Structure and Dynamics of Antifreeze Protein–Model Membrane

Jan 19, 2016 - Antifreeze proteins (AFPs) are the key biomolecules that enable species to survive under subzero temperature conditions. The physiologi...
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Structure and Dynamics of Antifreeze Protein−Model Membrane Interactions: A Combined Spectroscopic and Molecular Dynamics Study Rajiv K. Kar,† Kamal H. Mroue,‡ Dinesh Kumar,§ Bimo A. Tejo,∥ and Anirban Bhunia*,† †

Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700 054, India Biophysics and Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States § Center of Biomedical Magnetic Resonance, Sanjay Gandhi Post-Graduate Institute of Medical Sciences Campus, Lucknow 226014, India ∥ Department of Biotechnology, Surya University, Tangerang 15810, Indonesia ‡

S Supporting Information *

ABSTRACT: Antifreeze proteins (AFPs) are the key biomolecules that enable species to survive under subzero temperature conditions. The physiologically relevant activities of AFPs are based on the adsorption to ice crystals, followed by the inhibition of subsequent crystal layer growth of ice, routed with depression in freezing point in a noncolligative manner. The functional attributes governing the mechanism by which AFPs inhibit freezing of body fluids in bacteria, fungi, plants, and fishes are mainly attributed to their adsorption onto the surface of ice within the physiological system. Importantly, AFPs are also known for their application in cryopreservation of biological samples that might be related to membrane interaction. To date, there is a paucity of information detailing the interaction of AFPs with membrane structures. Here, we focus on elucidating the biophysical properties of the interactions between AFPs and micelle models that mimic the membrane system. Micelle model systems of zwitterionic DPC and negatively charged SDS were utilized in this study, against which a significant interaction is experienced by two AFP molecules, namely, Peptide 1m and wfAFP (the popular AFP sourced from winter flounder). Using low- and high-resolution biophysical characterization techniques, such as circular dichroism (CD) and NMR spectroscopy, a strong evidence for the interactions of these AFPs with the membrane models is revealed in detail and is corroborated by in-depth residue-specific information derived from molecular dynamics simulation. Altogether, these results not only strengthen the fact that AFPs interact actively with membrane systems, but also demonstrate that membrane-associated AFPs are dynamic and capable of adopting a number of conformations rendering fluidity to the system.



the biophysical and biochemical properties. 7,8 Multiple theoretical studies using conventional molecular dynamics (MD) and replica exchange molecular dynamics have been used as well to elucidate the structure−function relationships of AFPs.9,10 The classification system of AFPs (types I−V) is based on their structural construct in terms of amino acid content and secondary structure.11,12 The remarkable diversity and distribution of AFPs suggest that the evolution of different types of AFPs can be attributed to the adaptation owned after the sea-level glaciations;13 such independent expansion for adaptation refers to the convergent evolution of AFPs.14 It should be noted that different types of AFPs (structural variants) develop the potential to unravel the inhibition of ice

INTRODUCTION Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) are well-known for their property of lowering the freezing point of water in a noncolligative manner.1 This feature is primarily essential for the survival of species that have their ecological niche in the polar region in subzero environments.2 These AFPs are present in various biological living kingdoms, including vertebrates, bacteria, fishes, and insects, as well as in plants, fungi, and yeasts.3,4 Within the physiological system, AFPs bind to small ice crystals and inhibit the crystal growth without affecting the melting temperature, a phenomenon known as thermal hysteresis (TH).5 The molecular mechanisms underlying this phenomenon and the relevant structural insights have been deciphered with various experimental techniques such as X-ray crystallography and high-resolution NMR spectroscopy.6 In addition, low-resolution studies such as Fourier transformed infrared, circular dichroism (CD), thermal hysteresis, and ice recrystallization assay also help in elucidating © 2016 American Chemical Society

Received: November 14, 2015 Revised: January 17, 2016 Published: January 19, 2016 902

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The Journal of Physical Chemistry B crystal formation processes. Properties like α-helical construct, higher content of Ala residues in their sequence, and amphipathic nature are some of the remarkable features that fine-tune the structure−function activity relationships in AFPs.12,15 One of the simplest explanations regarding AFP activity can be attributed to the uniform composition of the hexagonal lattice structure of ice, as it is composed of oxygen and hydrogen only; it is hence possible for different types of AFPs to bind with various planes at ice surfaces.16 Nevertheless, the exact mechanism of action for AFPs is not well-understood to date, where hydrogen bonding and van der Waals surface complementary are two debatable concepts.17 Additionally, the activity of AFPs mediated by interaction with ice crystals also suggests another hypothesis that AFPs have a role in membrane binding.18 At lower temperatures, the rational function of the membrane is integrated and maintained under physiological conditions, and the fluidity of the membrane is preserved by the replacement of saturated/unsaturated fatty acids of the membrane.3 AFPs thereby bind to the cell walls and membranes to protect them from freezing damage. This phenomenon can also be referred to as acclimatization of the species to the freezing conditions.19,20 Studies of AFP (type I) interactions with model membrane systems have been summarized in a review by Inglis and co-workers.21 Earlier studies have suggested that the mechanism of action of AFPs is almost similar to antimicrobial peptides, as both interact with the biological membranes.22 In this context, Rubinsky and co-workers have found that fish AFPs are indeed successful in protecting the membranes in the low-temperature stress conditions.23 The phospholipid and cholesterol composition of cell membranes is one of the limiting factors for efficient cryopreservation. However, the protection of membrane from chilling in cold conditions is attributed to particular interactions of AFPs with membrane-bound integral proteins; the evidence for such interaction is reported by Hays et al.,24 showing that antifreeze glycoproteins (AFGPs) inhibit leakage of liposomes during thermotropic phase transition. Tablin et al. demonstrated the preservation of human blood platelets in the presence of AFGPs at 4 °C for about 4 days.25 On the other hand, the hypothesis by Tomczak et al. suggests that there exists a mechanism of membrane protection and corresponding stabilization by type I AFPs,18 where the feature might be linked with the peptide interaction and orientation “with” or “into” the membrane.26 Apart from these studies, there has been scarce evidence explaining the interaction properties of AFPs with membrane models in recent years.27 A previous report from our group focusing on engineering of Peptide 1m, an antifreeze peptide from psychrophilic yeast Glaciozyma antarctica, has demonstrated high antifreeze activity in the culture filtrate.28 Genomic analysis of G. antarctica showed that the protein has 30% similarity with an antifreeze protein sourced from Typhula ishikariensis.29 In an attempt to elucidate the mechanism of interaction between antifreeze peptides and membrane models, we have undertaken two different AFPs, namely, Peptide 1m28 and wfAFP (the popular AFP from winter flounder),30 with two micelle models. In particular, dodecylphosphocholine (DPC) micelle acts as a membrane model owing to the presence of phosphocholine head groups.31 Likewise, the anionic detergent sodium dodecyl sulfate (SDS) micelle provides a robust membrane environment for studying the association and dynamics of membrane binding proteins and polypeptides.32 It should be noted that bicelles have ordered arrangement (cylindrical geometry)

