Antifreeze Glycoproteins Bind Reversibly to Ice via Hydrophobic

Subscriber access provided by READING UNIV

Article

Antifreeze glycoproteins bind reversibly to ice via hydrophobic groups Kenji Mochizuki, and Valeria Molinero J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b13630 • Publication Date (Web): 02 Feb 2018 Downloaded from http://pubs.acs.org on February 2, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of the American Chemical Society is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 11 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

Journal of the American Chemical Society

Antifreeze glycoproteins bind reversibly to ice via hydrophobic groups Kenji Mochizuki1,2 and Valeria Molinero1* 1Department 2Institute

of Chemistry, The University of Utah, Salt Lake City, Utah 84112-0580, USA for Fiber Engineering, Shinshu University, Ueda, Nagano 386-8567, Japan

ABSTRACT: Antifreeze molecules allow organisms to survive in subzero environments. Antifreeze glycoproteins

(AFGPs), produced by polar fish, are the most potent inhibitors of ice recrystallization. To date, the molecular mechanism by which AFGPs bind to ice has not yet been elucidated. Mutation experiments cannot resolve whether the binding occurs through the peptide, the saccharides, or both. Here, we use molecular simulations to determine the mechanism and driving forces for binding of AFGP8 to ice, its selectivity for the primary prismatic plane, and the molecular origin of its exceptional ice recrystallization activity. Consistent with experiments, AFGP8 in simulations preferentially adopts the PPII helix secondary structure in solution. We show that the segregation of hydrophilic and hydrophobic groups in the PPII helix is vital for ice binding. Binding occurs through adsorption of methyl groups of the peptide and disaccharides to ice, driven by the entropy of dehydration of the hydrophobic groups as they nest in the cavities at the ice surface. The selectivity to the primary prismatic plane originates in the deeper cavities it has compared to the basal plane. We estimate the free energy of binding of AFGP8 and the longer AFGP4-6, and find them to be consistent with the reversible binding demonstrated in experiments. The simulations reveal that AFGP8 binds to ice through a myriad of conformations that it uses to diffuse through the ice surface and find ice steps, to which it strongly adsorbs. We interpret that the existence of multiple, weak binding sites is the key for the exceptional ice recrystallization inhibition activity of AFGPs.

1. Introduction Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) are essential for various organisms that survive in subzero environments.1-6 These natural proteins, as well as synthetic ice-binding polymers, are of interest for food and tissue cryopreservation.7-9 AFGPs are fully flexible molecules.10-11 Although flexibility of molecules in solution has been shown to be detrimental for their binding to ice,12-13 AFGPs are the most potent ice recrystallization inhibitors.14 Although the macroscopic antifreeze properties of AFGPs, such as thermal hysteresis (TH), dynamic ice shaping, and ice recrystallization inhibition (IRI), have been relatively well studied, the molecular mechanism by which they halt the growth of ice is still debated.4-6, 15-18 It has been proposed that AFGPs do not need to bind to ice to influence ice growth.19-22 Studies using terahertz spectroscopy20 and molecular dynamics simulation21 showed that AFGPs perturb the hydration dynamics over long distances and proposed that this is important for their antifreeze activity. The existence of a correlation between these properties, however, was disputed by a recent polarization-resolved femtosecond infrared spectroscopy study.23 Other studies indicate that

AFGPs are preferentially adsorbed at the primary prismatic and pyramidal planes of ice.24-25 It was initially proposed that AFGPs halt the growth of ice by binding irreversibly to growing ice steps at the ice surface.26 That model was later extended for non-stepwise ice growth, still assuming irreversible binding.27 Knight et al. proposed that irreversibility in binding arises from incorporation of OH groups of the disaccharides into the ice lattice.24 Although irreversibility in binding has been confirmed for several AFPs,28 Zepeda et al. later used confocal fluorescence microscopy to demonstrate that the binding of AFGPs to ice is reversible, and these molecules do not incorporate into ice crystals grown at low supercooling.29 To date, it has not been resolved what is the strength and origin of the driving force for the binding of AFGPs to ice. AFGPs are categorized by size, from AFGP1 (33 kDa) to AFGP8 (2.6 kDa).30-32 All of them consist of tri-peptide repeats of alanine-alanine-threonine (Ala-Ala-Thr) in which the hydroxyl group of the Thr is glycosylated with β-D-galactosyl-(1,3)-α-N-acetyl-D-galactosamine (Figure 1A). In the smaller AFGPs, the first Ala and the glycosylated Thr are occasionally substituted by proline and arginine, respectively.32-36 A key feature of AFGPs is their combination of a hydrophobic peptide and

ACS Paragon Plus Environment

Journal of the American Chemical Society 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

hydrogen-bonding saccharides substituents. Mutation studies using various AFGP analogues have been performed to obtain insights on the relationship between chemical and spatial structure of AFGPs and their antifreeze activity.16-18 (A)

(B) OH OH

HO

OH

O

OH

HO

O

O

O N H O H N

HN O

O

HN O

4

N O

Figure 1. Structure of AFGP8. (A) Sequence of AFGP8 is Ala-Ala-T*-Ala-Ala-T*-Pro-Ala-T*-Ala-Ala-T*-Pro-Ala, where T* denotes the glycosylated Thr. (B) Three dimensional structure of AFGP8, with the peptide backbone in the PPII helix conformation. The color coding for circles in (A) and balls in (B) is the same: CH3 of N-acetyl group in yellow, CH3 of 2-, 5-, 8-, 11-Ala in orange, CH3 of 4-, 10-Ala in green, CH2 of 7-, 13-Pro in green, and CH3 of Thr in pink.

Circular dichroism (CD) and nuclear magnetic resonance (NMR) studies indicate that the secondary structure of AFGPs in solution is a PPII helix.11, 37-42 Tachibana et al. combined ice-shaping experiments and measurements of thermal hysteresis of various mutants to conclude that the antifreeze activity strongly depends on the presence of PPII helix secondary structure.43 PPII helix is a left-handed helical conformation with three residues per turn. If all amino acids in AFGP8 adopt the PPII helix conformation, all four disaccharides face toward the same side, segregating the hydrophilic and hydrophobic domains of the biomolecule43 (Figure 1B). The role of hydrogen bonding and hydrophobic domains on the binding of AFGPs to ice has not yet been established. In a series of careful experiments, Budke et al. found low IRI activity of the AFGP backbone when the saccharide moieties were removed, and a reduction of efficacy when the disaccharides were replaced by monosaccharides.14 Experimental studies for C-linked AFGP analogues showed that small structural changes in the disaccharide moieties or the length of C-link impact their antifreeze activity.22, 44-47 These results point to a significant role of the disaccharide moieties in the interaction of AFGPs with ice. That inference is also supported by inactivation studies by boration of cis-hydroxyl groups of the AFGP galactose units.3, 20 Based on those results, it has been hypothesized that hydrogen bonding plays an important role in the binding of AFGPs to ice.18 However, proof of the role of hydrogen bonding in the binding is still elusive.

