Formation of ice-like water structure on the surface of an antifreeze protein

Antifreeze proteins (AFPs) are found in different species from polar, alpine, and subarctic regions where they serve to inhibit ice crystal growth by adsorption to ice surfaces. Computational methods have the power to investigate the antifreeze mechanism in atomic detail. Molecular dynamics simulations of water under different conditions have been carried out to test our water model for simulations of biological macromolecules in extreme conditions: very low temperatures (200 K) and at the ice/liquid water interface. We show that the flexible F3C water model reproduces properties of water in the solid phase (ice I(h)), the supercooled liquid phase, and at the ice/liquid water interface. Additionally, the hydration of the type III AFP from ocean pout was studied as a function of temperature. Hydration waters on the ice-binding surface of the AFP were less distorted and more tetrahedral than elsewhere on the surface. More ice-like hydrating water structures formed on the ice-binding surface of the protein such that it created an ice-like structure in water within its first hydration layer but not beyond, suggesting that this portion of the protein has high affinity for ice surfaces.     

Ice-surface adsorption enhanced colligative effect of antifreeze proteins in ice growth inhibition

This Communication describes a mechanism to explain antifreeze protein's function to inhibit the growth of ice crystals. We propose that the adsorption of antifreeze protein (AFP) molecules on an ice surface induces a dense AFP-water layer, which can significantly decrease the mole fraction of the interfacial water and, thus, lower the temperature for a seed ice crystal to grow in a super-cooled AFP solution. This mechanism can also explain the nearly unchanged melting point for the ice crystal due to the AFP's ice-surface adsorption. A mathematical model combining the Langmuir theory of adsorption and the colligative effect of thermodynamics has been proposed to find the equilibrium constants of the ice-surface adsorptions, and the interfacial concentrations of AFPs through fitting the theoretical curves to the experimental thermal hysteresis data. This model has been demonstrated by using the experimental data of serial size-mutated beetle Tenebrio molitor (Tm) AFPs. It was found that the AFP's ice-surface adsorptions could increase the interfacial AFP's concentrations by 3 to 4 orders compared with those in the bulk AFP solutions.     

Reversible binding of the HPLC6 isoform of type I antifreeze proteins to ice surfaces and the antifreeze mechanism studied by multiple quantum filtering-spin exchange NMR experiment

Antifreeze proteins (AFPs) protect organisms from freezing damage by inhibiting the growth of seed-ice crystals. It has long been hypothesized that irreversible binding of AFPs to ice surfaces is responsible for inhibiting the growth of seed-ice crystals as such a mechanism supports the popularly accepted Kelvin effect for the explanation of local freezing-point depression. However, whether the binding is reversible or irreversible is still under debate due to the lack of direct experimental evidence. Here, we report the first direct experimental result, by using the newly developed multiple quantum (MQ) filtering-spin exchange NMR experiment, that shows that the binding of HPLC6 peptides to ice surfaces is reversible. It was found that the reversible process can be explained by the model of monolayer adsorption. These results suggest that the Kelvin effect is not suitable for explaining the antifreeze mechanism, and direct interactions between the peptides and the ice-surface binding sites are the driving forces for the binding of AFPs to ice surfaces. We propose that there exists a concentration gradient of AFP from an ice-binding surface to the solution due to the affinity of ice surfaces to AFPs. This concentration gradient creates a dense layer of AFP in contact with the ice-binding surface, which depresses the local freezing point because of the colligative property, but not the Kelvin effect.     

