利用数字图象技术观察蛋白质的结晶过程

用汽相扩散法生长溶菌酶晶体并利用ＣＣＤ显微摄像系统记录了溶菌酶晶体的生长过程。由此图象序列,我们可以计算晶体的最大线度、生长速度,估计溶菌酶分子层的增长速度,了解蛋白质晶体在结晶室内的分布及其形态变化。得到结果如下：蛋白晶体生长初期最大线度与时间近似成线性关系;各晶面生长速度基本相等。     

不同重力条件下生长的溶菌酶晶体的结构

利用我国返回式卫星和国内研制的蛋白质结晶装置 ,先后进行了 2次空间蛋白质晶体生长实验 ,均获得了质量较好的溶菌酶晶体 .为了探索微重力对溶菌酶晶体结构的影响 ,对 2次空间生长的和地面实验室生长的溶菌酶晶体进行了高精度的晶体结构测定和研究     

离子液体1-丁基-3-甲基咪唑氯盐对溶菌酶结晶的影响

以亲水性离子液体1-丁基-3-甲基咪唑氯盐(BmimCl)为添加剂,研究离子液体对溶菌酶结晶的影响.分别考察了离子液体对溶菌酶晶体数量与尺寸、晶体形貌及蛋白质纯度的影响,并探讨了离子液体对结晶过程影响的作用机制.离子液体通过增大溶菌酶的溶解度和其自身低蒸气压两种途径,降低了溶菌酶在结晶过程中的过饱和度,更有利于晶体的成核和生长,得到更好的结果.如避免多晶态现象的发生,增大晶体的尺寸,降低溶菌酶样品纯度的要求.X-射线衍射分析表明,离子液体未改变晶体的晶型结构,但可提高晶体的衍射分辨率.     

离子液体1-丁基-3-甲基咪唑氯代盐对溶菌酶结晶的影响

以亲水性离子液体1-丁基-3-甲基咪唑氯盐(BmimCl)为添加剂,研究离子液体对溶菌酶结晶的影响.分别考察了离子液体对溶菌酶晶体数量与尺寸、晶体形貌及蛋白质纯度的影响,并探讨了离子液体对结晶过程影响的作用机制.离子液体通过增大溶菌酶的溶解度和其自身低蒸气压两种途径,降低了溶菌酶在结晶过程中的过饱和度,更有利于晶体的成核和生长,得到更好的结果.如避免多晶态现象的发生,增大晶体的尺寸,降低溶菌酶样品纯度的要求.X-射线衍射分析表明,离子液体未改变晶体的晶型结构,但可提高晶体的衍射分辨率.     

溶菌酶分子聚集状态对蛋白质晶体生长影响的研究

使用动态光散射仪及微批量法,实时测量了溶菌酶晶体生长过程中,蛋白质颗粒的聚集情况。实验表明当液滴中的溶菌酶分子单体形式占多数,同时也存在一定数目的聚集体时,将会产生晶体。另外还通过观察有无絮状物附着情况下,溶菌酶晶体的生长情况,研究了絮状物对晶体生长速度的影响。实验表明这种由高密度的蛋白质聚集体组成的絮状物附着在晶体表面时,晶体的生长受到抑制。而絮状物会逐渐解体,重构成四方晶体或球状结晶等更稳定的聚集状态。研究在一定程度上揭示了溶液中溶菌酶分子的聚集状态与结晶的关系。     

凝胶法生长溶菌酶晶体的研究

凝胶介质可以排除或削弱晶体生长过程中重力引起的对流和沉淀现象,用凝胶法生长生物大分子晶体是一种新的探索。使用类似于悬滴汽相扩散的方法,凝胶中生长出单个较大的外形发育完善且高度对称的鸡蛋清溶菌酶晶体。ＭＰＤ在凝胶中对溶菌酶结晶与溶液中具有相似的抑核作用。排循照像实验表明,凝胶法生长的晶体具有较强的衍射能力。     

