. (39)

Garnham, C. P.; Campbell, R. L.; Davies, P. L. Anchored clathrate waters bind

antifreeze proteins to ice. Proc. Natl. Acad. Sci. USA 2011, 108, 7363-7367.

38


Page 38 of 44

Page 39 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(40)

Sun, T.; Gauthier, S. Y.; Campbell, R. L.; Davies, P. L. Revealing surface waters

on an antifreeze protein by fusion protein crystallography combined with molecular dynamic simulations. J. Phys. Chem. B 2015, 119, 12808-12815. (41)

Celik, Y.; Drori, R.; Pertaya-Braun, N.; Altan, A.; Barton, T.; Bar-Dolev, M.;

Groisman, A.; Davies, P. L.; Braslavsky, I. Microfluidic experiments reveal that antifreeze proteins bound to ice crystals suffice to prevent their growth. Proc. Natl. Acad. Sci. USA 2013, 110, 1309-1314. (42)

Pertaya, N.; Marshall, C. B.; Celik, Y.; Davies, P. L.; Braslavsky, I. Direct

visualization of spruce budworm antifreeze protein interacting with ice crystals: basal plane affinity confers hyperactivity. Biophys. J. 2008, 95, 333-341. (43)

Chakraborty, S.; Jana, B. Conformational and hydration properties modulate ice

recognition by type I antifreeze protein and its mutants. Phys. Chem. Chem. Phys. 2017, 19, 11678-11689. (44)

Chakraborty, S.; Jana, B. Molecular insight into the adsorption of spruce

budworm antifreeze protein to an ice surface: a clathrate-mediated recognition mechanism. Langmuir 2017, 33, 7202-7214. (45)

Drori, R.; Celik, Y.; Davies, P. L.; Braslavsky, I. Ice-binding proteins that

accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics. J. R. Soc. Interface 2014, 11. (46)

Pertaya, N.; Marshall, C. B.; DiPrinzio, C. L.; Wilen, L.; Thomson, E. S.;

Wettlaufer, J. S.; Davies, P. L.; Braslavsky, I. Fluorescence microscopy evidence for quasipermanent attachment of antifreeze proteins to ice surfaces. Biophys. J. 2007, 92, 3663-3673.

39



(47)

Alireza Bagherzadeh, S.; Alavi, S.; Ripmeester, J. A.; Englezos, P. Why ice-

binding type I antifreeze protein acts as a gas hydrate crystal inhibitor. Phys. Chem. Chem. Phys. 2015, 17, 9984-9990. (48)

Van Der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.; Berendsen,

H. J. C. GROMACS: fast, flexible, and free. J. Comput. Chem. 2005, 26, 1701-1718. (49)

Pronk, S.; Páll, S.; Schulz, R.; Larsson, P.; Bjelkmar, P.; Apostolov, R.; Shirts, M.

R.; Smith, J. C.; Kasson, P. M.; van der Spoel, D.; Hess, B.; Lindahl, E. GROMACS 4.5: a highthroughput and highly parallel open source molecular simulation toolkit. Bioinformatics 2013. (50)

Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. GROMACS 4: algorithms

for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theory Comput. 2008, 4, 435-447. (51)

Berendsen, H. J. C.; van der Spoel, D.; van Drunen, R. GROMACS: a message-

passing parallel molecular dynamics implementation. Comp. Phys. Comm. 1995, 91, 43-56. (52)

Kaminski, G. A.; Friesner, R. A.; Tirado-Rives, J.; Jorgensen, W. L. Evaluation

and reparametrization of the opls-aa force field for proteins via comparison with accurate quantum chemical calculations on peptides. J. Phys. Chem. B 2001, 105, 6474-6487. (53)

Jorgensen, W. L.; Tirado-Rives, J. The OPLS [optimized potentials for liquid

simulations] potential functions for proteins, energy minimizations for crystals of cyclic peptides and crambin. J. Am. Chem. Soc. 1988, 110, 1657-1666. (54)

Berendsen, H. J. C.; Grigera, J. R.; Straatsma, T. P. The missing term in effective

pair potentials. J. Phys. Chem. 1987, 91, 6269-6271. (55)

Vega, C.; Sanz, E.; Abascal, J. L. F. The melting temperature of the most

common models of water. J. Chem. Phys. 2005, 122, 114507. 40


Page 40 of 44

Page 41 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(56)

Bryk, T.; Haymet, A. D. J. Ice 1h/water interface of the SPC/E model: Molecular

dynamics simulations of the equilibrium basal and prism interfaces. J. Chem. Phys 2002, 117, 10258-10268. (57)

Leinala, E. K.; Davies, P. L.; Doucet, D.; Tyshenko, M. G.; Walker, V. K.; Jia, Z.