compared to the conical curvature-like geometry exhibited by micelles, which makes bicelles suitable model systems for membrane studies as well.33 Importantly, the presence of a higher concentration of long chain lipids in bicelles reduces the molecular tumbling effect and renders the system suitable to study with solid-state NMR (and still amenable for solutionstate NMR studies). In contrast, micelles are suitable membrane models to study with solution-state NMR due to their faster molecular tumbling on the NMR time scale.33,34 In this study, micelle systems were thus selected as membrane models for elucidating the interaction with AFPs using solution-state NMR spectroscopy and molecular dynamics simulations. Specifically, we have performed the interaction analysis for AFPs with DPC and SDS micelles as membrane models, which further paves the way for important biomechanisms.



MATERIALS AND METHODS Peptides and Lipids. The antifreeze peptides considered for this study, namely, Peptide 1m and wfAFP, were purchased from China Peptides (Shanghai, China). Perdeuterated dodecyl phosphocholine (DPC-d38) and perdeuterated sodium dodecyl sulfate (SDS-d54) were purchased from Cambridge Isotope Inc., U.S.A. Dodecylphosphocholine (DPC) and sodium dodecyl sulfate (SDS) were obtained from Avanti Polar Lipids Inc. Spin-labeled lipids, 5-doxyl-stearic acid (5-DSA) or 16-doxylstearic acid (16-DSA), were purchased from Sigma (St. Louis, MO, U.S.A.). Circular Dichroism Experiments. Information pertaining to the globular secondary structure information on Peptide 1m and wfAFP in SDS and DPC micelles was monitored with circular dichroism (CD) analysis. Jasco J-815 spectrometer (Jasco International Co., Ltd. Tokyo, Japan), equipped with a Peltier cell holder and a temperature controller CDF-426 L at 37 °C, was used to perform the experiments. Samples were scanned from wavelength 195 to 260 nm. The concentration of peptides was 25 μM (pH 4.5), whereas the concentration of DPC and SDS was 20 mM. Baseline correction was performed with reference to distilled water, and data points were collected as an average of three repetitive scans. The data obtained were in millidegrees and were converted to molar ellipticity in units of (deg·cm2)/dmol. NMR Spectroscopy. Solution-state NMR experiments were carried out on a Bruker Avance 800 MHz spectrometer (CBMR, Lucknow, India) equipped with 5 mm cryoprobe, as well as on a Bruker Avance III 500 MHz spectrometer (Bose Institute, Kolkata, India) equipped with smart probe and zpulse field gradient. All NMR samples were prepared in 600 μL volumes with 90% H2O/10% D2O. The interactions of Peptide 1m and wfAFP with both zwitterionic DPC and with negatively charged SDS were inspected by recording one-dimensional proton NMR spectra with a spectral width of 10 ppm. The NMR samples contain peptides at 1 mM concentration, and that of DPC-d38 and SDS-d54 in the sample was 125 mM and 200 mM, respectively. The experiments were performed at pH of ∼4.5 at 37 °C. Two-dimensional 1H−1H TOCSY as well as NOESY spectra were acquired with 2048 (ω1) × 512 (ω2) points and WATERGATE for water suppression and StatesTPPI for quadrature detection in the t1 dimension.35 The mixing time for NOESY experiment was fixed to 150 ms, whereas the TOCSY spinlock mixing time was set to 80 ms. A data matrix of 4K (ω1) × 1K (ω2) was obtained after zero filling. 2,2-Dimethyl-2-silapentane-5-sulfonate sodium salt 903

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Figure 1. CD spectra of wfAFP (A) and Peptide 1m (B) in the presence of DPC and SDS micelles. The probability (percentage) of the secondary structures obtained by deconvolution of CD spectra using DicroWeb server is also shown for wfAFP (C) and Peptide 1m (D) in the presence of DPC (orange), SDS micelle (green), and in aqueous condition (blue).

preparation using Autogrid. External grid value was fixed to 1000, and all the remaining grids have constant energy-scoring grid points to cover the entire micelle. Lamarckian genetic algorithm was used (10 runs), which has a LUDI type of scoring function with the rates of gene mutation and crossover used as 0.02 and 0.8, respectively.38 Gasteiger-Marsili charges were used for the docking calculations.39 The remaining parameters were all kept at their default values. The lowest energy conformation was used for further analysis. The obtained complex was then subjected to energy minimization using Polak-Ribiere Conjugate Gradient (PRCG) with 2500 iteration steps. Truncated Newton Conjugate Gradient (TNCG) method was also used with gradient convergence method with a convergence threshold value of 0.05. The energy-minimized complex of AFPs and micelle system was then used for analysis. The obtained complexes from both approaches were processed for molecular dynamics simulation with a time period of 30 ns, making use of OPLS_2005 force field in Desmond.40 The model was solvated using TIP3P water model in an orthorhombic box of 10 Å edge length from any solute atom.41 The protocol used for simulation herein was similar to that used in our previous study on AFPs with NVT microcanonical system.42 A cutoff of 10 Å was used for the real-space part of electrostatic and Lennard−Jones interactions. RESPA integrator was used for long-range Coulomb interactions and the remaining interactions with a time step of 6 and 2 fs, respectively. The M-SHAKE algorithm was used to constrain the bond lengths of hydrogen. Note that in addition to the AFPs-DPC/SDS complexes, MD simulations of similar time scale were carried out on the peptides (wfAFP and Peptide 1m) in the absence of any micelle system to account for the variation of their dynamicity.