肺炎链球菌假想的溶菌酶样蛋白的活性分析和鉴定

目的 探索肺炎链球菌中一种假想的溶菌酶样蛋白的活性.方法 生物信息学分析该基因的功能结构域;利用长臂同源PCR对该基因进行敲除,观察D39野生菌和缺陷菌在生物学性状的改变;利用底物PNP-（GIcNAc）和溶壁微球菌,构建过表达载体,绘制生长曲线,对截断和全长蛋白的溶菌酶活性进行鉴定.结果 生物信息学分析结果显示该基因的编码产物为β-1,4-N-乙酰胞壁酸糖苷酶,属于糖基水解酶25家族;野生菌为长链生长,缺陷菌呈短链状;溶菌酶和假想的溶菌酶样蛋白均可使底物释放出游离的对硝基苯酚,A405nm吸光度值分别为1.166和0.792;同时也可使得溶壁微球菌发生溶解;含过表达质粒的肺炎链球菌较之野生菌,溶解较快.结论 假想的溶菌酶样蛋白具有溶菌酶活性,是一种新的溶菌酶.     

反刍动物溶菌酶研究进展

目前,抗生素滥用带来的副作用日益凸显,寻找抗生素的有效替代品显得尤为迫切。溶菌酶能水解细菌细胞壁肽聚糖中的β-1,4糖苷键,具有消化分解细菌、抑制外源微生物生长、增强机体免疫力的作用,在动物尤其是反刍动物体内广泛存在。本文讨论了反刍动物溶菌酶的来源与分布、基因序列、蛋白结构和酶学性质、蛋白功能及活性,对其耐酸分子机理进行了归纳总结;同时阐述了反刍动物溶菌酶基因的进化研究,最后对反刍动物溶菌酶研究进行了展望。研究反刍动物溶菌酶对于基础科学,并应用其转变成现实生产力意义重大。     

动物源溶菌酶研究进展

动物源溶菌酶是一种动物体内广泛存在的酶类,它可以水解细菌细胞壁肽聚糖中的β-1,4糖苷键,具有消化分解细菌、抑制外源微生物生长、增强机体免疫力的作用.目前溶菌酶已被用作研究蛋白功能、性质以及分子进化的模型.首先介绍了溶菌酶及其分子的晶体结构,溶菌酶基因及其蛋白研究进展,其次介绍了动物源溶菌酶的功能,包括溶菌酶生物学功能和重组蛋白功能活性,重点介绍了溶菌酶基因在转基因工程中的应用研究,最后对动物源溶菌酶研究进行了展望.研究动物源溶菌酶对于基础科学,并应用其转变成现实生产力具有重要的指导意义.     

溶菌酶对微小小单胞菌生物膜的影响

目的测定溶菌酶对微小小单胞菌及其生物膜的抑菌作用,并测出最小抑菌浓度(MIC)、最小杀菌浓度(MBC)和抑菌率。方法采用对倍稀释的方法,测定溶菌酶对微小小单胞菌的MIC、MBC;在96孔板中体外建立微小小单胞菌生物膜模型,采用MTT法检测溶菌酶对微小小单胞菌生物膜的影响;在六孔板中建立生物膜模型,使用激光共聚焦显微镜(CLSM)观察不同浓度溶菌酶对微小小单胞菌生物膜作用后的变化。结果溶菌酶对微小小单胞菌的MIC为0.0195 mg/mL,MBC为0.3125 mg/mL;CLSM观察结果显示,溶菌酶对微小小单胞菌生物膜的抑制作用随着浓度的增加而增强。结论溶菌酶对微小小单胞菌及其生物膜的生长和活性均具有抑制作用。     

