昆虫抗冻蛋白的研究

抗冻蛋白是具有热滞活性,能结合并抑制冰晶生长和抑制冰的重结晶的一类蛋白质。近几年来,昆虫抗冻蛋白的研究取得了较快的发展,本文通过分析昆虫抗冻蛋白的结构特点、抗冻活性、作用机制,并讨论了抗冻蛋白在食品工业、医学、基因工程方面的应用。结果表明,昆虫抗冻蛋白虽然结构呈多样性,但有很多关键的残基具有保守性,对维持抗冻蛋白结构和功能的完整性发挥着重要的作用;抗冻蛋白是由多基因家簇编码的。其作用机制主要是吸附一抑制机制,抗冻蛋白依靠氢键吸附到冰晶格,抑制冰晶生长;昆虫抗冻蛋白的应用具有很广阔的前景。     

抗冻蛋白在超低温保存中作用机制的新模型

在超低温保存(cryopreservation,-80℃—-196℃)中,抗冻蛋白(antifreeze porteins,AFPs)对细胞存活率和热力学特性的影响十分复杂。在一些实验条件下,抗冻蛋白表现出保护活性;在另一些实验条件下,它却表现出毒害性。换句话说,它既能阻遏冰晶生长,又能促使成冰核效应(ice nucle-ation effect)发生。在总结最新实验结果的基础上,结合抗冻蛋白进化和结构生物学方面的新进展,我们提出了一个新模型,该模型对抗冻蛋白功能的两面性作了较好的诠释。     

昆虫抗冻蛋白的结构与生物学特性研究

抗冻蛋白(antifreeze proteins AFPs)是一类抑制冰晶生长的蛋白质,它能以非依数性形式降低溶液的冰点而对其熔点影响甚微,因而也被称作热滞蛋白。近几年来对于昆虫抗冻蛋白的研究取得了较快的发展,已有20多种昆虫抗冻蛋白被分离纯化。就昆虫抗冻蛋白的结构特征、生物学特性以及在农业、医学和食品工业等方面的应用进行介绍。     

抗冻蛋白及其在植物抗冻生理中的作用

抗冻蛋白（ａｎｔｉｆｒｅｚｅｐｒｏｔｅｉｎｓ,ＡＦＰｓ）是一类抑制冰晶生长的蛋白质,能以非依数性形式降低水溶液的冰点,但对熔点影响甚微,从而导致水溶液的熔点和冰点之间出现差值。这种差值称为热滞活性（ｔｈｅｒｍａｌｈｙｓｔｅｒｅｓｉｓａｃｔｉｖｉｔｙ,...     

昆虫抗冻蛋白的研究进展

抗冻蛋白是具有热滞效应,能结合并抑制新的冰晶生长,能抑制冰的重结晶的一类蛋白质。近几年来,昆虫抗冻蛋白的研究取得了较快的发展,本文就昆虫抗冻蛋白的结构,活性的调控,功能与应用做一综述。     

沙冬青叶片热稳定抗冻蛋白特性分析

用双向电泳电泳回收法从沙冬青（ Ａｍｍｏｐｉｐｔａｎｔｈｕｓ ｍｏｎｇｏｌｉｃｕｓ（Ｍａｘｉｍ ．） Ｃｈｅｎｇｆ．） 叶片热稳定蛋白质中分离到一种抗冻蛋白ａｆｐ,其分子量为４０ ｋＤ,ｐＩ为９．０ ,热滞活性为０ ．９ ℃（２０ ｍｇ／ｍＬ） ,和其他抗冻蛋白进行比较,没有发现相同的类型。ａｆｐ Ｎ 端序列为ＳＤＤＬＳＦＴＦＮＫＦＶＰＣＱＴＤＩＬＦ。ａｆｐ 在沙冬青体内广泛分布且叶片含量较高,可能是沙冬青抗冻生理过程中的主要物质,对于沙冬青抵御冬季冷冻温度具有重要的作用。     