(DSS) was used as internal standard (0 ppm) for chemical shift referencing. Data processing was performed in TopSpin (Bruker, Switzerland) and was analyzed using SPARKY (T. D. Goddard and D. G. Kneller, University of California, San Francisco) running on Linux workstation. Spin Labeling NMR Experiments. The effect of line broadening was assessed with the help of paramagnetic relaxation NMR experiments for Peptide 1m. These experiments were performed using the successive addition of aliquots of 5-doxyl-stearic acid (5-DSA) and 16-doxyl-stearic acid (16DSA) in the NMR sample.36 The paramagnetic solution was prepared in deuterated methanol (D4-MeOH). NMR samples of peptides were the same as illustrated in the above section with similar concentrations for DPC and SDS micelles. After addition of 5-DSA or 16-DSA, the samples were allowed to equilibrate for a period of ∼10−15 min and then 1H−1H TOCSY spectra were recorded. All parameters were kept constant, except for the field shimming and probe tuning. The cross-peak intensities of Peptide 1m were measured before as well as after the addition of both paramagnetic solutions and were calculated according to established protocols.36 Molecular Modeling Studies. To investigate the interaction phenomena of Peptide 1m and wfAFP with DPC and SDS micelles, molecular modeling techniques were used. Molecular modeling is indeed one of the optimum techniques for elucidating atomistic-level information relevant to antifreeze proteins.10 We used Autodock software for obtaining a complex model of Peptide 1m with DPC and SDS micelles.37 The calculated coordinates of Peptide 1m (ligand) in both micelle systems were used for docking with respective to micelle models (receptor). The backbone of the peptide was kept rigid, and few of the side chains were kept flexible for the docking approach. A grid spacing of 0.37 Å was used for grid 904

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Figure 2. One-dimensional proton NMR spectra of wfAFP (A) and Peptide 1m (D) in water. The relative perturbation as indicated by the chemical shift can be seen in the proton spectra of wfAFP and Peptide 1m in DPC micelle (B, E) and SDS (C, F). All the experiments were performed at 37 °C using Bruker Avance 800 MHz spectrometer, equipped with a cryo-probe.

solution is α-helix (Figure S1),36 these spectra indicate that the conformational adaptation of this AFP is not perturbed due to its presence in a micelle environment. This indeed is a convincing result that shows the existence of interaction of AFPs with model micelle membranes. Likewise, the assessment of similar globular structure from the far-UV CD spectra of Peptide 1m in DPC and SDS micelles also reveals a similar αhelix conformation in the presence of both DPC as well as SDS membranes (Figure 1B). For Peptide 1m, we observe negative minima at 206 and 224 nm with DPC and SDS micelles, respectively. An in-depth secondary structure analysis was conducted by deconvolution of the CD spectra using the Dichro Web Server.48 The CD spectra obtained for the two AFPs in corresponding membrane micelle models were used for comparison against the reference set, which in turn provides the percentage estimation of secondary structure for helices, sheets, turns, and random coils. wfAFP in the presence of DPC shows higher α-helix (55%) content; on the contrary, more than 70% of the conformations are attributed to α-helix and 310-helix in the presence of SDS system, whereas 20% of conformation is attributed to turns (Figure 1C). In a similar manner, Peptide 1m also shows α-helicity in the presence of both DPC and SDC micelles. A minor population of the Peptide 1m also exhibits strand (10%) and turn (20%) conformations in DPC, in comparison to a 20% strand conformation in SDS micelles (Figure 1D). Overall, the CD results indicate that peptides wfAFP and Peptide 1m are both dominated by α-helical conformations in the presence of DPC and SDS micelles. For comparison, Figure 1C and D also include the deconvoluted secondary structure information of wfAFP and Peptide 1m in water in the absence of any micelle system (Figure 1C,D, blue bars). A majority of conformations appear to be α-helix with a value of 55% and 45% for wfAFP and Peptide 1m, respectively, which are comparable with the values obtained in the presence of micelle environments. In addition, both AFPs exist with minor populations, as turns and unordered secondary structures in aqueous solution. A contribution accounting to about 16.5% of turns is obtained for both peptides in water, and the unordered secondary

Trajectory Analysis. Trajectories were saved at 4 ps interval and were analyzed using VMD software.43 Principal components (PCs) for all atoms and selective Cα atoms for AFPs were analyzed using PCA suite (http://mmb.pcb.ub.edu/ software/pcasuite/). This helps in preparing average structures based on the principal component, which appears in the form of animated coordinates. Porcupine plots were prepared for the visual representation of the structures, using a Perl script.44 Mean squared displacement (MSD) was calculated using “g_msd” module of GROMACS.45 MSD helps in identifying the thermal displacement of each atom from the averaged center of mass based on eq 1: MSD(t ) = |r(t ) − rcm(t )|2 = |[ri(t ) − ri(0)] − [rcm(t ) − rcm(0)]|2

(1)

where ri(t) represents the position of the ith atom at time t, cm represents the center of mass, and ⟨ ⟩ represents the average values. The MSDs for Peptide 1m and wfAFP were calculated directly from the trajectory course based on the atoms of peptides; subsequent calculations were also performed on the basis of rmsd values. Cluster analyses were performed using MMTSB tools and were correlated with the MD simulation results.46