Page 2 of 11

Experiments and simulations indicate that AFP of types I and III bind to ice through hydrophobic groups.48-55 The sequences of these proteins have commonalities with those in AFGPs: e.g. type I AFP of winter flounder has Ala-Ala-Thr repeats in its ice binding site.52 The secondary structure of AFPs, however, is quite different from that of AFGPs. The type I AFP from winter flounder, for example, is a quite rigid α-helix.4 It has been proposed that methyl groups of the binding site of type I and III AFPs adsorb to ice assisted by matching in the distances between methyl groups and grooves at the ice surface.50, 53-55 The possibility that AFGPs bind to ice through hydrophobic groups was pointed out by Harding et al.,31 but has not been tested to date. Molecular simulations provide optimum resolution to unravel the microscopic mechanisms of ice-protein interactions.56-59 Although there have been some studies on AFGPs in solution,19, 21, 60 simulations of solvated AFGPs in the presence of ice have not yet been reported. Here, we perform all-atom molecular dynamics simulations of AFGP8 in solution and in ice-water systems to determine its mode of interaction with ice and the molecular forces that drive it. AFGP8 is the shortest AFGP, and consist of 14 residues with four disaccharide moieties. We first show that PPII helix is the dominant conformation in solution, and that its population becomes more pronounced on approaching the freezing temperature. We then demonstrate that AFGP8 directly binds to the primary prismatic plane of ice via methyl groups of the peptide and saccharide, and that the adsorption of AFGP8 inhibits ice growth. We compute the binding free energy of methane and the disaccharide to different ice surfaces to rationalize why AFGP8 binds to ice using hydrophobic groups and its selectivity for the primary prismatic plane. Furthermore, we show that AFGP8 binds to ice through a myriad of conformations that it uses to diffuse through the ice surface and find steps, where it is strongly adsorbed. To the best of our knowledge, this study provides the first molecular picture of the motifs and driving forces that account for the exceptional IRI activity of AFGPs. 2. Methods 2.1 Models The peptide (also called here the backbone) of AFGP8 is modeled with full atomistic detail using CHARMM2761-62 (CHARMM2263 plus CMAP64 for proteins). AFGP8 is represented by CHARMM27 and the CHARMM carbohydrate force field.65-71 The latter is also used for the modeling of the disaccharide. We use ref. 72 and the website of the MacKerell Lab73 to build up the force field. The Lennard-Jones, bond and angle parameters of C and H of CH4 for the free energy calculations are identical to those of CH3 in Thr, without partial charges. Water is represented by the TIP4P/Ice model.74 The melting temperature of hexagonal ice (ice Ih) in this model is 270 K,75 in very

2 ACS Paragon Plus Environment

Page 3 of 11 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


good agreement with the experimental value of 273.15 K.76 The intermolecular interactions are truncated at 0.85 nm. The Lennard-Jones parameters for cross-interactions are obtained through the Lorentz–Berthelot combination rules: εij=(εiiεjj)1/2 and σij=(σii+σjj)/2. The long-range Coulombic interactions are evaluated with the particle-mesh Ewald algorithm; dispersion corrections are implemented in the evaluation of the energy and pressure. 2.2 Molecular Dynamics Simulation Molecular dynamics (MD) simulations are carried out using GROMACS 2016.4,77-78 integrating the equations of motion with the leap-frog algorithm using a time step of 2 fs. The temperature T and pressure p for production runs are controlled with the Nosé-Hoover thermostat and Parrinello-Rahman barostat at 1 atm, with damping constants 1.0 and 2.0 ps, respectively. The Berendsen algorithm is used to control both T and p for equilibration. Periodic boundary conditions are applied in the three directions. 2.3 Replica exchange MD simulations Replica exchange MD (REMD) simulations79 are performed to enhance the conformational sampling for AFGP8 and the the peptide in bulk liquid water. An arbitrary initial conformation made with Pymol80 is placed into a cell with 1800 water molecules. The system is then relaxed through energy minimization using the steepest descent method. Then, a 10 ps MD simulation in the canonical (NVT) ensemble is followed by a 100 ps MD simulation in the isobaric isothermal (NpT) ensemble, both at 300 K. For the REMD simulation, 25 replica temperatures are used ranging from 268 to 398 K (more precisely, 268, 272, 276, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 346, 352, 358, 364, 370, 377, 384, 391, 398 K). Before the REMD simulations, the system is equilibrated at each temperature through a 500 ps NpT-MD simulation. Then, REMD simulations are performed in the NpT ensemble. The simulation length for each temperature is 400 ns; attempting to exchange temperature between replicas every 10 ps. Two sets of REMD simulations, starting from different initial conformations, are performed for AFGP8, while one set of REMD simulations is evolved for the peptide. The average exchange ratio between nearest neighbor temperatures is 16.0±2.7 % and 16.4±2.8 % for the solutions of AFGP8 and the plain backbone, respectively. 2.4 Conformational analysis. The conformations of AFGP8 and the peptide (AFGP8bb) in solutions are obtained from the REMD simulations, while those of AFGP8 bound to the primary prismatic plane of ice are averaged over 600 ns of ice-bound configurations prepared as described in subsection 2.5. The radius of gyration Rg is computed for the same C and N atoms in the protein backbone for all

the molecules. The Ramachandran plots are built with the statistics of dihedral angles along the simulation trajectories, computed with Gromacs. 2.5 Adsorption of AFGP8 on growing ice surface We perform non-equilibrium NVT simulations with cells containing slabs of vapor, liquid and ice. The simulation cells consist of 10000 water molecules and one AFGP8 molecule, as experiments indicate that AFGP8 exhibits TH activity even at concentrations below 20 mM.81-82 The size of the simulation box for the system in which the primary prismatic plane is exposed to liquid water is set to 6.34 × 5.92 × 11.0 nm. Two layers of proton disordered ice Ih (896 molecules) are generated with the program GenIce83 at the density of ice Ih for TIP4/Ice74, 0.909 g cm-3 (dark blue layers in Figures 4C-E). The oxygen atoms of the molecules in these two layers of ice are harmonically restrained at their original positions with a force constant 1000 kJ mol-1 nm-2. The remaining 9104 water molecules are free and placed on one side of the ice layer to let ice grow in a single direction. The system exposing the basal plane is prepared in the same way, with box size dimensions 6.34 × 6.28 × 11.0 nm. The center of mass of AFGP8 is initially placed approximately 1.2 nm above the ice surfaces. An energy minimization is followed by a 100 ps NVT-MD simulation at 275 K for equilibration. The production NVT-MD run is then evolved for 400 or 800 ns at 268 K, which is 2 K lower than the freezing temperature of TIP4P/Ice model.75 The molecules are identified as ice or liquid using the CHILL+ algorithm.84 The liquid hydration number of the methyl or methane groups is computed as the average number of liquid water molecules within 0.55 nm from the methyl or methane groups. The amount of liquid water released upon adsorption of these hydrophobic groups to water is computed as the difference between the liquid hydration number in solution and on the ice-adsorbed state. 2.6 Binding free energies The binding free energies of methane and the disaccharide β-D-galactosyl-(1,3)-N-acetyl-D-galactosamine to either the primary prismatic or basal ice planes is computed as the potential of mean force (PMF) using umbrella We prepare a three-phase sampling.85-86 vapor/liquid/ice system as discussed in subsection of 2.5, but the system size is smaller and only one ice layer is restrained. The system that exposed the primary prismatic plane contains 60 molecules in the restrained ice layer and 900 in the liquid phase, in a box with dimensions 2.27 × 2.22 ×10.0 nm. The one exposing the basal plane has also 60 molecules restrained into an ice layer plus 1020 free water molecules in a box of dimensions 2.27 × 2.35 × 10.0 nm. Just one solute molecule –methane or the disaccharide- is dissolved in the liquid phase. The temperature is set to 280 K, 10 K above the freezing point, in order to prevent growth of



dominant conformation, encompassing 67.1 % at T = 268 K, and its population is the most pronounced with decreasing temperature. A previous computational study of AFGP8 solvated with TIP4P/2005 water also found PPII helix to be dominant at 300 K.21 This indicates that the secondary structure of AFGP8 in solution is robust to changes in the water model. Consistent with CD and NMR spectroscopic studies,11, 37-41, 43 we find that PPII helix is the dominant but not unique conformation at the freezing point, due to the flexible nature of AFGP8. (A)

