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。     

第一类鱼抗冻蛋白对冰晶生长界面层结构的影响

根据冰晶在水溶液中生长的基本热力学性质,应用多层界面模型,分别得到了冰晶在纯水及抗冻蛋白溶液中生长界面层的吉布斯自由能.由冰晶生长界面层的吉布斯自由能,分析了冰晶在三种不同第一类鱼抗冻蛋白分子溶液中,热平衡状态下生长界面层的微观平衡结构,发现冰晶在抗冻蛋白溶液中生长与其在纯水中生长相比,界面层结构有明显变化,结合抗冻蛋...     

昆虫抗冻蛋白: 规则结构适应功能

抗冻蛋白在环境温度低于体液熔点时能够结合到生物体内的冰核表面,通过限制冰核生长和抑制冰晶重结晶而保护有机体免受结冰引起的伤害。与其他生物抗冻蛋白比较,昆虫抗冻蛋白有很强的活性,结构上具有显著特征,如一级结构规律重复,超二级结构为β-螺旋,可与冰晶发生相互作用,具有TXT基序等。该文综述了近年来关于昆虫抗冻蛋白的结构以及分子生物学等方面研究的新进展,讨论了其结构与功能的关系。     

昆虫抗冻蛋白的研究进展

抗冻蛋白是一类与冰晶有亲合力,能够与冰晶结合并抑制冰晶生长的蛋白或糖蛋白。自20世纪60年代以来,研究人员已经分别从鱼类、昆虫、植物、真菌和细菌中发现多种抗冻蛋白。其中已知鱼类抗冻蛋白有5种,也是研究最详细的。但是,近几年来发现昆虫抗冻蛋白活性普遍比较高,因此受到研究人员重视,研究取得了较快的发展。主要讨论昆虫抗冻蛋白的结构特点、抗冻活性、作用机制和应用,并分析目前的研究现状提出一些待解决的问题,以期望对昆虫抗冻蛋白的研究进行比较系统化的整理。     

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。     

甲虫抗冻蛋白热滞活性的二维吸附结合模型

甲虫抗冻蛋白是一种具有规则结构的昆虫抗冻蛋白。在相同浓度条件下,甲虫抗冻蛋白比鱼类抗冻蛋白有更高的热滞活性,目前已成为人们重点研究的一类抗冻蛋白。根据甲虫抗冻蛋白的结构特点及其在冰晶表面的吸附模式,应用二维吸附结合模型计算分析了具有6￣11个β-螺旋(β-helix)结构片段的甲虫抗冻蛋白变体分子,得到了它们的热滞活性随溶液浓度变化的规律,特别是热滞活性与甲虫抗冻蛋白的β-螺旋结构片段数的关系。结果显示,抗冻蛋白在冰晶表面的覆盖度是一个影响其热滞活性的重要因素。     

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。     

抗冻蛋白应用于水稻悬浮细胞超低温保存的研究

本文将鱼类抗冻蛋白应用于植物细胞的超低温保存。结果表明,在水稻悬浮细胞的两步法保存中,浓度为0.01mg/ml的抗冻蛋白具有特别的负作用,相对高浓度的抗冻蛋白则能减小细胞存活率的波动性。在玻璃化法保存中,浓度为0.2mg/ml的抗冻蛋白能改善保存效果,更高浓度的抗冻蛋白(>5mg/ml)反而会降低保存效果。环境冰晶量、抗冻蛋白浓度、低温保护剂浓度和细胞膜组成等是影响抗冻蛋白使用效果的几大因素。作者在机理分析中认为,一方面,抗冻蛋白能和冰晶作用,抑制重冰晶,防止去玻璃化;另一方面,抗冻蛋白也能和细胞膜作用,诱发膜表面冰晶形成。     

抗冻蛋白与植物低温胁迫反应

植物抗冻蛋白是从许多抗冻植物中分离的、参与植物抵御冻害反应的一类新型蛋白.这类抗冻蛋白具有多个亲水性缚冰域,能直接作用于冰晶,阻止冰晶在细胞间隙形成和再结晶.一些植物抗冻蛋白与致病相关蛋白有序列同源性,具有抗冻和抗病双重活性.植物抗冻蛋白的表达和积累,既受控于发育及转录因子调节,又受到低温、短日照、脱水及乙烯等因素的影响.异源超表达抗冻蛋白基因能赋予敏感宿主植物抗冻能力.文中论述了有关植物抗冻蛋白特性和鉴定,抗冻机制和表达调控,以及遗传转化等方面的研究进展.     