RESULTS AND DISCUSSION Globular Structure of AFPs in the Presence of Membrane Micelle Models. Previous studies indicate that both wfAFP and Peptide 1m, exhibits α-helical geometry in aqueous solution (Figure S1, Supporting Information).28,47 In this study, elucidation of the secondary structures of both peptides in the presence of SDS and DPC micelle systems was carried out using far-UV CD spectroscopy. Figure 1 represents the CD plots for wfAFP and Peptide 1m in the presence of DPC and SDS model membrane systems. Notably, wfAFP is used as a control peptide in this study because of its popular antifreeze activity.30 In both DPC and SDS micelles, the wfAFP spectra show two strong negative peaks, one at 208 and another at 222 nm, which are reflective of α-helical conformation (Figure 1A). Since the native structure of wfAFP in aqueous 905

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The Journal of Physical Chemistry B structural contribution is 20.4 and 27.4% for wfAFP and Peptide 1m, respectively. Overall, these results indicate that certain variations in secondary structure contribution persist for both AFPs in different micelle conditions, which is reflective of the existence of an interaction with membrane model systems. Structural Perturbation Observed by NMR Spectroscopy. Figure 2 displays the low-field region in the onedimensional (1D) proton (1H) NMR spectra of wfAFP and Peptide 1m in aqueous solution (Figure 2A,D) as well as in DPC and SDS micelles (top to bottom panel), respectively. Spectral dispersion was observed for wfAFP, where structural perturbation was more isolated and broadened in SDS micelles compared to DPC micelles (Figure 2B,C,E,F). Similarly, an indication of stable structural integrity for Peptide 1m conformation is being designated by these well-resolved NMR peaks in DPC and SDS micelles (Figure 2E,F). The dispersed one-dimensional 1H NMR spectra can thus be attributed to the unique conformational states of both wfAFP and Peptide 1m. The NMR spectrum of Peptide 1m is found to be more dispersed and broadened in the presence of SDS compared to the one obtained in the presence of DPC micelles. These characteristic spectra are indicative of relatively dynamic behavior in solution and the presence of various conformational states in the corresponding micelle system. The one-dimensional proton NMR spectra of both wfAFP and Peptide 1m also show a relative degree of perturbation, which implies the existence of an interaction of these AFPs with micelle systems. More importantly, the lack of severe signal overlap as revealed from the one-dimensional 1H NMR spectra is also indicative that the two AFPs do not adopt any random coil or unordered structure in the presence of DPC and SDS micelles. In the presence of micelles, a prominent line-shape broadening for the proton peaks is also observed in addition to reduced proton signal in wfAFP and Peptide 1m (Figure 2), suggesting that the peptides interact with the model membranes and exhibit certain structural perturbation that leads to conformational exchange due to the addition of micelle systems, which occurs from a fast to an intermediate time scale. We have also performed twodimensional (2D) TOCSY and NOESY NMR experiments on wfAFP and Peptide 1m in the presence of DPC and SDS micelles. Importantly, a severe overlap of NOEs is observed in the NOESY spectrum that makes it extremely challenging for assignment and calculation of the three-dimensional structures of the peptides in model membranes. The NOESY spectra of wfAFP and Peptide 1m are shown in Figures S2 and S3 of the Supporting Information. Localization of AFPs in Micelle Systems. The estimation of the positioning of Peptide 1m in both DPC and SDS micelles was conferred with the help of paramagnetic relaxation enhancement (PRE) experiments. Two paramagnetic lipids, namely, 5-doxyl-steric acid (5-DSA) and 16-doxyl-steric acid (16-DSA), were used to probe the localization of peptides into the micelle systems. These spin-labeled fatty acids have the potential to broaden the NMR signals by virtue of enhancement of the T2 transverse relaxation rate of the protons that are in close proximity to the doxyl steric acid. 5-DSA has the paramagnetic doxyl group at the fifth carbon position and 16DSA has the paramagnetic doxyl group at the 16th carbon position of the acyl chain. Hence, 5-DSA affects the NMR signals of the CαH protons that are positioned at either the micelle surface or at the 3−4 carbon atom positions with respect to the site of spin label. Similarly, 16-DSA perturbs the NMR signal for the CαH protons that are deeply buried/

inserted into the micelle or at the center of the micelle system. In this context, the perturbations of the NMR CαH/NH crosspeak signal intensities, upon addition of the paramagnetic lipids, were estimated with respect to the TOCSY cross-peaks. Particularly, the TOCSY cross-peaks along the F2 dimension for the amide resonances were used for calculating the changes in peak intensities. In the case of Peptide 1m with both DPC and SDS micelles, we found that both 5-DSA and 16-DSA nitro-oxide spin-labeled lipids induced perturbation of CαH/ NH cross-peak intensities. The inference for the peptide localization was further drawn on the basis of cumulative intensity reduction for the CαH/NH cross-peaks that was induced by successive addition of 5-DSA/16-DSA. The stepwise addition of 5-DSA and 16-DSA to the AFPs in conjunction with micelle models reveals a concurrent attenuation of intensities for the NOEs peak (Figure 3). In particular, the contour peaks of CαH region were used for analysis of the relative intensity decrease compared to the free solution of Peptide 1m in DPC/SDS micelles. The concurrent

Figure 3. Two-dimensional TOCSY spectra showing the CαH/NH cross-peaks of Peptide 1m in DPC (A and C) and SDS (B and D) micelles. The cross peaks are shown by red contours. The relative loss in peak intensities with respect to the perturbation induced by 0.5 mM 5-DSA in DPC (A), 16-DSA in SDS (B), 5-DSA in DPC (C), and 16DSA in SDS (D). The samples containing 5-DSA or 16-DSA are highlighted by green and blue color, respectively. All the experiments were performed at 37 °C using Bruker Avance III 500 MHz spectrometer. 906