(B) 67.1%

β

62.3%

PPII L-α α

ψ (degree)

ice. For each system, a series of NVT-MD simulations for 83 umbrella windows is performed with harmonic-restraint potentials, V(z)=k(z-d0)2, where the force constant k = 1000 kJ mol-1 nm-2, the equilibrium distance d0 spans from 0.36 to 2.00 nm every 0.02 nm, and z is the absolute position of the center of mass of the solute in the direction perpendicular to the ice surface. The target molecule can freely rotate around its center of mass and move parallel to the ice surface. For each window, an equilibration run of 200 ps is followed by a production run of length 10 ns for methane and 40 ns for the disaccharide. The resulting z distributions are then combined using the weighted histogram analysis method.87 The PMFs as a function of the absolute z value are later re-plotted against the distance from ice surface, where the zero is defined at the averaged coordinate of oxygen of the restrained ice molecules. The average PMF at distances from 1.4 to 1.7 nm, where liquid water is considered to be bulk liquid, is chosen as the baseline (i.e. the zero of the free energy profiles). To obtain the two-dimensional free energy profiles of the solute at contact and solvent-separated distances to the primary prismatic surface, we use the same simulation cells that for the PMFs and restrain the distance between methane and ice with a flat-bottomed potential. The potential does not introduce any force on the methane within the flat-bottomed region, while it adds a harmonic force with constant of 1000 kJ mol-1 nm-2 when the methane moves out of that region. The flat-bottomed region is z < 0.7 nm (0.5 nm from the ice surface) for the contact distance, and 0.65 < z < 0.95 nm (0.45 and 0.75 nm from the ice surface) for the solvent-separated distance. The production NVT-MD simulations are each 100 ns long. The lowest free energy on each two-dimensional PMF is set to zero. The molecular weights of AFGPn with n = 4, 5 and 6 are 18, 11 and 7.8 g mol-1, respectively.88 We estimate the free energy of binding of AFGPs4-6 in solution from the experimental ice surface coverage at -0.05 oC, θ = 0.04 for a concentration c = 5 μg mL-1 (corresponding to 5.4x10-7 mol L-1).29 As the coverage is low, we assume that the adsorption of the glycoprotein to ice follows the Langmuir isotherm: θ = cKA/(1+cKA), where the equilibrium constant for the adsorption KA is directly related to the free energy of binding by ΔG=-kBT lnKA, where kB is the Boltzmann constant.89 3. Results and Discussion 3.1 AFGP favors PPII helix conformations We first compute the conformations of AFGP8 in water from REMD simulations. Figures 2A-B show the Ramachandran plots for AFGP8 at 268 and 320 K. The plots represent the free energy as a function of the dihedral angles between aminoacids. PPII helix is the

Free Energy (kBT)

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

Page 4 of 11

(C)

(D) 64.3%

56.3%

φ (degree)

Figure 2. Ramachandran plots of AFGP8 and the corresponding peptide (without the saccharides) at 268 and 320 K. (A) AFGP8 at 268 K and (B) AFGP8 at 320 K, (C) peptide at 268 K, and (D) peptide at 320 K. The regions for PPII-helix (PPII), β-sheet (β), α-helix (α), and left-handed α-helix (L-α) secondary structures are labeled in the graphs. The populations (%) of PPII-helix, integrated inside the region highlighted with a black square, are also shown.

Figure 3. PPII helix dominates the secondary structure of AFGP8 in solution and the bound state. (A) Distribution of the radius of gyration Rg of AFGP8 in solution (green), bound to ice (orange) and the bare peptide in solution (black). (B) Population of PPII helix as a function of the Rg, colors as in (A). (C) Populations of PPII helix conformations for each residue. Data collected at 268 K. The ribbon in the inset of (C) displays a typical AFGP8 conformation, with a bent at Thr-6 (T6).


(A)

Population Population

AFGP8 in solution AFGP8 bound to Ice AFGP8bb in solution

0.4

PPII (%) PPII (%)

(B) 100

0.6

0.8

1.0

1.2

1.4

0.6

0.8

1.0

1.2

1.4

80 60 40 20 0.4

(nm) RRgg (nm) (C)100

T6

AFGP8

AFGP8bb

80

PPII (%)

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


60 40 20 0

A2 T3 A4 A5 T6 P7 A8 T9 A10A11T12P13

Residue

Figure 2C-D present the free energy landscape for the conformations of the non-functionalized AFGP8 peptide backbone. Like AFGP8, the peptide adopts PPII helix as a main local structure, and the population of PPII helix increases with decreasing temperature. Nevertheless, the global structures of AFGP8 and the peptide are very different. The peptide explores a larger conformational space and larger radii of gyration (Rg) than AFGP8 (Fig. 3A), and its Rg increases with the population of PPII helix (Fig. 3B). The peptide seems to have a random conformation, in which all residues except Pro have local PPII helix order about 60% of the time (Fig. 3C). This is consistent with the CD spectroscopic study that indicates that the peptide has a disordered structure.43, 90 On the other hand, AFGP8 is compact, with a heterogeneous pattern of prevalence of PPII helix along the residues: Figure 3C shows that Pro-7, Thr-9, Ala-11, and Pro-13 –all placed in the half of AFGP8 closer to its C endexhibit a very high probability of PPII helix (82-95 %), while that of Thr-3 and Thr-6 are very low. The small Rg of AFGP8 mainly arises from the bend at Thr-6 (see inset in Figure 3C). That bend in AFGP8 lets the hydrophobic groups face each other and the hydrophilic disaccharides be exposed to liquid water. We conclude that the disaccharide moieties make AFGP8 have a highly organized and compact PPII helix secondary structure. 3.2. PPII helix conformations are key for binding of AFGP8 to ice Figure 1B shows the spatial segregation between disaccharides and hydrophobic groups of the peptide

backbone in the PPII helix conformation of AFGP8. The nearest neighbor distance between methyl groups along each repetition of the three-fold helix (shown with same color in Figure 1B) is ~0.93 nm. This distance matches twice the lattice distance of ice in the primary prismatic face in the direction perpendicular to the c-axis, ~0.91 nm. Distance matching has been shown to be an important factor in modulating the free energy of binding to ice.12 We choose this PPII helix as the initial conformation for non-equilibrium MD simulations at 268 K in the presence of a growing primary prismatic plane. The Rg of the initial conformation is 1.23 nm with 100% population of PPII helix. We set AFGP8 with PPII helix conformation in the vicinity of the primary prismatic surface with 4 different orientations (Figure 4C): the disaccharide moieties either face liquid water or the ice surface, and the backbone is either parallel or perpendicular to the c-axis of the ice plane. We find that when the disaccharides face the ice surface, ice gradually grows, shoving the AFGP (Figures 4A and D). On the other hand, when the hydrophobic groups of the peptide face the ice, AFGP8 binds to the ice surface, halting the growth of the crystal (Figures 4A and E). AFGP8 binds to the primary prismatic plane exclusively through its hydrophobic groups.

