New simulation model of multicomponent crystal growth and inhibition

We review a novel computational model for the study of crystal structures both on their own and in conjunction with inhibitor molecules. The model advances existing Monte Carlo (MC) simulation techniques by extending them from modeling 3D crystal surface patches to modeling entire 3D crystals, and by including the use of "complex" multicomponent molecules within the simulations. These advances makes it possible to incorporate the 3D shape and non-uniform surface properties of inhibitors into simulations, and to study what effect these inhibitor properties have on the growth of whole crystals containing up to tens of millions of molecules. The application of this extended MC model to the study of antifreeze proteins (AFPs) and their effects on ice formation is reported, including the success of the technique in achieving AFP-induced ice-growth inhibition with concurrent changes to ice morphology that mimic experimental results. Simulations of ice-growth inhibition suggest that the degree of inhibition afforded by an AFP is a function of its ice-binding position relative to the underlying anisotropic growth pattern of ice. This extended MC technique is applicable to other crystal and crystal-inhibitor systems, including more complex crystal systems such as clathrates.     

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.     

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.     

Source of the ice-binding specificity of antifreeze protein type I

Antifreeze proteins (AFPs) are a group of structurally very diverse proteins with the unique capability of binding to the surface of seed ice crystals and inhibiting ice crystal growth. The AFPs bind with high affinity to specific planes of the ice crystal. Previously, this affinity of AFPs has been ascribed to the formation of multiple hydrogen bonds across the protein-ice interface, but more recently van der Waals interactions have been suggested to be the dominant energetic factors for the adsorption. To determine whether van der Waals interactions are also responsible for the binding specificities of AFPs, the protein-ice interaction of the helical AFP Type I from winter flounder (HPLC6) was studied using a Monte Carlo rigid body docking approach. HPLC6 binds in the [1102] direction of the [2021] plane, with the Thr-Ala-Asn surface comprising the protein's binding face. The binding of HPLC6 to this ice plane is highly preferred, but the protein is also found to bind favorably to the [1010] prism plane using a different protein surface comprised of Thr and Ala residues. The results show that van der Waals interactions, despite accounting for most of the intermolecular energy (>80%), are not sufficient to completely explain the AFP binding specificity.     

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.     

Structures of hydrocarbon hydrates during formation with and without inhibitors

The formation of hydrates from a methane-ethane-propane mixture is more complex than with single gases. Using nuclear magnetic resonance (NMR) and high-pressure powder X-ray diffraction (PXRD), we have investigated the structural properties of natural gas hydrates crystallized in the presence of kinetic hydrate inhibitors (KHIs), two commercial inhibitors and two biological ice inhibitors, or antifreeze proteins (AFPs). NMR analyses indicated that hydrate cage occupancy was at near saturation for controls and most inhibitor types. Some exceptions were found in systems containing a new commercial KHI (HIW85281) and a recombinant plant AFP, suggesting that these two inhibitors could impact the kinetics of cavity formation. NMR analysis confirmed that the hydrate composition varies during crystal growth by kinetic effects. Strikingly, the coexistence of both structures I (sI) and II (sII) were observed in NMR spectra and PXRD profiles. It is suggested that sI phases may form more readily from liquid water. Real time PXRD monitoring showed that sI hydrates were less stable than sII crystals, and there was a conversion to the stable phase over time. Both commercial KHIs and AFPs had an impact on hydrate metastability, but transient sI PXRD intensity profiles indicated significantly different modes of interaction with the various inhibitors and the natural gas hydrate system.     

Comparative Analysis of Hydration Layer Reorientation Dynamics of Antifreeze Protein and Protein Cytochrome P450

Antifreeze proteins (AFPs) inhibit ice recrystallization by a mechanism remaining largely elusive. Dynamics of AFPs' hydration water and its involvement in the antifreeze activity have not been identified conclusively. We herein, by simulation and theory, examined the water reorientation dynamics in the first hydration layer of an AFP from the spruce budworm, Choristoneura fumiferana, compared with a protein cytochrome P450 (CYP). The increase of potential acceptor water molecules around donor water molecules leads to the acceleration of hydrogen bond exchange between water molecules. Therefore, the jump reorientation of water molecules around the AFP active region is accelerated. Due to the mutual coupling and excitation of hydrogen bond exchange, with the acceleration of hydrogen bond exchange, the rearrangement of the hydrogen bond network and the frame reorientation of water are accelerated. Therefore, the water reorientation dynamics of AFP is faster than that of CYP. The results of this study provide a new physical image of antifreeze protein and a new understanding of the antifreeze mechanism of antifreeze proteins.     