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The Journal of Physical Chemistry B reduction in peak intensities indicates a potential interaction of Peptide 1m with the zwitterionic micelle DPC, with a relative decrease in peak intensity of 83.1 and 74.5% for 5-DSA and 16DSA, respectively (Figure 3A,C). Similarly, in the presence of anionic surfactant SDS, a reduction in peak intensity of 88.6% and 86.6% for 5-DSA and 16-DSA, respectively, is observed (Figure 3B,D). Note that the reduction percentage of peak intensity for 5-DSA is almost comparable to that of 16-DSA in the presence of both DPC and SDS micelles. These data thus put forward an inference that Peptide 1m is neither present completely at the micellar interface nor it penetrates to the core/center of the micelle. Rather, it must be located at the interface of micelle to form salt bridge or electrostatic interaction between phosphate/anionic groups of micelle with positively charged amino acids of Peptide 1m; there might also exist a hydrophobic interaction between acyl chains of lipids with the aliphatic/aromatic groups of Peptide 1m. We have subsequently used these assumptions for molecular modeling investigations, in which AutoDock was used to derive the complexes that were selected based on the positioning of peptides over the micelles. AFP Complexes with DPC and SDS Micelles in Explicit Solvent Condition. The starting structures for docking of AFPs with membrane models were taken from the protein data bank (wfAFP PDB code: 1WFB, and Peptide 1m PDB code: 2LQ0). As discussed in the previous section, the complexes of wfAFP and Peptide 1m with DPC and SDS micelles were obtained using Autodock with a presumption from PRE experiments (Figure 3). The residues of wfAFP such as Asn16, Lys18, Glu22, Thr24, and Asn27 were found to be at the interface of the DPC micelle. Furthermore, Thr13, Asn16, Lys18, Glu22, and Asn27 of wfAFP were also located at the interface of SDS micelles. Similarly, hydrophilic residues Ser11, Arg15, Glu19, and Arg23 of Peptide 1m were next to either DPC or SDS micelle (Figures S4 and S5). A more detailed illustration pertaining to the atomistic information for the interaction phenomena between AFPs and micelle systems is discussed below. It is noteworthy that these electrophilic residues can either form hydrogen bonds or electrostatic interactions with the negatively charged headgroup of DPC and SDS micelles. The corresponding distance between the proton donor and acceptor in such analysis was about 4 Å, and the angle between acceptor atom, hydrogen, and donor atom was ∼120°. Unfortunately, the experimental NOEs were ambiguous due to a limitation in the spectral resolution, which precludes the use of NMR restraints in molecular dynamics (MD) simulations. MD simulations of the complex systems were performed using OPLS_2005 force field in Desmond module of Schrodinger.40 The stability parameter and the optimum time scale requisite for the convergence of trajectory were analyzed precisely based on the potential energy of the system (Figure S6). The stability of peptide structures in the DPC and SDS micelles were further analyzed with the help of all-atom RMSD (Figure 4). The convergence/stability of the AFPs was found to be more stable in the presence of SDS micelle compared to that of the DPC micelle. A similar analysis of the backbone RMSD was also carried out to identify the convergence criteria for the peptide conformation in MD simulations (Supporting Information, Figure S7). This is evidenced by the fact that wfAFP in DPC was stabilized after 10 ns of MD simulation (Figure 4A). In a similar context, Peptide 1m was stable after 15 ns of MD simulation (Figure 4C). On the contrary, the stability

Figure 4. Convergence criteria for AFPs in the presence of membrane models revealed with the help of all-atom RMSD and radius of gyration (Rg) in DPC micelles for wfAFP (A) and Peptide 1m (C); and in SDS micelles for wfAFP (B) and Peptide 1m (D). MSD plots based on the RMSD profile of AFPs representing the thermal fluctuation for conformations in DPC for wfAFP (E) and Peptide 1m (G); and in SDS micelles for wfAFP (F) and Peptide 1m (H).

of wfAFP and Peptide 1m were found to be stabilized by SDS within the first 5 ns of MD simulation (Figure 4B,D). Taking into account the dynamical features of AFPs in micelle systems, structural snapshots at various time points are shown in the Supporting Information (Figure S8: WfAFP-DPC micelle; Figure S9: WfAFP-SDS micelle; Figure S10: Peptide 1m-DPC micelle; and Figure S11: Peptide 1m-SDS micelle). Briefly, in the presence of DPC and SDS micelles, the straightforward geometry of WfAFP and the spherical shape of micelle are believed to be responsible for bending of peptide during the course of simulation. In the case of WfAFP with DPC micelle, an unstructured segment spanning residues A19-A25 is apparent, which gives more deviation in the 7.5 ns snapshot compared to the 1 ns snapshot (Figure S8). A similar bending of the peptide structure is reflective for wfAFP with SDS micelle (Figure S9). Importantly, both deviations seem to be converged at the 30 ns time point, as indicated with the snapshots. The bending pattern for Peptide 1m in DPC micelle is more pronounced, which shows a deviation up to 15 ns and then is stabilized until 30 ns (Figure S10). The dynamic behavior of Peptide 1m is found to be more conserved in SDS micelle, which shows only minimal fluctuation until 5 ns, after which it attains stability (Figure S11). The partial stability of both AFPs in the presence of SDS (a negatively charged micelle system) can also be accounted for by counterion stabilization (Na+ ions). Detailed investigation of counterion stabilization has not been performed in the present study, since counterions were incorporated at random positions in both DPC and SDS systems. We have also accounted for the radius of gyration (Rg) that reflects the bending of peptide conformation in the model membrane (see “Rg”, Figure 4A−D). A decrease in Rg was found to be higher for wfAFP in DPC micelle (∼1 nm), whereas that for Peptide 1m in DPC micelle was 0.85 nm. This 907

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Figure 5. All-atom and Cα-atom MSD plots of AFPs representing the diffusion property for wfAFP in the presence of DPC (A) and SDS (B) micelles, and for Peptide 1m in the presence of DPC (C) and SDS (D) micelles. The diffusive behavior of wfAFP and Peptide 1m in water is shown in (E) and (F), respectively.

was calculated with the consideration of atomistic fluctuations within the corresponding micelle system (Figure 5 and Figure S12, Supporting Information); and last, the MSD were taken into account using the Eigen vectors obtained from the principal component analysis (PC1 and PC2; Figure S13, Supporting Information). Based on the RMSD profile of AFPs, a prominent distinction was found with the MSD profiles in the presence of DPC and SDS micelles (Figure 4E−H). In particular, for both wfAFP and Peptide 1m, the convergence criterion was found to be smoothly achieved in the presence of SDS micelle compared to the DPC system. The MSD along the RMSD profile of wfAFP and Peptide 1m in the presence of DPC micelle reveal no convergence, which confirms that the Brownian motion dominates the subsequent conformation steps (Figure 4E,G). In other words, it can also be said that the cosine content of trajectories in DPC micelle for AFPs is higher (Table S1, Supporting Information). In contrast, the presence of SDS micelles reveals rapid convergence within the first 5 ns of trajectory for wfAFP, whereas a low profile convergence is found for Peptide 1m (Figure 4F,H). Notably, the difference of MSD plot based on RMSD for DPC and SDS system varies within a 4-fold margin (as indicated by the Y-axis).