Number of Ice Molecules

Page 5 of 11

(A) 6000

Prismatic

Sugar Down

5000

(B) 6000 5000

D

4000

Basal

Sugar Up

3000

4000 3000

C

E 2000

2000 1000 0

(C)

200

400

(D)

600

Time (ns)

1000 800 0

400

800

(E)

Figure 4. AFGP8 binds to ice through the methly groups of the peptide and saccharides, halting its growth. (A) Influence of AFGP8 on the growth the primary prismatic surfaces at 268 K. AFGP8 with PPII helix conformation is initially set in four different ways: the disaccharide facing liquid water with the backbone parallel (thick red line) or perpendicular (thin red line) to the c-axis, and with the disaccharide facing ice with the backbone parallel (thin blue line) or perpendicular (thick blue line) to the c-axis. (B) AFGP8, initially with PPII helix conformation and with the disaccharide facing the liquid does not stop the growth of the basal plane at 268 K. (C-E) shows snapshots of the simulation cells of ice growth. The bottom two layers of ice,


Journal of the American Chemical Society colored in dark blue, are restrained by a harmonic potential. The free ice and liquid water molecules are shown by light blue lines and gray dots, respectively. (C) and (D) show snapshots of trajectory shown with a thin blue line in (A) at 1 and 400 ns, respectively. (E) shows the simulation cell at 800 ns in the trajectory shown with a thin red line in (A).

AFGP8 binds to ice in extended conformations, unlike the compact ones it preferentially adopts in solution (Figure 3A). The population of PPII helix in the bound state is even higher than in solution (Figure 3B). It has been previously noted that the conformation of the peptides at the ice surface needs not to be identical to the one in solution, due to the flexible nature of AFGPs.10, 91 To further test whether the PPII conformation matters for the binding, we prepare an α-helix conformation of AFGP8 (Figure 5A) and place it close to the ice primary prismatic surface. We find that the α-helix does not halt the growth of ice (Figure 5B), and is eventually released to the liquid (Figure 5C). We interpret that this is because the α-helix is a four-fold conformation in which the methyl groups are exposed to water but their access to the surface is hindered by the disaccharide moieties, which radiate from the backbone in all directions (Figure 5A). We conclude that adoption of PPII helix conformation is key for the binding of AFGPs to ice.

growth (Figure 4B). In next section we investigate the origin of this selectivity. 3.4 Hydrophobic dehydration provides the driving force for binding AFGPs to ice The simulations indicate that binding of AFGP8 to ice occurs through adsorption of methyl groups to the ice surface. To understand what drives the binding, we compute the binding free energies of methane (CH4) and the disaccharide (without the peptide) as a function of the distance to the basal and primary prismatic ice surfaces. We find that the disaccharide has a negligible binding free energy to both the primary prismatic and basal planes of ice (Figures 6A). This indicates that the stabilization the saccharide could acquire through hydrogen bonding or adsorption through the methyl group does not compensate for the loss of rotational entropy upon binding. The rotational entropy accessible to saccharides in AFGPs, however, is smaller, allowing for contributions to the binding by adsorption through their methyl groups (left panel of Fig. 6B, yellow balls in Fig. 7). Based on the lack of binding of the AFGP when the sugars point to the surface and also in the disturbance hydrogen bonding produces on the ice structure (e.g. Fig. 7B), we expect the hydrogen bonding contribution to the binding of AFGPs to be very small. (A) 4

(B) Prismatic Basal

2

(C)

Binding Free Energy (kJ/mol)

(B)5000 Number of Ice Molecules

(A)

4000

C 3000

2000

1000

0

350

700

Time (ns)

0 -2 0.2

0.6

1.0

(C)4

1.4

(D)

2 0

w

w

Figure 5. AFGP8 in a α-helix conformation cannot bind to ice. (A) α-helix conformation of AFGP8; same color code is used as Figure 1B. (B) Growth of the primary prismatic plane of ice at 268 K in the presence of α-helix AFGP8 initially lying on the ice surface. (C) Snapshot of the system at 670 ns, showing the α-helix conformation partially unfolded.

-2

contact -4 s&w 0.2

w

w

s

s

solvent-separated 0.6

1.0

1.4

Distance from Ice Surface (nm)

(E) Contact

w

3.3 AFGP8 binds to the primary prismatic, but not the basal plane of ice Experiments indicate that AFGP8 binds to the primary prismatic plane, not the basal plane of ice.15 We investigate whether AFGP8 with PPII helix conformations binds to the basal plane of ice when the hydrophobic moieties of AFGP8 initially face the ice surface. The simulations concur with the experiments that AFGP8 does not bind to the basal plane nor halts its

w

c-axis

s

s

9 8 7 6 5 4 3 2 1 0

(F) Solvent-separated

10 9 8 7 6 5 4 3 2 1 0

Free Energy (kJ mol-1)

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

Page 6 of 11

Figure 6. Driving force for binding of AFGP8 to ice is provided by the adsorption of methyl groups to cavities on the ice surface. (A) Binding free energy of the disaccharide to the primary prismatic (red) and basal (black)


Page 7 of 11 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


planes. (B) Configurations showing the contact interaction of the saccharide with the prismatic plane through the methyl of the N-acetyl group (yellow ball) and through hydrogen bonds (dashed lines). (C) Binding free energy of methane to the primary prismatic (red) and basal (black) planes. The binding free energies at the contact and solvent-separated distances for methane on the primary prismatic plane are -1.7 ± 0.2 and -2.0 ± 0.2 kJ mol-1, respectively. Panel (D) shows the position of free energy minima for methane (green ball) adsorbed at contact distances to ice (cyan lines). The “s” labels indicate the loci of the most stable minima at the contact distance, “w” the weaker ones. (E-F) Two dimensional free energy profiles for methane on top of the primary prismatic surface at (E) contact and (F) solvent-separated distances, plotted on a unit cell surface conformation corresponding to the dashed region in the panel (D). The sampling regions for the contact and solvent separated distances are shown in the up pc central figure between panels E and F. (A) (A) 200ns 200ns

(B) (B) 500ns 500ns

(C) (C) 800ns 800ns

(D)

(E)

(F)

200ns 200ns

500ns 500ns

800ns 800ns

Figure 7. Examples of binding configurations of AFGP8 to the primary prismatic plane of ice. The snapshots correspond to times along the two binding trajectories of Fig 4A. In each panel, the top and bottom snapshots correspond to the side and top views, respectively. In the top views, only one ice layer bellow AFGP8 is shown (gray lines). The methyl groups bound to ice are shown as bold spheres, with the color-coding of Fig. 1.