Effect of antifreeze proteins on the nucleation, growth, and the memory effect during tetrahydrofuran clathrate hydrate formation

The inhibition activities of two antifreeze proteins (AFPs) on the formation of tetrahydrofuran (THF) clathrate hydrate have been tested. AFPs from fish (wfAFP) and insect (CfAFP) changed the morphology of growing THF hydrate crystals. Also, both AFPs showed higher activities in inhibiting the formation THF hydrate than a commercial kinetic inhibitor, poly(vinylpyrrolidone) (PVP). Strikingly, both AFPs also showed the ability to eliminate the "memory effect" in which the crystallization of hydrate occurs more quickly after the initial formation. This is the first report of molecules that can inhibit the memory effect. Since the homogeneous nucleation temperature for THF hydrate was measured to be 237 K, close to that observed for ice itself, the action of kinetic inhibitors must involve heterogeneous nucleation. On the basis of our results, we postulate a mechanism for heterogeneous nucleation, the memory effect and its elimination by antifreeze proteins.     

Database and search techniques for two-dimensional gel protein data: a comparison of paradigms for exploratory data analysis and prospects for biological modeling

Two-dimensional (2-D) polyacrylamide gel electrophoresis can detect thousands of polypeptides, separating them by apparent molecular weight (Mr) and isoelectric point (pI). Thus it provides a more realistic and global view of cellular genetic expression than any other technique. This technique has been useful for finding sets of key proteins of biological significance. However, a typical experiment with more than a few gels often results in an unwiedly data management problem. In this paper, the GELLAB-II system is discussed with respect to how data reduction and exploratory data analysis can be aided by computer data management and statistical search techniques. By encoding the gel patterns in a "three-dimensional" (3-D) database, an exploratory data analysis can be carried out in an environment that might be called a "spread sheet for 2-D gel protein data". From such databases, complex parametric network models of protein expression during events such as differentiation might be constructed. For this, 2-D gel databases must be able to include data from other domains external to the gel itself. Because of the increasing complexity of such databases, new tools are required to help manage this complexity. Two such tools, object-oriented databases and expert-system rule-based analysis, are discussed in this context. Comparisons are made between GELLAB and other 2-D gel database analysis systems to illustrate some of the analysis paradigms common to these systems and where this technology may be heading.     

A molecular orbital explanation of bond distance variation caused by hydrogen bond formation

For hydrogen bond systems X–D–HA–Y, a simple molecular orbital model is proposed to understand the mechanism of the bond distance variations caused by the hydrogen bond formation. This model explains the bond distance variations for X–D and A–Y as follows. Electrostatic potential that the electrons in a molecule receive from other molecules causes the changes in atomic orbital energy differences between the bonded atoms. Then, the changes in the orbital energy differences make the bond orders larger or smaller and consequently the bond distances vary. The validity of this model has been confirmed by the effective fragment potential method, using the test systems of (HCOOH)₂, HCONH₂ (formamide) crystal and BF₃·2H₂O crystal.     

自由能计算揭示苏氨酸对抗冻蛋白与冰晶结合能力的影响

采用分子动力学模拟和自由能计算研究了中等活性黑麦草抗冻蛋白(Lolium perenne antifreeze protein, LpAFP)冰结合位点(Ice-binding site, IBS)上苏氨酸(Thr)含量对其吸附冰晶能力的影响. 构建了一系列LpAFP突变体结构, 使其IBS上苏氨酸含量逐步增加, 其中包括一个对IBS上11个位点的突变, 使每个β片段均具有Thr-x-Thr基序(x是非保守的氨基酸, 主要是疏水氨基酸). 利用重要性采样算法(WTM-eABF)计算了LpAFP及其突变体与冰晶结合过程的自由能变化, 该算法结合了Well-tempering metadynamics的“填谷”和扩展拉格朗日自适应偏置力方法的“削峰”的优点, 显著提高了算法的采样效率. 结果表明, LpAFP突变体的IBS苏氨酸含量越高, 其与冰的结合在能量上越有利. 当突变体具有重复Thr-x-Thr基序时, 其与冰的结合能力最强. 进一步分析表明, 苏氨酸含量越高, IBS结合的液态水分子越多, 与冰晶结合时锚定包合水稳定存在的时间就越长, 抗冻蛋白的IBS与冰面之间的氢键网络也越稳定, 从而提高了抗冻蛋白与冰的结合能力. 增加苏氨酸残基的含量是提高中等活性抗冻蛋白抗冻活性的方法.     