bending in the 3D conformation of wfAFP attains stability within 5 ns time period (Figure 4B) and that for Peptide 1m is minimal, which is indicative of an almost straight line for the SDS micelle system (Figure 4D). These data correspond to the structural snapshots discussed in the previous section (Figures S8−S11). Taken together, these results indicate that the convergence criteria for structural stability of AFPs have been achieved with membrane models during the course of the simulation. Diffusion Characteristics of AFPs in the Presence of Micelles. The trajectory of conformations with a 3D hyperspace always proceeds with a displacement of solute atoms. Notably, the path followed in the associated system always corresponds to random collision (Figure 4E−H). More importantly, the stochastic nature of conformational sampling renders the sequential steps in trajectory always independent of one another. This infers that the account of thermal fluctuation of atoms with respect to the averages center of mass is crucial in governing the molecular interaction. In order to elucidate such atomistic information from the trajectory course, MSD analysis that reflects the diffusion behavior of the AFPs was performed using three individual approaches. First, MSD was calculated based on RMSD profile of AFPs (Figure 4E−H); second, MSD 908

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Figure 6. Account of motional fluctuations for AFPs represented by residue-specific fluctuations (RMSF) for wfAFP (A−C, upper panel) and Peptide 1m (D−F, lower panel). The left panel corresponds to the system in the presence of DPC micelle (A, D), and the middle panel corresponds to those in SDS micelle (B, E). The motional fluctuation corresponding to dynamicity in water, in the absence of micelle system is shown for wfAFP (C) and Peptide 1m (F). Porcupine plots corresponding to the motional fluctuations based on principal component analysis (PCA) are also presented for each AFP.

In the second approach, MSD plots were analyzed taking into consideration all-atoms and Cα-atoms of the AFPs (Figure 5). Although the MSD plots calculated for respective atoms displayed similar meaningful pattern as those derived on the basis of RMSD profile, some unique differences can be found, which add to our understanding regarding the AFP conformations. The convergence criteria for wfAFP and Peptide 1m are poorly achieved in the presence of DPC micelles (Figure 5A,C), which indicate the dominance of Brownian movement. A steady slope of the diffusion peak in the presence of DPC micelle after 10 ns implies that the subdiffusive motion encounters a barrier of potential energy. These MSD plots were also indicative of the external protein diffusion (translational and rotational motions) and internal protein diffusion (anharmonic motions). Thus, the difference in MSD plots indicates more fluctuations of side chain atoms of the peptides in DPC than SDS micelles. On the contrary, the trapping time (or water retention time) for wfAFP and Peptide 1m is more prominent in the presence of SDS micelles, and it falls in the 15−30 ns time scale range (Figure 5B,D). The MSD of AFPs in SDS micelles indicates that the diffusive property (both internal and external protein diffusion) is higher (Figure 5B,D). Furthermore, a steady state convergence was achieved at 12 and 15 ns for wfAFP and Peptide 1m, respectively. Similar to Figure 5, representative MSD contributions from main-chain and side-chain atoms are shown in Figure S12. We have also accounted for the water retention behavior of these AFPs in the absence of any micelle system, with the help of MD simulation surrounded by TIP3P water models. In particular, the diffusive behavior of water is more rigorous for Peptide 1m (2-fold higher) compared to that of wfAFP (Figure 5E,F). Considering the behavior of wfAFP in water, the convergence criteria is found to be achieved within 10−15 ns time period and is indicative of relative stability for the conformation (Figure 5E). In contrast, the dynamicity of Peptide 1m is found to be more robust, which fails to attain any convergence in 30 ns (Figure 5H). Thus, wfAFP can be considered more stable in water, as compared to its behavior in DPC and SDS systems. On the other hand, the conclusion is contrary for Peptide 1m, where convergence to diffusive behavior is found to be achieved in the

presence of DPC and SDS systems (15 ns onward), whereas the same convergence is found to be absent in water. In the third approach for accounting MSD of AFPs in the presence of micelle systems, the analysis was performed based on the Eigen vector projection points from the principal components (PC-analysis) that govern the molecular motion (Figure S13). Note that principal components 1 and 2 (PC1 and PC2) account for more than 70% of the statistical behavior that governs the molecular motion.42 With this notation, we have analyzed the MSD using PC1 and PC2, which in turn serves as a tool to validate the previously described inference. Briefly, the PC1 component attains similar plot profile for wfAFP in DPC and SDS micelles (Figure S13A,B), indicating that molecular motion experiences strong Brownian movement. Similar behavior is also accounted in case of Peptide 1m with reference to the MSD analysis based on PC1 (Figure S13C,D). In contrast, the MSD variation along the PC2 is more valuable, which indicates convergence and water retention time. The convergence criterion for wfAFP is achieved in SDS micelle after 10 ns, whereas Peptide 1m achieves the same in SDS micelle after 20 ns. Similar convergence criteria were found to be lacking in the DPC micelle system for both AFPs. The MSD analysis derived from PCA is thus in good accordance with the atomistic MSD results, where internal and external diffusive property is found higher for AFPs in SDS micelle system. Residue-Specific Fluctuation Analysis of AFPs. The conformational characteristics of the AFPs in the micelle systems have been analyzed with respect to RMSD and radius of gyration, as shown in the previous section (Figure 4). Our next approach was to use RMSF analysis for obtaining valuable information on residue-specific motional fluctuations in each peptide in the presence and absence of the micelle systems. The extent of residues motional fluctuations was also investigated on the basis of principal component analysis (PCA) that are projected in the form of porcupine plots. Figure 6 represents the motional fluctuation of backbone atoms and the RMSF for each residue of the two AFPs used in this study. It is evident that these fluctuations are more conserved in the presence of SDS micelles relative to those in DPC (Figure 6B,E). Moreover, the terminal residues are found to exhibit more fluctuation in comparison to the residues present in the middle 909