Adsorption of the methyl groups to the natural cavities or grooves of the ice surface has been previously proposed to be the mode of binding of type III AFP.55 The free energy gain due to this interaction, however, had not been reported in the literature. Figure 6C shows

that the binding free energy of methane to primary prismatic planes has two minima with comparable stability, corresponding to methane in contact with ice and separated from ice by a monolayer of water. Adsorption of methane to the basal plane also displays two minima, but the contact one does not provide any stabilization with respect to the molecule in solution. The existence of contact and solvent-separated minima is typical of interactions between hydrophobic molecules in water.92-94 We note that the position and attenuation of the minima in the free energy profiles is the same as for methane-methane interactions.95 We conclude that in the interaction of ice with the AFGP8, the ice surface plays a role akin to that of a hydrophobic wall. We identify two preferential loci for adsorption of methane in contact with the primary prismatic face, which we label as s and w in Figure 6D. The two-dimensional free energy profile for methane at contact distances shows a strong adsorption at the s site and a weak adsorption at the w site (Figure 6E). The difference in these binding minima arises from the geometry of the primary prismatic surface, which displays “cavities” that can fit a methane or methyl group.50, 55 Methane in the s-cavity is nested in a hexagonal ring of waters in a flipped boat conformation (Figure 6D), while methane in a w-cavity sits in the edge between two boat rings. We interpret that the adsorption to these cavities results from the entropic gain when water that solvates the hydrophobic group in solution is replaced by a pre-formed cavity at the crystal surface.96 The weaker binding of methane to the basal plane (Figure 6C) can be understood in terms of the flatter topology of the basal surface, which exposes chair configurations that cannot “hold” the hydrophobic group. We conclude that the topography of the ice surface is key for the binding affinity of the small hydrophobic groups to ice surfaces. Figure 6F shows that the free energy landscape at the solvent-separated distance is not modulated by the underlying structure of the ice surface. This explains why the binding free energies of methane at the solvent-separated distance are the same for the basal and primary prismatic planes (Figure 6C). The adsorption of methane to contact distances to the primary prismatic surface releases 6.2 water molecules from the hydration shell to the solution. The total number of water molecules around methane is 20 in both the liquid and the surface. Using the experimental entropy of hydration of methane (-66.7 JK-1mol-1),97 the entropic gain of releasing 6.2 water molecules out of a solvation shell of 20 should be about -5.8 kJ mol-1 at 280 K (the temperature of the free energy profiles in Figure 6). This is over twice more favorable than the actual free energy of binding measured in the simulations. This difference could arise from an unfavorable enthalpy of binding or a reduced entropic gain due to the mismatch



between the size of methane and the shallow cavities at the ice surface. The latter was previously shown to decrease the adsorption of hydrophobic particles to the clathrate/water interface.96 Our analysis suggests that the entropy of dehydration drives the binding of small hydrophobic groups to the ice surface. 3.5 AFGP8 walks on the ice surface until it finds ice steps Empowered by the understanding of the binding of individual hydrophobic groups and the saccharide to ice, we now focus on the binding of the glycoprotein. Figure 7 shows snapshots along two AFGP8 binding trajectories, wherein the methyl groups that are bound to ice are shown with bold spheres, colored as in Figure 1. First, we note that AFGP8 has many binding configurations. Nevertheless, binding always occurs through hydrophobic groups. The methyl groups of both the backbone and disaccharide bind to ice at contact and solvent separated distances. We note that even when AFGP8 is only partly bound to ice (Figures 7B-C and E-F) it still halts the growth of the crystal (Figure 4A). These results are consistent with the experimental observations that synthetic AFGP analogues shorter than AFGP8 also exhibit thermal hysteresis,43 ice-shaping,43 and ice recrystallization inhibition.14 To estimate the binding free energy of AFGP8 to a flat ice surface, we first assume the binding free energy per methyl group (or methylene of Pro) is the same as for methane. The simulations indicate that AFGP8 has an average of 5 to 6 of these groups at the contact distance and 2 to 4 at solvent-separated distance, from which we estimate a binding free energy of AFGP8 around -15 kJ mol-1. The binding may be weaker, as AFGP8 loses some configurational entropy when it transfers from the solution to the surface. Our prediction for AFGP8 is consistent with the -25 kJ mol-1 binding free energy we estimate for AFGPs4-6 (which is, on average, about 3.6 times longer than AFGP8) using the experimental surface coverage by the AFGP,29 and assuming that the adsorption follows a Langmuir isotherm.89 This analysis further supports that AFGPs bind to ice through their hydrophobic groups, driven by an increase in the entropy of water upon hydrophobic desolvation. The relatively weak binding free energy of AFGPs to ice is consistent with the reversibility of its binding in experimental time scales.29 As the barriers between bound states are comparable to the thermal energy (Figure 6E), the molecules are free to move through the ice surface between adsorption and desorption events. As a result, AFGP8 “walks” through the ice surface (Figures 7A-C) using a myriad of conformations in which different sets of methyl groups bind to ice with the backbone oriented along various directions. We conjecture that the mobility of AFGP8 allows it to reach steps at the ice surface and -by adsorbing to them- halt the growth of the crystal. Indeed, we find that AFGP8

Page 8 of 11

strongly adsorbs to steps at the ice surface (Figure 8), from which it does not desorb over the hundreds of nanoseconds of the simulations (Figures 7E-F). The steps provide additional stabilization, as they replace more liquid by ice in the solvation shell of the methyl groups, which increases the entropic driving force for binding. Thus, the binding free energy to a step should be more favorable than to a flat surface. Free wandering of the AFGPs through the ice surface until they bind to and pin the growing steps may be at the root of the exceptional ice recrystallization ability of these biomolecules.

Figure 8. Adsorption of methyl groups of AFGP8 to an ice step in the primary prismatic ice face. The snapshots correspond to two details of the configuration shown in Fig. 7F. Hexagonal rings of water molecules near the bound methyl groups are shown with gray lines. Three colors are used to clarify the different layers of ice. The top ice layer in the right panel is slightly hindered but still forms hexagonal rings.

4. Conclusions We use all-atom molecular dynamics simulations of AFGP8 in solution and in liquid/ice systems to elucidate the selectivity of the glycoprotein for the primary prismatic plane, the mechanism and driving force for its binding to ice, and the origin of its exceptional ice recrystallization inhibition activity. To the best of our knowledge, this is the first report of molecular simulations of binding of an antifreeze glycoprotein to ice. Our simulations predict that AFGP8 in solution has a highly preserved PPII helix secondary structure, consistent with NMR and CD spectroscopic studies.11, 37-42 It has been previously noted that adoption of PPII helix leads to spatial segregation of hydrophobic and hydrophilic moieties.43 We find that this segregation is key for the binding of AFGPs to ice. The simulations indicate that the binding of AFGPs to ice is driven by the adsorption of methyl groups of the peptide and saccharides to cavities on the ice surface. Interestingly, this mechanism was previously suggested for the binding of type I and III AFPs,50, 55 which also share with AFGPs the matching in the distances between methyl groups in the binding site and the cavities or grooves in the ice surfaces to which these biomolecules bind. This suggests that the combination of distance