Study of triacylglycerol polymorphs by nuclear magnetic resonance: effects of temperature and chain length on relaxation parameters

It is very important to monitor the characteristics of triacylglycerol crystal network in fats, as these crystals have an impact on many food properties such as texture, sensory taste, and extended shelf life. Although time-domain NMR (TD-NMR) is now the reference technique to determine the solid fat index in food, the entire possibilities of this technique are not used. Some NMR studies have been performed to determine its power for the discrimination of polymorphism. In this study, extended investigations proved that TD-NMR could evaluate triacylglycerol (TA) polymorphism, independently from temperature and chain length. Study of the dipolar interactions through second moment M(2), which is characteristic of proton mobility in solid-state samples, provided a new understanding of the structural organization of crystal molecules. Proton spin-lattice relaxation, which has been proved to be a true probe of polymorphism, has provided information on crystal networks. Combination of the two techniques revealed two very interesting kinds of results, i.e. the presence of a minimum spin-lattice relaxation time T(1) for tristearin alpha, which is a characteristic of a dynamic molecular process, and differences in behavior between long and short chain lengths, both at a molecular and a crystal level.     

Construction of interesting organic supramolecular structures with synthons cooperation in the cocrystals of 1H-benzotriazole and hydroxybenzoic acids

Multifunctional molecules are capable of assembling via different supramolecular synthons,or hydrogen bond motifs,between the same or different functional groups,leading to the possibility of cocrystal.Utilization of the interplay of dimensionality(1-D,2-D and 3-D),orientation of functional groups of the building blocks,influence of rigid/flexible linking groups,and weak interactions provides an interesting route for the creation of novel supramolecular architectures in the crystal lattice.N-unsubstituted 1H-benzotriazole and carboxylic acid,being self-complementary molecules,offer a broad scope of study of binary compounds based on the complementary combination of H-bonding/donating sites.We report here the construction of three extended molecular networks in cocrystals of the carboxylic acid group of the acid and the 1H-benzotriazole triazole moiety.We have been able to identify four major supramolecualr synthons that would be helpful in the prediction of structural motifs for these kinds of studies.Interestingly,these heterosynthons are strikingly similar,to those of the homosynthons of the individual functional groups.The nature of the aza groups helps to enhance the overall volume of the crystal lattice thus leading to the formation of various supramolecular assemblies.Thermal stability of these compounds has been investigated by thermogravimetric analysis(TGA) of mass loss.     

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.     

Studying molecular interactions via capillary electrophoresis and microscale thermophoresis: A review

The process of choosing the most proper technique for studying the molecular interactions is based on critical factors such as instrumentation complexity, automation, experimental procedures, analysis time, consumables, and cost-value. This review has tracked the use of affinity capillary electrophoresis (ACE) and microscale thermophoresis (MST) techniques in the evaluation of molecular binding among different molecules during the 5 years 2016–2021. ACE has proved to be an attractive technique for biomolecular characterization with high resolution efficiency where small variations in several controlling factors can much improve such efficiency compared to other analytical techniques. Meanwhile, MST has proved its higher sensitivity for smaller amounts of complex non-purified biosamples without affecting its robustness while providing high through output. However, the main motivation to review both techniques in the proposed review was their capability to carry out all experiments without the need for immobilizing one interacting partner, besides a great flexibility in the use of buffering systems. The proposed review demonstrates the importance of both techniques in different areas of life sciences. Moreover, the recent advances in exploiting ACE and MST in other research interests have been discussed.     

Ab-initio calculations on large molecules and solids by desirable computational procedures

Desirable computational procedures developed here recently for ab-initio calculations on large molecules are outlined. Effective core model potentials (MODPOT) permit calculations of valence electrons only explicitly, yet accurately; a charge-conserving integral prescreening evaluation to decide whether a block of integrals will be larger than a preset threshold and thus be calculated explicitly is effective for spatially extended systems; an efficient MERGE technique to save and reuse common invariant skeletal integrals is useful for geometry variations and for adding basis fcuntions, substituent groups and molecules; and an effective configuration interaction (CI) Hamiltonian into which are folded the effects of the occupied molecular orbitals from which no excitations are allowed is useful for molecular decompositions and intermolecular reactions. These techniques have been extended for CI calculations on breaking a chemical bond in a molecule in a crystal or solid; atom-class/atomic-class potential functions and dispersion calculations have been added. In a new program, POLY-CRYST, all the integral strategies for large molecules are meshed.     

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.