Molecular basis for antifreeze activity difference of two insect antifreeze protein isoforms

The insect spruce budworm(Choristoneura fumiferana) produces antifreeze protein(AFP) to assist in the protection of the over-wintering larval stage and contains multiple isoforms. Structures for two isoforms,known as CfAFP-501 and CfAFP-337,show that both possess similar left-handed β-helical structure,although thermal hysteresis activity of the longer isoform CfAFP-501 is three times that of CfAFP-337. The markedly enhanced activity of CfAFP-501 is not proportional to,and cannot be simply accounted for,by the increased ice-binding site resulting from the two extra coils in CfAFP-501. In or-der to investigate the molecular basis for the activity difference and gain better understanding of AFPs in general,we have employed several different computational methods to systematically study the structural properties and ice interactions of the AFPs and their deletion models. In the context of intact AFPs,a majority of the coils in CfAFP-501 has better ice interaction and causes stronger ice lattice disruption than CfAFP-337,strongly suggesting a cooperative or synergistic effect among β-helical coils. The synergistic effect would play a critical role and make significant contributions to the anti-freeze activity β-helical antifreeze proteins. This is the first time that synergistic effect and its implica-tion for antifreeze activity are reported for β-helical antifreeze proteins.     

Mirror image forms of snow flea antifreeze protein prepared by total chemical synthesis have identical antifreeze activities

The recently discovered glycine-rich snow flea antifreeze protein (sfAFP) has no sequence homology with any known proteins. No experimental structure has been reported for this interesting protein molecule. Here we report the total chemical synthesis of the mirror image forms of sfAFP (i.e., L-sfAFP, the native protein, and D-sfAFP, the native protein's enantiomer). The predicted 81 amino acid residue polypeptide chain of sfAFP contains Cys residues at positions 1, 13, 28, and 43 and was prepared from four synthetic peptide segments by sequential native chemical ligation. After purification, the full-length synthetic polypeptide was folded at 4 degrees C to form the sfAFP protein containing two disulfides. Chemically synthesized sfAFP had the expected antifreeze activity in an ice recrystallization inhibition assay. Mirror image D-sfAFP protein was prepared by the same synthetic strategy, using peptide segments made from d-amino acids, and had an identical but opposite-sign CD spectrum. As expected, D-sfAFP displays the same antifreeze properties as L-sfAFP, because ice presents an achiral surface for sfAFP binding. Facile synthetic access to sfAFP will enable determination of its molecular structure and systematic elucidation of the molecular basis of the antifreeze properties of this unique protein.     

Growth inhibition mechanism of an ice-water interface by a mutant of winter flounder antifreeze protein: a molecular dynamics study

Molecular dynamics (MD) simulations of a growing ice-water interface of a pyramidal {2021} plane in the presence of a mutant of winter flounder antifreeze protein (AFP) were conducted. Simulation results indicated that the AFP was partially surrounded by ice grown at the pyramidal interface. The AFP stably bound to the interface only when AFP hydrophobic residues bound to ice. Simulation results also indicated a drastic decrease in the growth velocity of the ice surrounding the stably bound AFP, in agreement with ice growth inhibition processes that have been observed in real systems. We confirmed that the decrease in the growth velocity of ice was attributable to the melting point depression caused by the Gibbs-Thomson effect. Simulation results suggested that the growth of ice surrounding the AFP is needed to promote stable AFP binding to the interface and subsequent ice growth inhibition. MD simulations of a growing ice-water interface of a prismatic {10_10} plane were also conducted. Neither the stable binding of the AFP to the interface nor the decrease in the growth velocity occurred for the prismatic plane. These results agree with the fact that AFPs inhibit the growth of ice only on the pyramidal planes in real systems.     