Protein crystallization under high pressure

Pressure is expected to be an important parameter to control protein crystallization, since hydrostatic pressure affects the whole system uniformly and can be changed very rapidly. So far, a lot of studies on protein crystallization have been done. Solubility of protein depends on pressure. For instance, the solubility of tetragonal lysozyme crystal increased with increasing pressure, while that of orthorhombic crystal decreased. The solubility of subtilisin increased with increasing pressure. Crystal growth rates of protein also depend on pressure. The growth rate of glucose isomerase was significantly enhanced with increasing pressure. The growth rate of tetragonal lysozyme crystal and subtilisin decreased with increasing pressure. To study the effects of pressure on the crystallization more precisely and systematically, hen egg white lysozyme is the most suitable protein at this stage, since a lot of data can be used. We focused on growth kinetics under high pressure, since extensive studies on growth kinetics have already been done at atmospheric pressure, and almost all of them have explained the growth mechanisms well. The growth rates of tetragonal lysozyme decreased with pressure under the same supersaturation. This means that the surface growth kinetics significantly depends on pressure. By analyzing the dependence of supersaturation on growth rate, it was found that the increase in average ledge surface energy of the two-dimensional nuclei with pressure explained the decrease in growth rate. At this stage, it is not clear whether the increase in surface energy with increasing pressure is the main reason or not. Fundamental studies on protein crystallization under high pressure will be useful for high pressure crystallography and high pressure protein science.     

Protein crystallization in microgravity.

A space experiment involving protein crystallization was conducted in a microgravity environment using the space shuttle "Endeavour" of STS-47, on a 9-day mission from September 12th to 20th in 1992. The crystallization was carried out according to a batch method, and 5 proteins were selected as flight samples for crystallization. Two of these proteins: hen egg-white lysozyme and co-amino acid: pyruvate aminotransferase from Pseudomonas sp. F-126, were obtained as single crystals of good diffraction quality. Since 1992 we have carried out several space experiments for protein crystallization aboard space shuttles and the space station MIR. Our experimental results obtained mainly from hen egg-white lysozyme are described below, focusing on the effects of microgravity on protein crystal growth.     

The effect of protein impurities on lysozyme crystal growth

While bulk crystallization from impure solutions is used industrially as a purification step for a wide variety of materials, it is a technique that has rarely been used for proteins. Proteins have a reputation for being difficult to crystallize and high purity of the initial crystallization solution is considered paramount for success in the crystallization. Although little is written on the purifying capability of protein crystallization or of the effect of impurities on the various aspects of the crystallization process, recent published reports show that crystallization shows promise and feasibility as a purification technique for proteins. To further examine the issue of purity in macromolecule crystallization, this study investigates the effect of the protein impurities, avidin, ovalbumin, and conalbumin at concentrations up to 50%, on the solubility, crystal face growth rates, and crystal purity of the protein lysozyme. Solubility was measured in batch experiments while a computer controlled video microscope system was used to measure the ?110? and ?101? lysozyme crystal face growth rates. While little effect was observed on solubility and high crystal purity was obtained (>99.99%), the effect of the impurities on the face growth rates varied from no effect to a significant face specific effect leading to growth cessation, a phenomenon that is frequently observed in protein crystal growth. The results shed interesting light on the effect of protein impurities on protein crystal growth and strengthen the feasibility of using crystallization as a unit operation for protein purification.     

The binding of precipitant ions in the tetragonal crystals of hen egg white lysozyme

Abstract

The bonds between lysozyme molecules and precipitant ions in single crystals grown with chlorides of several metals are analysed on the basis of crystal structure data. Crystals of tetragonal hen egg lysozyme (HEWL) were grown with chlorides of several alkali and transition metals (LiCl, NaCl, KCl, NiCl₂ and CuCl₂) as precipitants and the three-dimensional structures were determined at 1.35?Å resolution by X-ray diffraction method. The positions of metal and chloride ions attached to the protein were located, divided into three groups and analysed. Some of them, in accordance with the recently proposed and experimentally confirmed crystal growth model, provide connections in protein dimers and octamers that are precursor clusters in the crystallization lysozyme solution. The first group, including Cu⁺², Ni⁺² and Na⁺¹ cations, binds specifically to the protein molecule. The second group consists of metal and chloride ions bound inside the dimers and octamers. The third group of ions can participate in connections between the octamers that are suggested as building units during the crystal growth. The arrangement of chloride and metal ions associated with lysozyme molecule at all stages of the crystallization solution formation and crystal growth is discussed.