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Figure 7. Tracing the secondary structural analysis of AFPs in the presence of micelle systems from MD simulation. The secondary structure of wfAFP is shown in the upper panel in the presence of DPC micelle (A) and SDS micelle (B). Similarly, the secondary structure of Peptide 1m is shown in the lower panel in the presence of DPC micelle (C) and SDS micelle (D). The color codes used in the plots are shown in the figure.

segment in each of wfAFP and Peptide 1m. Notably, the fluctuation at the N- and C-terminal residues of wfAFP show a fluctuation of ∼>0.6 and ∼ SDS micelle ≫ DPC micelle. Likewise, for Peptide 1m, the order of dynamic stability is in the order, SDS micelle > DPC micelle ≫ water. Conversely, a noticeable bending motion is observed for the central region of wfAFP (residues 13−23) in DPC, as indicated by the RMSF plot (Figure 6A). Trapping of the motional fluctuations using PCA is one of the valuable techniques that can provide atomistic information based on the major driving forces (first principal component) that govern the overall trajectory. The corresponding porcupine plots are also shown in Figure 6, where the shapes of the porcupines reflect the fluctuation ranges from the lowest point to highest fluctuation point. Overall, it is revealed that the dynamicity of the AFPs is more restricted in the presence of SDS micelles relative to that in DPC. In addition, the inference drawn from the RMSF correlates well with the MSD analysis in Figure 4. The convergence criteria, as found to be achieved with AFPs in the

presence of SDS micelles, reflect a lower degree of freedom. Likewise, the MSD analysis for the AFPs simulations in absence of micelle systems (Figure 5E and 5F) correlates well with the RMSF and porcupine plots (Figure 6C and 6F). Furthermore, the lower fluctuations of AFPs were also reflected with the water retention time, which was longer for the AFPs in the presence of SDS micelles compared to that in DPC. Insights into AFP−Micelle Interactions and Secondary Structure Analyses. Molecular dynamics studies of Peptide 1m and wfAFP in the presence of micelle systems help in analyzing the impact of the environment on the dynamic features of the corresponding peptides. Peptide 1m was found to maintain the structural rigidity with respect to the backbone atoms and overall secondary structure. On the contrary, the helical straightforwardness of wfAFP was found to be much perturbed in the presence of both micelle systems (see “porcupine plots”, Figure 6). Both AFPs were found to be located at the bulk of micelle−water interface for the entire 30 ns simulation time scale. In Peptide 1m, the positively charged residues like Arg15 and Arg22 were observed to interact with the negatively charged phosphate headgroup of SDS moiety, whereas Arg2 and Arg22 are found to interact with negatively charged phosphate head groups in DPC micelle (Figure S10). The hydrophobic residues (such as the Ala9, Ala10, Ile13, Val14, Cys16, and Al17) were found to interact with the acyl chains of SDS moiety (Figure S11). It was also observed that the spherical shape of the DPC micelles (starting structure) was found to be distorted during the simulation time course, while this shape was found to be well-conserved in the case of SDS 910

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Figure 8. Comparison of docked posed of AFPs to ice-binding region for wfAFP (A) and Peptide 1m (B); with DPC micelle for wfAFP (C) and Peptide 1m (D); with SDS micelle for wfAFP (E) and Peptide 1m (F). Key residues of AFPs involved in making contact with ice, DPC, and SDC moieties are highlighted with stick representation.

similar to that indicated by the deconvolution of the CD spectrum (Figure 1). Note that the indication of α-helix, 310helix, turn, and unordered secondary structure, as inferred from CD deconvolution, matches well with the residue-wise phi-psi calculation from MD simulations (Figure 7). However, some variations such as the presence of strandlike secondary structure, as reflected in Figure 1C,D, are found not to be indicative from MD simulations. Such differences can be attributed to limitations of computational techniques and due to errors in force-field terms that fail to match with experimental results. The effect of the environmental perturbation on the secondary structure of AFPs are welldepicted by the figure, where a discrete patch for the presence of helicity is evidenced in wfAFP and Peptide 1m due to the presence of DPC micelles (Figure 7A,C). Similarly, it is also evident that the structural stability of wfAFP and Peptide 1m is more prominent with the SDS micelle system compared to DPC micelle system (Figure 7B,D). Finally, the association of AFPs to the ice surface and those to micelles were compared using docked models (Figure 8). The adsorption of AFPs to ice surface is believed to be governed mainly by van der Waals interactions, where the hydrophobic residues alanine and threonine are found to occupy the voids between the orderly packed tetragonal geometry of ice.10 This is illustrated in Figure 8A, where wfAFP interacts with sequential alanine residues and three threonine residues (Thr2, Thr13, and Thr35). On the other hand, the de novo designed AFP Peptide 1m is found to interact with various hydrophobic residues (Figure 8B), which is also

micelles (Figures S8−S11). It was also evidenced from the MD simulation trajectory that the extent of helical conformation for Peptide 1m was more conserved in SDS micelles than in DPC. Mainly, the Peptide 1m residues that are present in the hydrophobic core of DPC micelle (viz., Ile13, Cys16, and Glu19) and the residues spanning around them preserve the helical content (Figure S10). The terminal residues were found to be perturbed over the course of simulation and adopt random coil or turn-like structures. Similarly, in the case of SDS micelles, the Peptide 1m residues that were found to be in the hydrophobic core retain the helical characteristics; these residues include Ala9, Ile13, Val14, Arg15, Cys16, Ala17, and Arg22 (Figure S11). The account of secondary structure analysis of AFPs in the presence of micelle models is shown in Figure 7, whereas structural snapshots of AFPs with micelle system from MD simulation time scale can be found in Figures S8−S11. In the case of wfAFP, a break in the helical conformation was observed in the sequential region Ala19 to Ala25 due to the presence of DPC micelle (Figures 6A, 7A, and S8). A secondary structural transition from helical conformation to turn-like structure was observed in this region. A similar observation of such secondary structure transformation was also found in the case of SDS micelle, but the extent was less compared to that in DPC micelle system (Figures 6B, 7B, and S9). In the presence of SDS, the loss of helical structure was observed in the region of Ala19 to Glu22 and in the terminal region of wfAFP. Interestingly, the perturbation of secondary structure, as inferred from the trajectory analysis of MD simulation, was 911