差示扫描量热法直接测定沙冬青抗冻蛋白的热滞效应

用差示扫描量热法直接测定了从沙冬青中提取的一种抗冻蛋白（ＡＦＰ）组分的低温热行为。结果表明,该组分的低温热行为远较文献报道的各种抗冻蛋白复杂。在降、升温过程中,在低和高温侧都给出两个放或吸热峰,两个峰表现出相互独立而又相互依存的热滞行为。低温峰的热滞活性远高于高温岭。我们认为,这种ＡＦＰ分子对水及冰晶很可能有两种不同的相互作用和影响。     

使用差示扫描量热仪测定抗冻蛋白热滞活性方法的研究

抗冻蛋白因具有独特的抗冻活性而被研究者广泛关注。但是,目前抗冻活性的检测没有一个标准的、统一的检测方法,这严重制约了该方面的研究进展。作者详细研究了采用差示扫描量热仪测定样品热滞活性的方法,并对该方法的稳定性、专一性和精密度进行评价。结果显示,采用差示扫描量热仪测定样品的热滞活性具有较高的稳定性、重复性和精密度。因此,差示扫描量热仪法可以作为一种通用的方法进行抗冻蛋白热滞活性的检测。     

Ice nucleation inhibition: mechanism of antifreeze by antifreeze protein

The effect of antifreeze protein type III (one type of fish antifreeze protein) on ice crystallization was examined quantitatively based on a "micro-sized ice nucleation" technique. It was found for the first time that antifreeze proteins can inhibit the ice nucleation process by adsorbing onto both the surfaces of ice nuclei and dust particles. This leads to an increase of the ice nucleation barrier and the desolvation kink kinetics barrier, respectively. Based on the latest nucleation model, the increases in the ice nucleation barrier and the kink kinetics barrier were measured. This enables us to quantitatively examine the antifreeze mechanism of antifreeze proteins for the first time.     

Energy-optimized structure of antifreeze protein and its binding mechanism.

A combination of Monte Carlo simulated annealing and energy minimization was utilized to determine the conformation of the antifreeze protein from the fish winter flounder. It was found from the energy-optimized structure that the hydroxyl groups of its four threonine residues, i.e. Thr2, Thr13, Thr24, Thr35, are aligned on almost the same line parallel to the helix axis and separated successively by 16.1, 16.0 and 16.2 A, respectively, very close to the 16.6 A repeat spacing along [0112] in ice. Based on such a space match, a zipper-like model is proposed to elucidate the binding mechanism of the antifreeze protein to ice crystals. According to the current model, the antifreeze protein may bind to an ice nucleation structure in a zipper-like fashion through hydrogen bonding of the hydroxyl groups of these four Thr residues to the oxygen atoms along the [0112] direction in ice lattice, subsequently stopping or retarding the growth of ice pyramidal planes so as to depress the freeze point. The calculated results and the binding mechanism thus derived accord with recent experimental observations. The mechanistic implications derived from such a special antifreeze molecule might be generally applied to elucidate the structure-function relationship of other antifreeze proteins with the following two common features: (1) recurrence of a Thr residue (or any other polar amino acid residue whose side-chain can form a hydrogen bond with water) in an 11-amino-acid period along the sequence concerned; and (2) a high percentage of Ala residue component therein. Further experiments are suggested to test the ice binding model.     

The mechanism of the type III antifreeze protein action: a computational study

The random network model of water quantitatively describes the different hydration heat capacities of polar and apolar solutes in terms of differential distortions of the water-water hydrogen bonding angle in the first hydration shell. This method of hydration analysis is applied here to study the hydration of the wild type III thermal hysteresis protein from eel pout and three mutations at residue 16. Wild type and one mutant have full activity, the other two mutants have little or no anti-freeze (thermal hysteresis) activity. The analysis reveals significant differences in the hydration structure of the ice-binding site (centered on residue 16) among four proteins. For the A16T and A16Y mutants with reduced activity, polar groups have a typical polar-like hydration. For the wild type and mutant A16C with 100% of the wild type activity, polar groups have unusual, very apolar-like hydration. In the latter case, hydrating water molecules form a more ice-like pattern of hydrogen bonding on the ice-binding face, while in the former case water-water H-bonds are more distorted and more heterogenous. Overall, the binding surface of active protein strongly enhances the water tetrahedral structure, i.e. promotes ice-like hydration. It is concluded that the specific shape, residue size and clustering of both polar/apolar groups are essential for the binding surface to recognize, and preferentially interact with nascent ice crystals forming in liquid water.     