Page 9 of 11 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


matching to ice (which prevents building up stress in the biomolecules that would disfavor its binding12, 59) and weak hydrophobic interactions with the ice surface is a successful strategy for reversible ice binding that nature has evolved independently using a variety of protein sequences and secondary structures. Mutation studies have indicated that the N-acetyl group in galactosamine and the Thr γ-methyl group in AFGPs are necessary for antifreeze activity,43 because these elements contribute to maintain the PPII helix conformation.21, 98 The simulations demonstrate the importance of PPII helix for ice binding and the role of the saccharides in stabilizing those configurations, and reveal that the methyl groups of the disaccharides contribute to the adsorption of the glycoprotein to ice. We do not find, however, any indication that hydrogen bonding between the saccharides and ice contribute to the direct binding of AFGP8. In agreement with experiments,24-25 the simulations predict that AFGP8 binds to the primary prismatic but not the basal planes of ice. We demonstrate that the selectivity originates in the different topography of the ice surfaces. The primary prismatic face provides deeper cavities to adsorb a methyl group. The replacement of methyl groups by other hydrophobes that fit more tightly to the cavities on the ice surface might strengthen the adsorption.96 Our analysis indicates that the adsorption of small hydrophobic groups to ice is driven by the entropy gain upon partial replacement of the liquid solvation shell by a pre-formed ice cavity. We conclude that entropy is the main driving force for the “hydrophobic binding” of AFGPs to ice. We estimate the binding free energy of AFGP8 to be no stronger than -15 kJ mol-1, consistent with the -25 kJ mol-1 we derive from experimental data for the 3.6 longer (on average) AFGPs4-6. The relatively small binding free energies are consistent with the finding that the binding is reversible in experimental time scales.29 Reversibility in its binding to ice sets AFGPs apart from insect antifreeze proteins,99 which –different from AFGPs- display good thermal hysteresis but low ice recrystallization inhibition activity.100 This suggests that weak, reversible binding, might be an asset for optimum inhibition of ice recrystallization. Different from hyperactive AFPs and polyvinyl alcohol (PVA), which experience strong cooperative hydrogen bonding to ice facilitated by lattice matching,56, 59, 101 AFGPs bind to ice not only weakly but –judging from the scaling of the binding free energies with length – also non cooperatively. Cooperativity in binding of PVA to ice has been attributed to the loss of configurational entropy of the flexible ice binding molecule at the ice surface.59 We conjecture that the lack of cooperativity in binding of AFGPs is due to a significant retention of flexibility of the molecule in its bound state.

Indeed, we find that AFGP8 binds to ice through a myriad of conformations involving different methyl groups of the peptide and saccharides. The simulations reveal that rapid exchange between bound conformations let AFGPs “walk” on flat ice surfaces. The glycoproteins wander through the surface until they find and adsorb to a step at the ice surface. The pinning of growing steps by the AFGPs explains why, although the primary prismatic surface is typically rough, it becomes smooth and develops layer-by-layer growth in AFGP solutions.29 We note that the most potent synthetic IRI agent,102 PVA, is orders of magnitude less effective than even the shortest AFGP.14 PVA is a fully-flexible molecule that binds strongly to the primary prismatic plane of ice but does not display significant flexibility and mobility in the bound state.59 This suggests that the existence of multiple ice binding modes may be key for the step-pinning mechanism and exceptional IRI activity of AFGPs and could be hold the key for the design of synthetic analogues that rival them in potency. AUTHOR INFORMATION Corresponding Author

[email protected] Funding Sources

No competing financial interests have been declared. Work funded by the Japan Society for the Promotion of Science through award 15H05474 and the United States. National Science Foundation through award CHE-1305427. ACKNOWLEDGMENT

We gratefully acknowledge Yoshinori Furukawa and Patricio Carvajal for insightful discussions. K.M. was supported by the Japan Society for the Promotion of Science through award 15H05474 and an oversea research fellowship. V.M. was supported by the United States National Science Foundation through award CHE-1305427 “Center for Aerosol Impacts on Climate and the Environment”. Most calculations were performed at the Research Center for Computational Science in Okazaki, Japan. We thank the Center of High Performance Computing at The University of Utah for technical support. REFERENCES 1. Feeney, R. E.; Burcham, T. S.; Yeh, Y., Annu. Rev. Biophys. Biophys. Chem. 1986, 15, 59-78. 2. DeVries, A. L.; Wohlschlag, D. E., Science 1969, 163, 1073-1075. 3. Devries, A. L., Science 1971, 172, 1152-1155. 4. Voets, I. K., Soft Matter 2017, 13 (28), 4808-4823. 5. Dolev, M. B.; Braslavsky, I.; Davies, P. L., Annu. Rev. Biochem. 2016, 85, 515-542. 6. Oude Vrielink, A. S.; Aloi, A.; Olijve, L. L. C.; Voets, I. K., Biointerphases 2016, 11 (1), 018906. 7. Gibson, M. I.; Barker, C. A.; Spain, S. G.; Albertin, L.; Cameron, N. R., Biomacromolecules 2008, 10 (2), 328-333.