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SDS micelles is also responsible for perturbing the secondary structure and inducing unordered structure in the case of wfAFP. It is worth mentioning that further work on AFP interaction with other model membrane systems like bicelles is necessary, as bicelles are of particular significance because they are devoid of acute curvatures that are found with detergent micelles (conical curvature geometry).34 Based on physical insights from our present study, a bend in molecular structure of AFP attributed to the micelle curvature is evidence, which might not be the case in the presence of bicelles. On the other hand, the relevance of this study with micelles is that AFPs are observed to have dynamic stability (less atomic fluctuations) in the presence of negatively charged membrane models compared to zwitterionic membranes; this atomistic information is based on the charge of the system and not on molecular architecture, which can be found preserved in real biological conditions. Collectively, the biological relevance of this study indeed lies in that it paves the way for a thorough understanding of the mechanism(s) of AFP, membrane interactions for the purpose of exploring robust means of cryopreservation.

one of the key reasons for its lower antifreeze activity compared to wfAFP.28 In the present study, the initial binding phenomenon for AFPs was investigated with CD and NMR techniques. The PRE data was also used to evaluate the interaction of AFPs with DPC and SDS systems using AutoDock, followed by MD simulation. Interesting differences of membrane interaction in AFPs is found to be mediated with polar as well as hydrophobic residues both in wfAFP and Peptide 1m. In particular, residues like Leu12, Asn16, Ala19, Leu22, Thr24, and Asn27 of wfAFP were found to be involved in binding with DPC micelle (Figure 8C). Likewise, wfAFP interacts with SDS system through residues like Thr13, Asn16, Lys18, Thr22, and Asn27 (Figure 8E). Importantly, the bending of wfAFP, which is evident in DPC and SDS systems, is found to be absent in MD simulation in aqueous systems only. Moreover, based on the structural snapshot, it was also reflective that such bending in molecular geometry is due to micelle circumference (larger geometry of wfAFP compared to diameter of micelle systems). Considering Peptide 1m, the interaction with DPC micelle is found to be mediated by Arg2, Phe5, Ala9, Phe12, Cys6, His20, and Arg23 (Figure 8D). The extent of interacting residues in this case was further found to be varying in the course of MD simulation. Likewise, the interaction of Peptide 1m in SDS system was found to be mediated by Pro7, Ser11, Arg15, Phe18, Arg22, and Arg23 (Figure 8F). Thus, with these docked results, the approximation of binding residues from AFP can be correlated to have different contributions for its antifreeze activity and crucial biological relevance for membrane interactions.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b11164. CD spectra of wfAFP and Peptide 1m (AFPs) (Figure S1); 2D NOESY NMR spectra of AFPs in DPC and SDS micelles (Figures S2 and S3); View of AFPs docked complex to DPC and SDS micelle (Figures S4 and S5); Potential energy of systems from MD trajectories (Figure S6); Backbone RMSDs for AFPs from MD simulation (Figure S7); Structural snapshot of AFPs with micelle systems from MD simulation (Figures S8−S11); MSD plot for main-chain and side atoms of AFPs in the presence of micelle systems (Figure S12); MSD based on PCA analysis for AFPs in the presence of micelle systems (Figure S13); Cosine content and relevant characteristics of AFPs trajectory in micelle systems (Table S1; PDF).



CONCLUSIONS The present study provides strong evidence for the interactions of the two AFPs, wfAFP and Peptide 1m, with membrane model systems. Zwitterionic and anionic detergents are crucial in unraveling the membrane interaction of proteins, since they mimic the lipid environment.31,32 Using biophysical techniques such as CD and NMR spectroscopy, in conjunction with molecular dynamics, it is evident that both AFPs potentially bind with zwitterionic as well as negatively charged model membranes. Importantly, the secondary structure of type I AFPs was found to preserve its helical conformation in the presence of both membrane models, which imparts relevance for similar cellular phenomena in plants, fungi, bacteria, and fish. Though the interaction details of antifreeze protein with ice are widespread in literature, elucidation of the physiological relevance with cellular membranes is undeveloped to date. It is believed that antifreeze proteins have significant cryopreservation activity with erythrocytes,49 ram spermatozoa,50 bovine sperm,51 pig oocytes,52 and whole rat livers.53 Our study provides evidence that wfAFP and Peptide 1m also undergo significant interactions with DPC and SDS systems, with the adoption of α-helical secondary structure. More importantly, the localization of AFPs was found to be at the interface of micelle and solvent systems, where an electrostatic interaction of residues Arg15 and Arg22 of Peptide 1m exists with the head groups of phospholipids. Interestingly, the three-dimensional structures of both wfAFP and Peptide 1m were found to be well conserved, with SDS micelles relative to those with the DPC micelles. Hydrophobic interaction (particularly involving Ala9, Ala10, Ile13, Val14, and Ala17) was found to exist for Peptide 1m, whereas the residues Ala19, Ala20, Ala21, Thr24, and Ala25 were found to exhibit a similar hydrophobic interaction with the micelles in wfAFP. This interaction with DPC and



AUTHOR INFORMATION

Corresponding Author

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

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The research was supported by Council of Scientific and Industrial Research (CSIR; Grant No. 02(0005)/11/EMR-II), Government of India. R.K.K. thanks CSIR, Government of India, for senior research fellowship (SRF; Grant No. 09/ 015(0467)/2014-EMR-I).



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DOI: 10.1021/acs.jpcb.5b11164 J. Phys. Chem. B 2016, 120, 902−914