Subzero nonfreezing cryopresevation of rat hearts using antifreeze protein I and antifreeze protein III

The purpose of the present study was to evaluate whether AFPs protect the heart from freezing and improve survival and viability in subzero cryopreservation. Hearts were subject to 5 preservation protocols; University of Wisconsin solution (UW) at 4 degrees C, UW at -1.3 degrees C without nucleation, UW at -1.3 degrees C with nucleation, UW AFP I (15 mg/cm(3)) at -1.3 degrees C with nucleation, and in UW AFP III (15 mg/cm(3)) at -1.3 degrees C with nucleation. Hearts were preserved for 24, 28, and 32 h, rewarmed and connected to the working isolated perfusion system. Data [heart rate (HR), coronary flow (CF), and developed pressure (dP)] was collected 30 and 60 min after reperfusion. Hearts preserved at -1.3 degrees C without AFPs froze, while hearts preserved with AFP did not freeze when nucleation was initiated and survived. Survival and dP of hearts preserved for 24h at -1.3 degrees C using AFP III was better than those preserved at 4 degrees C, (dP; 1.4 vs. 0.8, p<0.05). Four of six hearts and six of six hearts died when preserved at 4 degrees C for 28 and 32 h, respectively, all of the hearts that were preserved at -1.3 degrees C with or without AFPs survived after 28 h (n=18) and 32 h (n=18). CF was higher in UW -1.3 degrees C group without attempted nucleation than in AFP I and AFP III groups after 28 and 32 h (3.4 vs. 1.7, p<0.05, and 3.4 vs. 1.7, p<0.05, respectively). In conclusion, AFPs were found to protect the heart from freezing and improve survival and dP (AFP III) in prolonged subzero preservation.     

抗冻蛋白的研究进展

本文概述了抗冻蛋白的种类、生成和性质,总结了抗冻蛋白基因及基因转化方面的研究情况。     

Expression, purification, and antifreeze activity of carrot antifreeze protein and its mutants

Antifreeze proteins (AFPs) enable organisms to survive under freezing or sub-freezing conditions. AFPs have a great potential in the low temperature storage of cells, tissues, organs, and foods. This process will require a large number of recombinant AFPs. In the present study, the recombinant carrot AFP was highly expressed in Escherichia coli strain BL21 (DE3). The activity of the purified and refolded recombinant proteins was analyzed by measurement of thermal hysteresis (TH) activity and detection of in vitro antifreeze activity by measuring enhanced cold resistance of bacteria. Two carrot AFP mutants generated by site-directed mutagenesis were also expressed and purified under these conditions for use in parallel experiments. Recombinant DcAFP displayed a TH activity equivalent to that of native DcAFP, while mutants DcAFP-N130Q and rDcAFP-N130V showed 32 and 43% decreases in TH activity, respectively. Both the recombinant DcAFP and its mutants were able to enhance the cold resistance of bacteria, to degrees consistent with their respective TH activities.     