抗冻蛋白研究进展

抗冻蛋白是一类具有热滞效应、冰晶形态效应和重结晶抑制效应的蛋白质。简单介绍了各种抗冻蛋白的生化特征、作用机制及其应用研究 ,并对抗冻蛋白的基因和基因工程研究作了较为系统的综述。     

Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site

The grass Lolium perenne produces an ice-binding protein (LpIBP) that helps this perennial tolerate freezing by inhibiting the recrystallization of ice. Ice-binding proteins (IBPs) are also produced by freeze-avoiding organisms to halt the growth of ice and are better known as antifreeze proteins (AFPs). To examine the structural basis for the different roles of these two IBP types, we have solved the first crystal structure of a plant IBP. The 118-residue LpIBP folds as a novel left-handed beta-roll with eight 14- or 15-residue coils and is stabilized by a small hydrophobic core and two internal Asn ladders. The ice-binding site (IBS) is formed by a flat beta-sheet on one surface of the beta-roll. We show that LpIBP binds to both the basal and primary-prism planes of ice, which is the hallmark of hyperactive AFPs. However, the antifreeze activity of LpIBP is less than 10% of that measured for those hyperactive AFPs with convergently evolved beta-solenoid structures. Whereas these hyperactive AFPs have two rows of aligned Thr residues on their IBS, the equivalent arrays in LpIBP are populated by a mixture of Thr, Ser and Val with several side-chain conformations. Substitution of Ser or Val for Thr on the IBS of a hyperactive AFP reduced its antifreeze activity. LpIBP may have evolved an IBS that has low antifreeze activity to avoid damage from rapid ice growth that occurs when temperatures exceed the capacity of AFPs to block ice growth while retaining the ability to inhibit ice recrystallization.     

Lessons from nature for preservation of mammalian cells, tissues, and organs

The study of mechanisms by which animals tolerate environmental extremes may provide strategies for preservation of living mammalian materials. Animals employ a variety of compounds to enhance their survival, including production of disaccharides, glycerol, and antifreeze compounds. The cryoprotectant glycerol was discovered before its role in amphibian survival. In the last decade, trehalose has made an impact on freezing and drying methods for mammalian cells. Investigation of disaccharides was stimulated by the variety of organisms that tolerate dehydration stress by accumulation of disaccharides. Several methods have been developed for the loading of trehalose into mammalian cells, including inducing membrane lipid-phase transitions, genetically engineered pores, endocytosis, and prolonged cell culture with trehalose. In contrast, the many antifreeze proteins (AFPs) identified in a variety of organisms have had little impact. The first AFPs to be discovered were found in cold water fish; their AFPs have not found a medical application. Insect AFPs function by similar mechanisms, but they are more active and recombinant AFPs may offer the best opportunity for success in medical applications. For example, in contrast to fish AFPs, transgenic organisms expressing insect AFPs exhibit reduced ice nucleation. However, we must remember that nature's survival strategies may include production of AFPs, antifreeze glycolipids, ice nucleators, polyols, disaccharides, depletion of ice nucleators, and partial desiccation in synchrony with the onset of winter. We anticipate that it is only by combining several natural low temperature survival strategies that the full potential benefits for mammalian cell survival and medical applications can be achieved.     

Positive Darwinian Selection Promotes Heterogeneity Among Members of the Antifreeze Protein Multigene Family

A variety of organisms have independently evolved proteins exhibiting antifreeze activity that allows survival at subfreezing temperatures. The antifreeze proteins (AFPs) bind ice nuclei and depress the freezing point by a noncolligative absorption–inhibition mechanism. Many organisms have a heterogeneous suite of AFPs with variation in primary sequence between paralogous loci. Here, we demonstrate that the diversification of the AFP paralogues is promoted by positive Darwinian selection in two independently evolved AFPs from fish and beetle. First, we demonstrate an elevated rate of nonsynonymous substitutions compared to synonymous substitutions in the mature protein coding region. Second, we perform phylogeny-based tests of selection to demonstrate a subset of codons is subjected to positive selection. When mapped onto the three-dimensional structure of the fish antifreeze type III antifreeze structure, these codons correspond to amino acid positions that surround but do not interrupt the putative ice-binding surface. The selective agent may be related to efficient binding to diverse ice surfaces or some other aspect of AFP function. Received: 27 February 2001 / Accepted: 12 September 2001     