Electrochemical, microscopic, and spectroscopic characterization of prevesicle nanostructures and vesicles on mixed cationic surfactant systems

Several experimental techniques (conductivity, zeta potential, transmission electronic microscopy, and steady-state fluorescence spectroscopy) have been used to study the formation of mixed colloidal aggregates consisting of a cationic double-chain surfactant, di-dodecyldimethylammonium bromide (di-C12DMAB), and a single-chain alkyltrimethylammonium bromide with 10 and/or 14 carbon atoms (decyltrimethylammonium bromide, C10TAB, and/or tetradecyltrimethylammonium bromide, C14TAB). Special interest has been devoted to the prevesicle domain, within which the formation of aggregated nanostructures was first reported in our laboratory. For that purpose, studies have been carried out on the very dilute region by means of conductivity experiments, confirming the existence of two critical aggregation concentrations in that concentration domain: the so-called mixed critical aggregate concentration, CAC, and the mixed critical vesicle concentration, CVC. By carrying out TEM experiments on negatively stained samples, we were surprised to find a number of aggregates without a clear aggregation pattern and with a variety of sizes and shapes at concentrations below CAC, where only monomers were expected. However, the nanoaggregates found at concentrations between CAC and CVC, also by TEM microscopy, show a clear and ordered "fingerprint"-like aggregation pattern similar to the liquid-crystalline phases reported for DNA-liposome complexes and/or DNA packed with viral capsids. Finally, at total surfactant concentrations above CVC, the aggregates were confirmed, by means of cryo-TEM micrographs and zeta potential measurements, to be essentially unilamellar spherical vesicles with a medium polydispersity and a net-averaged surface density charge of around 12 x 10(-3) C m(-2). The fluorescence emission of two probes, TNS (anionic) and PRODAN (nonionic), allows for the analysis of the micropolarity and microviscosity of the different microenvironments present in aqueous surfactant solutions where the above-mentioned vesicle and prevesicle aggregates are present.     

Stoichiometry and physical chemistry of promiscuous aggregate-based inhibitors

Many false positives in early drug discovery owe to nonspecific inhibition by colloid-like aggregates of organic molecules. Despite their prevalence, little is known about aggregate concentration, structure, or dynamic equilibrium; the binding mechanism, stoichiometry with, and affinity for enzymes remain uncertain. To investigate the elementary question of concentration, we counted aggregate particles using flow cytometry. For seven aggregate-forming molecules, aggregates were not observed until the concentration of monomer crossed a threshold, indicating a "critical aggregation concentration" (CAC). Above the CAC, aggregate count increased linearly with added organic material, while the particles dispersed when diluted below the CAC. The concentration of monomeric organic molecule is constant above the CAC, as is the size of the aggregate particles. For two compounds that form large aggregates, nicardipine and miconazole, we measured particle numbers directly by flow cytometry, determining that the aggregate concentration just above the CAC ranged from 5 to 30 fM. By correlating inhibition of an enzyme with aggregate count for these two drugs, we determined that the stoichiometry of binding is about 10,000 enzyme molecules per aggregate particle. Using measured volumes for nicardipine and miconazole aggregate particles (2.1 x 10(11) and 4.7 x 10(10) A(3), respectively), computed monomer volumes, and the observation that past the CAC all additional monomer forms aggregate particles, we find that aggregates are densely packed particles. Finally, given their size and enzyme stoichiometry, all sequestered enzyme can be comfortably accommodated on the surface of the aggregate.     

A two-dimensional adsorption kinetic model for thermal hysteresis activity in antifreeze proteins

Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs), collectively abbreviated as AF(G)Ps, are synthesized by various organisms to enable their cells to survive in subzero environments. Although the AF(G)Ps are markedly diverse in structure, they all function by adsorbing to the surface of embryonic ice crystals to inhibit their growth. This adsorption results in a freezing temperature depression without an appreciable change in the melting temperature. The difference between the melting and freezing temperatures, termed thermal hysteresis (TH), is used to detect and quantify the antifreeze activity. Insights from crystallographic structures of a number of AFPs have led to a good understanding of the ice-protein interaction features. Computational studies have focused either on verifying a specific model of AFP-ice interaction or on understanding the protein-induced changes in the ice crystal morphology. In order to explain the origin of TH, we propose a novel two-dimensional adsorption kinetic model between AFPs and ice crystal surfaces. The validity of the model has been demonstrated by reproducing the TH curve on two different beta-helical AFPs upon increasing the protein concentration. In particular, this model is able to accommodate the change in the TH behavior observed experimentally when the size of the AFPs is increased systematically. Our results suggest that in addition to the specificity of the AFPs for the ice, the coverage of the AFPs on the ice surface is an equally necessary condition for their TH activity.     