Ice-binding mechanism of winter flounder antifreeze proteins

We have studied the winter flounder antifreeze protein (AFP) and two of its mutants using molecular dynamics simulation techniques. The simulations were performed under four conditions: in the gas phase, solvated by water, adsorbed on the ice (2021) crystal plane in the gas phase and in aqueous solution. This study provided details of the ice-binding pattern of the winter flounder AFP. Simulation results indicated that the Asp, Asn, and Thr residues in the AFP are important in ice binding and that Asn and Thr as a group bind cooperatively to the ice surface. These ice-binding residues can be collected into four distinct ice-binding regions: Asp-1/Thr-2/Asp-5, Thr-13/Asn-16, Thr-24/Asn-27, and Thr-35/Arg-37. These four regions are 11 residues apart and the repeat distance between them matches the ice lattice constant along the (1102) direction. This match is crucial to ensure that all four groups can interact with the ice surface simultaneously, thereby, enhancing ice binding. These Asx (x = p or n)/Thr regions each form 5-6 hydrogen bonds with the ice surface: Asn forms about three hydrogen bonds with ice molecules located in the step region while Thr forms one to two hydrogen bonds with the ice molecules in the ridge of the (2021) crystal plane. Both the distance between Thr and Asn and the ordering of the two residues are crucial for effective ice binding. The proper sequence is necessary to generate a binding surface that is compatible with the ice surface topology, thus providing a perfect "host/guest" interaction that simultaneously satisfies both hydrogen bonding and van der Waals interactions. The results also show the relation among binding energy, the number of hydrogen bonds, and the activity. The activity is correlated to the binding energy, and in the case of the mutants we have studied the number of hydrogen bonds. The greater the number of the hydrogen bonds the greater the antifreeze activity. The roles van der Waals interactions and the hydrophobic effect play in ice binding are also highlighted. For the latter it is demonstrated that the surface of ice has a clathratelike structure which favors the partitioning of hydrophobic groups to the surface of ice. It is suggested that mutations that involve the deletion of hydrophobic residues (e.g., the Leu residues) will provide insight into the role the hydrophobic effect plays in partitioning these peptides to the surface of ice.     

A mechanism for stabilization of membranes at low temperatures by an antifreeze protein.

Polar fish, cold hardy plants, and overwintering insects produce antifreeze proteins (AFPs), which lower the freezing point of solutions noncolligatively and inhibit ice crystal growth. Fish AFPs have been shown to stabilize membranes and cells in vitro during hypothermic storage, probably by interacting with the plasma membrane, but the mechanism of this stabilization has not been clear. We show here that during chilling to nonfreezing temperatures the alpha-helical AFP type I from polar fish inhibits leakage across model membranes containing an unsaturated chloroplast galactolipid. The mechanism involves binding of the AFP to the bilayer, which increases the phase transition temperature of the membranes and alters the molecular packing of the acyl chains. We suggest that this change in acyl chain packing results in the reduced membrane permeability. The data suggest a hydrophobic interaction between the peptide and the bilayer. Further, we suggest that the expression of AFP type I in transgenic plants may be significant for thermal adaptation of chilling-sensitive plants.     

抗冻蛋白特征、作用机理与预测新进展

抗冻蛋白（antifreezeproteins,AFPs）可以通过抑制冰晶生长保护生物体免受低温冻害,具有重要的生物学意义和应用价值。现在在鱼类、节肢动物、植物及微生物中均发现有AFPs的存在。基于对已有研究文献和相关网络数据的系统调查统计,详细描述了AFPs数据的类别特征,并对其作用机理的研究历史和最新取得的突破性进展作了较为系统的阐述,并对AFPs预测所取得的成果作了介绍,还对AFPs研究的现状和未来研究方向作了讨论和展望。     

Structure and dynamics of a beta-helical antifreeze protein

Antifreeze proteins (AFPs) protect many types of organisms from damage caused by freezing. They do this by binding to the ice surface, which causes inhibition of ice crystal growth. However, the molecular mechanism of ice binding leading to growth inhibition is not well understood. In this paper, we present the solution structure and backbone NMR relaxation data of the antifreeze protein from the yellow mealworm beetle Tenebrio molitor (TmAFP) to study the dynamics in the context of structure. The full (15)N relaxation analysis was completed at two magnetic field strengths, 500 and 600 MHz, as well as at two temperatures, 30 and 5 degrees C, to measure the dynamic changes that occur in the protein backbone at different temperatures. TmAFP is a small, highly disulfide-bonded, right-handed parallel beta-helix consisting of seven tandemly repeated 12-amino acid loops. The backbone relaxation data displays a periodic pattern, which reflects both the 12-amino acid structural repeat and the highly anisotropic nature of the protein. Analysis of the (15)N relaxation parameters shows that TmAFP is a well-defined, rigid structure, and the extracted parameters show that there is similar restricted internal mobility throughout the protein backbone at both temperatures studied. We conclude that the hydrophobic, rigid binding site may reduce the entropic penalty for the binding of the protein to ice. The beta-helical fold of the protein provides this rigidity, as it does not appear to be a consequence of cooling toward a physiologically relevant temperature.