A β-helical antifreeze protein isoform with increased activity: structural and functional insights. J. Biol. Chem. 2002, 277, 33349-33352. (58)

Humphrey, W.; Dalke, A.; Schulten, K. VMD: visual molecular dynamics. J. Mol.

Graph. 1996, 14, 33-38. (59)

Mashiach, E.; Schneidman-Duhovny, D.; Peri, A.; Shavit, Y.; Nussinov, R.;

Wolfson, H. J. An integrated suite of fast docking algorithms. Proteins: Struct. Func. Bioinform. 2010, 78, 3197-3204. (60)

Schneidman-Duhovny, D.; Inbar, Y.; Nussinov, R.; Wolfson, H. J. PatchDock and

SymmDock: servers for rigid and symmetric docking. Nucleic Acids Res. 2005, 33, W363W367. (61)

Hub, J. S.; de Groot, B. L.; van der Spoel, D. g_wham—a free weighted

histogram analysis implementation including robust error and autocorrelation estimates. J. Chem. Theory Comput. 2010, 6, 3713-3720. (62)

Bonomi, M.; Branduardi, D.; Bussi, G.; Camilloni, C.; Provasi, D.; Raiteri, P.;

Donadio, D.; Marinelli, F.; Pietrucci, F.; Broglia, R. A.; Parrinello, M. PLUMED: a portable plugin for free-energy calculations with molecular dynamics. Comp. Phys. Comm. 2009, 180, 1961-1972. (63)

Barducci, A.; Bonomi, M.; Parrinello, M. Metadynamics. Wiley Interdis. Rev.

Comput. Mol. Sci. 2011, 1, 826-843. 41



(64)

Alessandro, L.; Francesco, L. G. Metadynamics: a method to simulate rare events

and reconstruct the free energy in biophysics, chemistry and material science. Rep. Prog. Phys. 2008, 71, 126601. (65)

Moraca, F.; Amato, J.; Ortuso, F.; Artese, A.; Pagano, B.; Novellino, E.; Alcaro,

S.; Parrinello, M.; Limongelli, V. Ligand binding to telomeric G-quadruplex DNA investigated by funnel-metadynamics simulations. Proc. Natl. Acad. Sci. USA 2017, 114, E2136-E2145. (66)

Drori, R.; Davies, P. L.; Braslavsky, I. When are antifreeze proteins in solution

essential for ice growth inhibition? Langmuir 2015, 31, 5805-5811. (67)

Fidler, J.; Rodger, P. M. Solvation structure around aqueous alcohols. J. Phys.

Chem. B 1999, 103, 7695-7703. (68)

Jiménez-Ángeles, F.; Firoozabadi, A. Nucleation of methane hydrates at moderate

subcooling by molecular dynamics simulations. J. Phys. Chem. C 2014, 118, 11310-11318. (69)

Walsh, M. R.; Koh, C. A.; Sloan, E. D.; Sum, A. K.; Wu, D. T. Microsecond

simulations of spontaneous methane hydrate nucleation and growth. Science 2009, 326, 1095. (70)

Jana, B.; Pal, S.; Bagchi, B. Enhanced tetrahedral ordering of water molecules in

minor grooves of dna: relative role of dna rigidity, nanoconfinement, and surface specific interactions. J. Phys. Chem. B 2010, 114, 3633-3638. (71)

Biswal, D.; Jana, B.; Pal, S.; Bagchi, B. Dynamical transition of water in the

grooves of dna duplex at low temperature. J. Phys. Chem. B 2009, 113, 4394-4399. (72)

Luzar, A.; Chandler, D. Hydrogen-bond kinetics in liquid water. Nature 1996,

379, 55.

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(73)

Parui, S.; Jana, B. Pairwise hydrophobicity at low temperature: appearance of a

stable second solvent-separated minimum with possible implication in cold denaturation. J. Phys. Chem. B 2017, 121, 7016-7026.

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Optimum Number of Anchored Clathrate Water and Its Instantaneous

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