Dual function of the hydration layer around an antifreeze protein revealed by atomistic molecular dynamics simulations

Atomistic molecular dynamics simulations are used to investigate the mechanism by which the antifreeze protein from the spruce budworm, Choristoneura fumiferana, binds to ice. Comparison of structural and dynamic properties of the water around the three faces of the triangular prism-shaped protein in aqueous solution reveals that at low temperature the water structure is ordered and the dynamics slowed down around the ice-binding face of the protein, with a disordering effect observed around the other two faces. These results suggest a dual role for the solvation water around the protein. The preconfigured solvation shell around the ice-binding face is involved in the initial recognition and binding of the antifreeze protein to ice by lowering the barrier for binding and consolidation of the protein:ice interaction surface. Thus, the antifreeze protein can bind to the molecularly rough ice surface by becoming actively involved in the formation of its own binding site. Also, the disruption of water structure around the rest of the protein helps prevent the adsorbed protein becoming covered by further ice growth.     

A new model for simulating 3-d crystal growth and its application to the study of antifreeze proteins

A novel computational technique for modeling crystal formation has been developed that combines three-dimensional (3-D) molecular representation and detailed energetics calculations of molecular mechanics techniques with the less-sophisticated probabilistic approach used by statistical techniques to study systems containing millions of molecules undergoing billions of interactions. Because our model incorporates both the structure of and the interaction energies between participating molecules, it enables the 3-D shape and surface properties of these molecules to directly affect crystal formation. This increase in model complexity has been achieved while simultaneously increasing the number of molecules in simulations by several orders of magnitude over previous statistical models. We have applied this technique to study the inhibitory effects of antifreeze proteins (AFPs) on ice-crystal formation. Modeling involving both fish and insect AFPs has produced results consistent with experimental observations, including the replication of ice-etching patterns, ice-growth inhibition, and specific AFP-induced ice morphologies. Our work suggests that the degree of AFP activity results more from AFP ice-binding orientation than from AFP ice-binding strength. This technique could readily be adapted to study other crystal and crystal inhibitor systems, or to study other noncrystal systems that exhibit regularity in the structuring of their component molecules, such as those associated with the new nanotechnologies.     

Molecular interactions of cationic and anionic surfactants in mixed monolayers and aggregates

The properties of anionic-rich and cationic-rich mixtures of CTAB (cetyltrimethylammonium bromide) and SDS (sodium dodecyl sulfate) were investigated with conductometry and surface tension measurements and by determining the surfactant NMR self-diffusion coefficients. The critical aggregate concentration (CAC), surface tension reduction effectiveness(gamma(CAC)), surface excess(Gamma(max)), and mean molecular surface area (A(min)) were determined from plots of the surface tension (gamma) as a function of the total surfactant concentration. The compositions of the adsorbed films (Z) and aggregates (chi) were estimated by using regular solution theory, and then the interaction parameters in the aggregates (beta) and the adsorbed film phases (beta(sigma)) were calculated. The results showed that the synergism between the surfactants enhances the formation of mixed aggregates and reduces the surface tension. Further, the nature and strength of the interaction between the surfactants in the mixtures were obtained by calculating the values of the following parameters: the interaction parameter, beta, the size parameter, rho, and the nonrandom mixing parameter, P*. These results indicate that in ionic surfactant mixtures the optimized packing parameter has the highest value and that the size parameter can be used to account for deviations from the predictions of regular solution theory. It was concluded that, for planar air/aqueous interfaces and aggregation systems, this nonideality increases as the temperature increases. This trend is attributed to the increased dehydration of the surfactant head groups that results from increases in temperature. Further, our conductometry measurements show that the counterion binding number of mixed micelles formed in mixtures with a high CTAB content is different to those with a high SDS content. This difference is due to either their different aggregation sizes or the different interactions between the head groups and the counterions.     