8. Carpenter, J. F.; Hansen, T. N., Proc Natl Acad Sci USA 1992, 89 (19), 8953-8957. 9. Liang, S.; Yuan, B.; Jin, Y.-X.; Zhang, J.-B.; Bang, J. K.; Kim, N.-H., Reprod Fertil Dev 2017, 29, 2140-2148 10. Tsvetkova, N. M.; Phillips, B. L.; Krishnan, V. V.; Feeney, R. E.; Fink, W. H.; Crowe, J. H.; Risbud, S. H.; Tablin, F.; Yeh, Y., Biophys. J. 2002, 82 (1), 464-473. 11. Lane, A. N.; Hays, L. M.; Tsvetkova, N.; Feeney, R. E.; Crowe, L. M.; Crowe, J. H., Biophys. J. 2000, 78 (6), 3195-3207. 12. Qiu, Y.; Odendahl, N.; Hudait, A.; Mason, R.; Bertram, A. K.; Paesani, F.; DeMott, P. J.; Molinero, V., J. Am. Chem. Soc. 2017, 139 (8), 3052-3064. 13. Daley, M. E.; Spyracopoulos, L.; Jia, Z.; Davies, P. L.; Sykes, B. D., Biochemistry 2002, 41 (17), 5515-5525. 14. Budke, C.; Dreyer, A.; Jaeger, J.; Gimpel, K.; Berkemeier, T.; Bonin, A. S.; Nagel, L.; Plattner, C.; DeVries, A. L.; Sewald, N.; Koop, T., Cryst. Growth Des. 2014, 14 (9), 4285-4294. 15. Ben, R. N., Chem. Bio. Chem. 2001, 2 (3), 161-166. 16. Urbańczyk, M.; Góra, J.; Latajka, R.; Sewald, N., Amino Acids 2017, 49 (2), 209-222. 17. Biggs, C. I.; Bailey, T. L.; Graham, B.; Stubbs, C.; Fayter, A.; Gibson, M. I., Nat. Commun. 2017, 8 (1), 1546. 18. Balcerzak, A. K.; Capicciotti, C. J.; Briard, J. G.; Ben, R. N., RSC Adv. 2014, 4 (80), 42682-42696. 19. Krishnamoorthy, A. N.; Holm, C.; Smiatek, J., J. Phys. Chem. B 2014, 118 (40), 11613-11621. 20. Ebbinghaus, S.; Meister, K.; Born, B.; DeVries, A. L.; Gruebele, M.; Havenith, M., J. Am. Chem. Soc. 2010, 132 (35), 12210-12211. 21. Mallajosyula, S. S.; Vanommeslaeghe, K.; MacKerell, A. D., J. Phys. Chem. B 2014, 118 (40), 11696-11706. 22. Czechura, P.; Tam, R. Y.; Dimitrijevic, E.; Anastasia V Murphy, a.; Ben, R. N., J. Am. Chem. Soc. 2008, 130 (10), 2928-2929. 23. Groot, C. C. M.; Meister, K.; DeVries, A. L.; Bakker, H. J., J. Phys. Chem. Lett. 2016, 7 (23), 4836-4840. 24. Knight, C. A.; Driggers, E.; DeVries, A. L., Biophys. J. 1993, 64 (1), 252-259. 25. Furukawa, Y.; Nagashima, K.; Nakatsubo, S.-i.; Yoshizaki, I.; Tamaru, H.; Shimaoka, T.; Sone, T.; Yokoyama, E.; Zepeda, S.; Terasawa, T.; Asakawa, H.; Murata, K.-i.; Sazaki, G., Sci. Rep. 2017, 7, 43157. 26. Raymond, J. A.; DeVries, A. L., Proc. Natl. Acad. Sci. U.S.A. 1977, 74 (6), 2589-2593. 27. Knight, C. A.; Cheng, C. C.; DeVries, A. L., Biophys. J. 1991, 59 (2), 409-418. 28. Bar Dolev, M.; Braslavsky, I.; Davies, P. L., Ann. Rev. Biochem. 2016, 85 (1), 515-542. 29. Zepeda, S.; Yokoyama, E.; Uda, Y.; Katagiri, C.; Furukawa, Y., Cryst. Growth Des. 2008, 8 (10), 3666-3672. 30. DeVries, A. L.; Komatsu, S. K.; Feeney, R. E., J. Biol. Chem. 1970, 245 (11), 2901-2908. 31. Harding, M. M.; Anderberg, P. I.; Haymet, A. D. J., Eur. J. Biochem. 2003, 270 (7), 1381-1392. 32. Lin, Y.; Duman, J. G.; DeVries, A. L., Biochem. Biophys. Res. Commun. 1972, 46 (1), 87-92. 33. Hew, C. L.; Slaughter, D.; Fletcher, G. L.; Joshi, S. B., Can. J. Zool. 1981, 59 (11), 2186-2192. 34. Burcham, T. S.; Osuga, D. T.; Rao, B. N.; Bush, C. A.; Feeney, R. E., J. Biol. Chem. 1986, 261 (14), 6384-6389. 35. Osuga, D. T.; Feeney, R. E.; Yeh, Y.; Hew, C.-L., Comp. Biochem. Physiol. B 1980, 65 (2), 403-406. 36. O'Grady, S. M.; Schrag, J. D.; Raymond, J. A.; Devries, A. L., J. Exp. Zool. 1982, 224 (2), 177-185. 37. Bush, C. A.; Feeney, R. E.; Osuga, D. T.; Ralapati, S.; Yeh, Y., Int. J. Pept. Protein Res. 1981, 17 (1), 125-129. 38. Wilkinson, B. L.; Stone, R. S.; Capicciotti, C. J.; Thaysen-Andersen, M.; Matthews, J. M.; Packer, N. H.; Ben, R. N.; Payne, R. J., Angew. Chem. Int. Ed. 2012, 51 (15), 3606-3610. 39. Bush, C. A.; Feeney, R. E., Int. J. Pept. Protein Res. 1986, 28 (4), 386-397.

Page 10 of 11

40. Lane, A. N.; Hays, L. M.; Feeney, R. E.; Crowe, L. M.; Crowe, J. H., Protein Sci. 1998, 7 (7), 1555-1563. 41. Rao, B. N. N.; Bush, C. A., Biopolymers 1987, 26 (8), 1227-1244. 42. Franks, F.; Morris, E. R., Biochim. Biophys. Acta 1978, 540 (2), 346-356. 43. Tachibana, Y.; Fletcher, G. L.; Fujitani, N.; Tsuda, S.; Monde, K.; Nishimura, S. I., Angew. Chem. 2004, 116 (7), 874-880. 44. Eniade, A.; Purushotham, M.; Ben, R. N.; Wang, J. B.; Horwath, K., Cell Biochem. Biophys. 2003, 38 (2), 115-124. 45. Liu, S.; Ben, R. N., Org. Lett. 2005, 7 (12), 2385-2388. 46. Tam, R. Y.; Rowley, C. N.; Petrov, I.; Zhang, T.; Afagh, N. A.; Woo, T. K.; Ben, R. N., J. Am. Chem. Soc. 2009, 131 (43), 15745-15753. 47. Tam, R. Y.; Ferreira, S. S.; Czechura, P.; Chaytor, J. L.; Ben, R. N., J. Am. Chem. Soc. 2008, 130 (51), 17494-17501. 48. Haymet, A. D. J.; Leanne G Ward, a.; Harding, M. M., J. Am. Chem. Soc. 1999, 121 (5), 941-948. 49. Fairley, K.; Westman, B. J.; Pham, L. H.; Haymet, A. D. J.; Harding, M. M.; Mackay, J. P., J. Biol. Chem. 2002, 277 (27), 24073-24080. 50. Nada, H.; Furukawa, Y., J. Phys. Chem. B 2008, 112 (23), 7111-7119. 51. Battle, K.; Salter, E. A.; Edmunds, R. W.; Wierzbicki, A., J. Cryst. Growth 2010, 312 (8), 1257-1261. 52. Wierzbicki, A.; Dalal, P.; Cheatham III, T. E.; Knickelbein, J. E.; Haymet, A. D. J.; Madura, J. D., Biophys. J. 2007, 93 (5), 1442-1451. 53. Yang, D. S.; Hon, W. C.; Bubanko, S.; Xue, Y.; Seetharaman, J.; Hew, C. L.; Sicheri, F., Biophys. J. 1998, 74 (5), 2142-2151. 54. Sönnichsen, F. D.; DeLuca, C. I.; Davies, P. L.; Sykes, B. D., Structure 1996, 4 (11), 1325-1337. 55. Howard, E. I.; Blakeley, M. P.; Haertlein, M.; Haertlein, I. P.; Mitschler, A.; Fisher, S. J.; Siah, A. C.; Salvay, A. G.; Popov, A.; Dieckmann, C. M.; Petrova, T.; Podjarny, A., J. Mol. Recognit. 2011, 24 (4), 724-732. 56. Kuiper, M. J.; Morton, C. J.; Abraham, S. E.; Gray-Weale, A., Elife 2015, 4, e05142. 57. Nada, H.; Furukawa, Y., Polym. J. 2012, 44 (7), 690-698. 58. Mochizuki, K.; Qiu, Y.; Molinero, V., J. Am. Chem. Soc. 2017, 139 (47), 17003-17006. 59. Naullage, P. M.; Lupi, L.; Molinero, V., J. Phys. Chem. C 2017, 121, 26949–26957. 60. Nguyen, D. H.; Colvin, M. E.; Yeh, Y.; Feeney, R. E.; Fink, W. H., Biophys. J. 2002, 82 (6), 2892-2905. 61. aFeller, S. E.; MacKerell, A. D., J. Phys. Chem. B 2000, 104 (31), 7510-7515. 62. Klauda, J. B.; Brooks, B. R.; MacKerell, A. D.; Venable, R. M.; Pastor, R. W., J. Phys. Chem. B 2005, 109 (11), 5300-5311. 63. MacKerell, A. D.; Bashford, D.; Bellott, M.; Dunbrack, R. L.; Evanseck, J. D.; Field, M. J.; Fischer, S.; Gao, J.; Guo, H.; Ha, S.; Joseph-McCarthy, D.; Kuchnir, L.; Kuczera, K.; Lau, F. T.; Mattos, C.; Michnick, S.; Ngo, T.; Nguyen, D. T.; Prodhom, B.; Reiher, W. E.; Roux, B.; Schlenkrich, M.; Smith, J. C.; Stote, R.; Straub, J.; Watanabe, M.; Wiórkiewicz-Kuczera, J.; Yin, D.; Karplus, M., J. Phys. Chem. B 1998, 102 (18), 3586-3616. 64. MacKerell Jr., A. D.; Feig, M.; Brooks III, C. L., J. Comput. Chem. 2004, 25 (11), 1400-1415. 65. Guvench, O.; Greene, S. N.; Kamath, G.; Brady, J. W.; Venable, R. M.; Pastor, R. W.; MacKerell Jr., A. D., J. Comput. Chem. 2008, 29 (15), 2543-2564. 66. Guvench, O.; Hatcher, E.; Venable, R. M.; Pastor, R. W.; MacKerell Jr., A. D., J. Chem. Theory Comput. 2009, 5 (9), 2353-2370. 67. Hatcher, E.; Guvench, O.; MacKerell Jr., A. D., J. Phys. Chem. B 2009, 113 (37), 12466-12476. 68. Hatcher, E. R.; Guvench, O.; MacKerell Jr., A. D., J. Chem. Theory Comput. 2009, 5 (5), 1315-1327. 69. Raman, E. P.; Guvench, O.; MacKerell Jr., A. D., J. Phys. Chem. B 2010, 114 (40), 12981-12994.