Purification and Structure Analysis of Antifreeze Proteins from Ammopiptanthus mongolicus

Abstract

We purified many kinds of antifreeze proteins with high activity from the leaves of Ammopiptanthus mongolicus by several biochemical techniques. The antifreeze activities of these AFPs were measured by both osmometry and differential scanning calorimetry, and the inhibition of growth of ice crystals by the AFPs was obvious. Additionally, the antifreeze proteins were analyzed by sequencing, glycosylation reaction, mass spectroscopy, and circular dichroism spectroscopy. Both samples have some other unique structures different from those of fishes and of insects. It was suggested that plant AFPs might have a particular antifreeze mechanism in comparison with that of fish and insects.     

利用伪氨基酸组分和支持向量机预测抗冻蛋白

抗冻蛋白是一类具有提高生物抗冻能力的蛋白质。抗冻蛋白能够特异性的与冰晶相结合,进而阻止体液内冰核的形成与生长。因此,对抗冻蛋白的生物信息学研究对生物工程发展。提高作物抗冻性有重要的推动作用。本文采用由400条抗冻蛋白序列和400条非抗冻蛋白序列构成数据集,以伪氨基酸组分为特征,利用支持向量机分类算法预测抗冻蛋白,对训练集预测精度达到91．3％,对测试集预测精度达到78．8％。该结果证明伪氨基酸组分能够很好的反映抗冻蛋白特性,并能够用于预测抗冻蛋白。     

Structure and evolutionary origin of Ca(2+)-dependent herring type II antifreeze protein

In order to survive under extremely cold environments, many organisms produce antifreeze proteins (AFPs). AFPs inhibit the growth of ice crystals and protect organisms from freezing damage. Fish AFPs can be classified into five distinct types based on their structures. Here we report the structure of herring AFP (hAFP), a Ca(2+)-dependent fish type II AFP. It exhibits a fold similar to the C-type (Ca(2+)-dependent) lectins with unique ice-binding features. The 1.7 A crystal structure of hAFP with bound Ca(2+) and site-directed mutagenesis reveal an ice-binding site consisting of Thr96, Thr98 and Ca(2+)-coordinating residues Asp94 and Glu99, which initiate hAFP adsorption onto the [10-10] prism plane of the ice lattice. The hAFP-ice interaction is further strengthened by the bound Ca(2+) through the coordination with a water molecule of the ice lattice. This Ca(2+)-coordinated ice-binding mechanism is distinct from previously proposed mechanisms for other AFPs. However, phylogenetic analysis suggests that all type II AFPs evolved from the common ancestor and developed different ice-binding modes. We clarify the evolutionary relationship of type II AFPs to sugar-binding lectins.     

昆虫抗冻蛋白的分离纯化及特性分析

昆虫抗冻蛋白具有很高的热滞活性,可保护机体免受结冰引起的伤害。昆虫抗冻蛋白的分离纯化多采用凝胶过滤层析、离子交换层析及HPLC等技术,已用于鱼类抗冻蛋白纯化的冰亲和纯化(IAP)技术也可考虑应用于昆虫抗冻蛋白的分离提纯。昆虫抗冻蛋白具有高活性,规则的一级结构及类似的冰晶结合表面等特性。     

Effect of a mutation on the structure and dynamics of an alpha-helical antifreeze protein in water and ice

One strategy of psychrophilic organisms to survive subzero temperature is to produce antifreeze protein (AFPs), which inhibit the growth of macromolecular ice. To better understand the binding mechanism, the structure and dynamics of several AFPs have been studied by nuclear magnetic resonance (NMR) and X-ray crystallography. The results have shown that different organisms can use diverse structures (alpha-helix, beta-helix, or different globular folds) to achieve the same function. A number of studies have focused on understanding the relationship between the alpha-helical structure of fish type I AFP and its function as an inhibitor of ice growth. The results have not explained whether the 90% activity loss caused by the conservative mutation of two threonines to serines (Thr13Ser/Thr24Ser) is attributable to a change in protein structure in solution or in ice. We examine here the structure and dynamics of the winter flounder type I AFP and the mutant Thr13Ser/Thr24Ser in both solution and solid states using a wide range of NMR approaches. Both proteins remain fully alpha-helical at all temperatures and in ice, demonstrating that the activity change must therefore not be attributable to changes in the protein fold or dynamics but differences in surface properties.