Applications of type I antifreeze proteins: studies with model membranes & cryoprotectant properties

Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs), found in the body fluids of many species of polar fish allow them to survive in waters colder than the equilibrium freezing point of their blood and other internal fluids. Despite their structural diversity, all AF(G)Ps kinetically depress the temperature at which ice grows in a non-colligative manner and hence exhibit thermal hysteresis. AF(G)Ps also share the ability to interact with and protect mammalian cells and tissues from hypothermic damage (e.g., improved storage of human blood platelets at low temperatures), and are able to stabilize or disrupt membrane composition during low temperature and freezing stress (e.g., cryoprotectant properties in stabilization of sperm and oocytes). This review will summarize studies of AFPs with phospholipids and plant lipids, proposed mechanisms for inhibition of leakage from membranes, and cryoprotectant studies with biological samples. The major focus will be on the alpha-helical type I antifreeze proteins, and synthetic mutants, that have been most widely studied. For completeness, data on glycoproteins will also be presented. While a number of models to explain stabilization and destabilization of different lipid systems have been proposed, it is currently not possible to predict whether a particular AFP will stabilize or destabilize a given lipid system. Furthermore the relationship between the antifreeze property of thermal hysteresis and membrane stabilization is unknown. This lack of detailed knowledge about how AFPs function in the presence of different types of materials has hampered progress toward the development of antifreezes for cold storage of cells, tissues, and organs.     

Mimicking the properties of antifreeze glycoproteins: synthesis and characterization of a model system for ice nucleation and antifreeze studies

Synthesis of beta-D-Gal-(1 --> 3)-beta-D-GalNAc coupled to HOC2H4NHCOC15H30SH is described. This compound was coadsorbed at various proportions with C2H5OC2H4NHCOC15H30SH to form statistically mixed self-assembled monolayers (SAMs) on gold in an attempt to mimic the properties of the active domain in antifreeze glycoproteins (AFGPs). The monolayers were characterized by null ellipsometry, contact angle goniometry, X-ray photoelectron spectroscopy, and infrared reflection-absorption spectroscopy. The disaccharide compound adsorbed preferentially, and SAMs prepared at a solution molar ratio >0.3 displayed total wetting. The mixed SAMs showed well-organized alkyl chains up to a disaccharide surface fraction of 0.8. The amount of gauche conformers in the alkyls increased rapidly above this point, and the monolayers became disordered and less densely packed. Furthermore, the generated mixed SAMs were subjected to water vapor at constant relative humidity and the subsequent ice crystallization on a cooled substrate was monitored via an optical microscope. Interestingly, rapid crystallization occurred within a narrow range of temperatures on mixed SAMs with a high disaccharide content, surface fraction >0.3. The reported crystallization temperatures and the ice layer topography were compared with results obtained for a much simpler reference system composed of -OH/-CH3 terminated n-alkanethiols in order to account for changes in topography of the water/ice layer with surface energy. Although preliminary, the obtained results can be useful in the search for the molecular mechanism behind the antifreeze activity of AFGPs.     

萘和丹酰基修饰的扇形树枝状分子聚集体的能量转移行为

分别对1-3代聚(酰胺-胺)(PAMAM)结构的dendron分子的外端基和focal point进行了修饰,得到了外端基为萘(给体)色团、焦点(focal point)为丹酰(受体)色团的树枝状化合物Dan-ABπ-Nap(n=2,4,8).利用荧光光谱测定了不同浓度下所得一系列树枝状分子在水中的荧光强度,并计算了它...     