Page 11 of 11 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


70. Guvench, O.; Mallajosyula, S. S.; Raman, E. P.; Hatcher, E.; Vanommeslaeghe, K.; Foster, T. J.; Jamison II, F. W.; MacKerell Jr., A. D., J. Chem. Theory Comput. 2011, 7 (10), 3162-3180. 71. Mallajosyula, S. S.; Guvench, O.; Hatcher, E.; MacKerell Jr., A. D., J. Chem. Theory Comput. 2012, 8 (2), 759-776. 72. Mallajosyula, S. S.; Jo, S.; Im, W.; MacKerell, A. D., Methods Mol Biol. 2015, 1273 (Chapter 25), 407-429. 73. MacKerell, A. D. Website of MacKerell Lab. http://mackerell.umaryland.edu/charmm_ff.shtml (accessed Sept. 1 2017). 74. Abascal, J. L. F.; Sanz, E.; García Fernández, R.; Vega, C., J. Chem. Phys. 2005, 122 (23), 234511-10. 75. García Fernández, R.; Abascal, J. L. F.; Vega, C., J. Chem. Phys. 2006, 124 (14), 144506. 76. Preston-Thomas, H., Metrologia 1990, 27 (1), 3. 77. Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E., J. Chem. Theory Comput. 2008, 4 (3), 435-447. 78. Abraham, M. J.; Murtola, T.; Schulz, R.; Páll, S.; Smith, J. C.; Hess, B.; Lindahl, E., SoftwareX 2015, 1-2, 19-25. 79. Sugita, Y.; Okamoto, Y., Chem. Phys. Lett. 1999, 314 (1-2), 141-151. 80. Schrodinger, LLC, The PyMOL Molecular Graphics System, Version 1.8. 2015. 81. Bouvet, V. R.; Lorello, G. R.; Ben, R. N., Biomacromolecules 2006, 7 (2), 565-571. 82. Younes-Metzler, O.; Robert N Ben, a.; Giorgi, J. B., Langmuir 2007, 23 (23), 11355-11359. 83. Matsumoto, M.; Yagasaki, T.; Tanaka, H., J. Comput. Chem. 2018, 39 (1), 61-64. 84. Nguyen, A. H.; Molinero, V., J. Phys. Chem. B 2015, 119 (29), 9369-9376. 85. Mochizuki, K.; Sumi, T.; Koga, K., Sci. Rep. 2016, 6, 24657. 86. Mochizuki, K.; Sumi, T.; Koga, K., Phys. Chem. Chem. Phys. 2016, 18 (6), 4697-4703. 87. Hub, J. S.; de Groot, B. L.; van der Spoel, D., J. Chem. Theory Comput. 2010, 6 (12), 3713-3720. 88. Feeney, R. E., Am. Sci. 1974, 62, 712-719. 89. Butt, H.-J.; Graf, K.; Kappl, M., Physics and Chemistry of Interfaces. Wiley-VCH: Weinheim, Germany, 2013. 90. Nagel, L.; Budke, C.; Erdmann, R. S.; Dreyer, A.; Wennemers, H.; Koop, T.; Sewald, N., Chem. A Eur. J. 2012, 18 (40), 12783-12793. 91. Uda, Y.; Zepeda, S.; Kaneko, F.; Matsuura, Y.; Furukawa, Y., J. Phys. Chem. B 2007, 111 (51), 14355-14361. 92. Sobolewski, E.; Makowski, M.; Czaplewski, C.; Liwo, A.; Stanisław Ołdziej, a.; Scheraga, H. A., J. Phys. Chem. B 2007, 111 (36), 10765-10774. 93. Mochizuki, K.; Sumi, T.; Koga, K., Phys. Chem. Chem. Phys. 2017, 19 (35), 23915-23918. 94. Wallqvist, A.; Berne, B. J., Chem. Phys. Lett. 1988, 145 (1), 26-32. 95. Song, B.; Molinero, V., J Chem Phys 2013, 139 (5), 054511. 96. Yagasaki, T.; Matsumoto, M.; Tanaka, H., J. Am. Chem. Soc. 2015, 137 (37), 12079-12085. 97. Lazaridis, T.; Paulaitis, M. E., J. Phys. Chem. 1992, 96 (9), 3847-3855. 98. Mimura, Y.; Yamamoto, Y.; Inoue, Y.; Chujo, R., Int. J. Biol. Macromol. 1992, 14 (5), 242-248. 99. Celik, Y.; Drori, R.; Pertaya-Braun, N.; Altan, A.; Barton, T.; Bar Dolev, M.; Groisman, A.; Davies, P. L.; Braslavsky, I., Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (4), 1309-1314. 100. Olijve, L. L. C.; Meister, K.; DeVries, A. L.; Duman, J. G.; Guo, S.; Bakker, H. J.; Voets, I. K., Proc. Natl. Acad. Sci. U.S.A. 2016, 113 (14), 3740-3745. 101. Garnham, C. P.; Campbell, R. L.; Davies, P. L., Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (18), 7363-7367. 102. Congdon, T.; Notman, R.; Gibson, M. I., Biomacromolecules 2013, 14 (5), 1578-1586.

TOC figure


Antifreeze Glycoproteins Bind Reversibly to Ice via Hydrophobic

Recommend Documents