Communicated by Ramaswamy H. Sarma     

A common mechanism underlying amyloid fibrillation and protein crystallization revealed by the effects of ultrasonication

Protein crystals form in supersaturated solutions via a nucleation and growth mechanism. The amyloid fibrils of denatured proteins also form via a nucleation and growth mechanism. This similarity suggests that, although protein crystals and amyloid fibrils are distinct in their morphologies, both processes can be controlled in a similar manner. It has been established that ultrasonication markedly accelerates the formation of amyloid fibrils and simultaneously breaks them down into fragmented fibrils. In this study, we investigated the effects of ultrasonication on the crystallization of hen egg white lysozyme and glucose isomerase from Streptomyces rubiginosus. Protein crystallization was monitored by light scattering, tryptophan fluorescence, and light transmittance. Repeated ultrasonic irradiations caused the crystallization of lysozyme and glucose isomerase after cycles of irradiations. The size of the ultrasonication-induced crystals was small and homogeneous, and their numbers were larger than those obtained under quiescent conditions. Switching off ultrasonic irradiation when light scattering or tryptophan fluorescence began to change resulted in the formation of larger crystals due to the suppression of the further nucleation and fractures in preformed crystals. The results indicate that protein crystallization and amyloid fibrillation are explained on the basis of a common phase diagram in which ultrasonication accelerates the formation of crystals or crystal-like amyloid fibrils as well as fragmentation of preformed crystals or fibrils.     

Control of nucleation in the crystallization of lysozyme.

This work investigates the influence of storage of lysozyme in solution on its crystallization. The crystallization of hen egg-white lysozyme exhibits a storage effect (aging) that depends on the length of time the lysozyme solution is stored, after dissolving from freeze-dried powder, before being brought to crystallization conditions. The number of crystals obtained increases, while their size decreases, as the solution ages. Observations suggest that this effect is due to the presence of fungi that multiply in the stored protein solution. This aging effect was used to control nucleation and determine the number and size of lysozyme crystals to be formed in a given sample.     

On the crystallization of proteins

We report on theoretical and experimental work aimed at a systematic approach to the crystallization of proteins. Successful crystallization depends on the competition between the growth rates for compact three-dimensional structures and long-chain structures leading to an amorphous precipitate. Quasi-elastic light scattering was used to monitor the size and shape distribution of small aggregates in a model system (lysozyme) during the pre-nucleation stage. With the aid of a simple model, the line-width of the scattered light was used to predict whether crystals or an amorphous precipitate would result. Once visible crystals appeared, the lysozyme concentration near the crystal surface was monitored and the kinetic parameters for growth obtained. A peculiar self-limiting phenomenon causes crystals to stop growing after a certain size has been reached. When these terminal size crystals were cleaved, growth occurred at the surface until the original size was approximately restored.     

Structural study on hen egg-white lysozyme crystals grown in gravity and microgravity

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Comparison of two different lysozyme types under native and crystallization conditions using two-dimensional NMR and dynamic light scattering

In order to elucidate differences observed in the aggregation kinetics of hen-egg white lysozyme under crystallization conditions we have undertaken a comparative study of the enzyme marketed by Seikagaku and Sigma companies. When the crystallization of the two lysozyme preparations is followed by time-resolved dynamic light scattering, the structural differences are also observed under native conditions in the early nucleation kinetics. The differences are manifested in the formation rates of macroscopic crystals, but do not influence the morphology of the typical tetragonal lysozyme crystal. Using two-dimensional NMR we have followed the differences in the native-like solution structure of the two preparations, while the primary sequence and molecular mass are identical. According to the published structure of tetragonal lysozyme crystal the largest deviations were found for the residues involved in the intermolecular interactions in crystal structure.