Molecular dynamics study of the interactions of ice inhibitors on the ice {001} surface

The interactions of antifreeze protein (AFP) type I, antifreeze glycoproteins, polyvinyl pyrrolidone (PVP), and various amino acids with ice are investigated using Cerius2, a molecular modelling tool. Binding energies of these additives to a major ice crystal face {001} are computed. Binding energy comparison of threonine molecules (by themselves) and as threonine residues within AFP type I demonstrate their role in improving AFP's binding ability to the ice crystal face. The shifts in onset points of ice crystallization with AFP type I, PVP, and amino acids are measured using differential scanning calorimetry. These values when correlated with their respective binding energies reveal a direct proportionality and demonstrate AFP's effectiveness in inhibiting growth and nucleation of ice, over amino acids.     

Growth inhibition at the ice prismatic plane induced by a spruce budworm antifreeze protein: a molecular dynamics simulation study

A molecular dynamics simulation was conducted to investigate the growth kinetics at the ice prismatic interface to which a spruce budworm antifreeze protein was bound. Two initial binding conformations of the protein at the interface--one energetically stable and the other energetically unstable--were examined. For both binding conformations, the growth of ice was observed around the protein. A sharp decrease in the rate of ice growth was observed around the protein that initially had the energetically stable binding conformation. Simulation results suggest that the observed decrease in the ice growth rate was attributable to melting point depression caused by the Gibbs-Thomson effect. The protein that initially had the energetically unstable binding conformation markedly relaxed so as to stably bind to the prismatic plane interface of the grown ice; thereafter, a decrease in the ice growth rate was observed as well. However, the binding conformation that the protein approached during the relaxation was different from that of the protein that initially had the energetically stable binding conformation. Thus, the simulation indicates the existence of two binding conformations for inducing a decrease in the ice growth rate. The results are possibly related to the hyperactivity of a spruce budworm antifreeze protein in real systems.     

Aggregation of polymer-grafted nanoparticles in good solvents: a hierarchical modeling method

Brownian dynamics simulations are carried out to study the aggregation behavior of polymer-grafted nanoparticles (NPs) in good solvents by using the coarse-grained model derived from the all-atom force field, according to the hierarchical modeling strategy, and here PEG-grafted gold nanoparticles (GNPs) were taken as an example. Generally, grafting PEG to the surface of GNPs is to protect them from aggregation in the solution. However, our results reveal that PEG-grafted GNPs may also aggregate when concentration increases. Our simulations indicate that there exists a critical aggregating concentration (CAC), beyond which the PEG-grafted GNPs will aggregate. We further check the effects of grafting density and the length of grafted chains on the aggregation behavior of the grafted GNPs, and find that there exists an optimized length of grafted chain, at which the system has the maximal CAC. Furthermore, the aggregate size of self-assembled mesostructures formed by the grafted GNPs increases with the concentration. Interestingly, it is observed that the aggregation favors to form linear gold nanowires rather than compact gold nanoclusters, and the corresponding mechanism is also addressed. It is expected that this work would provide useful information for the fabrication of metal nanowires and the surface modification of metal nanoparticles.     

Influence of structural features on the self‐assembly of short ionic oligopeptides

We investigated the self‐aggregation of 12 short ionic oligopeptides constituted by 4–7 amino acid residues to establish useful structure–property relationships that might be exploited in the biomedical field by using the concept of molecular Lego. We show that the critical aggregation concentration (CAC) of tetrapeptides decreases with increasing hydrophobicity of neutral residues. Additionally, the dependence of the CAC of isomeric oligopeptides on the distribution of amino acid residues confirms the high tendency to self‐organization of molecules with alternating ionic and neutral residues. Indeed, atomic force microscopy (AFM) images recorded on oligopeptide solutions above the CAC show the presence of either fibrillar or spherical aggregates depending on oligopeptide structure and concentration, steric hindrance, solution pH, and time. The potential of the investigated oligopeptides in tissue engineering applications is supported by their in vitro cytocompatibility. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 889–897, 2010