蛋白质结晶的研究进展

概括了蛋白质结晶的基本过程,阐述了蛋白质结晶的早期发展历程,重点介绍了蛋白质结晶的近期研究状况,主要包括:形核机理的研究、结晶条件的筛选和结晶技术的优化以及基于结构的药物设计技术。特别是对离子液体在蛋白质结晶过程中的应用及发展前景进行了讨论。     

电场中的蛋白质结晶

人们一直致力于寻求提高蛋白质晶体质量的方法,利用电场诱导蛋白质结晶即是诸多方法中的一种。已有文献报道显示,电场对蛋白质结晶的影响是积极的。我们从直流电场、交流电场、内置电场、外置电场对蛋白质结晶的影响及相关结晶设备,电场中生长的蛋白质晶体质量的评估,电场中蛋白质结晶的原理及影响因素等方面,对已报道的电场中的蛋白质结晶研究工作进行了总结。     

溶菌酶的结晶研究

介绍了溶菌酶结晶的意义,通过对晶核形成、晶体生长和停止生长三个阶段的论述,详细地阐述了溶菌酶的结晶机理;并综述了结晶过程中的各个影响因素:蛋白质浓度、pH值、添加剂、生长温度及重力场、磁场等;展望了溶菌酶结晶研究的发展趋势和发展前景。     

制备用于结晶的蛋白质样品

制备高质量蛋白质晶体是通过X射线衍射解析蛋白质分子三维结构的关键环节,是结构生物学领域中的瓶颈问题之一。蛋白质的结晶受多因素控制,其中蛋白质样品自身的质量是影响蛋白质能否结晶及晶体质量好坏的关键因素。我们从蛋白质纯度、可溶性、均一性及表面修饰等方面介绍了如何获得适于结晶的蛋白质样品,以及如何借助相关仪器检测蛋白质样品的质量,预测蛋白质的可结晶性。     

一种简便灵活的蛋白质结晶的接种技术

介绍一种简便灵活的蛋白质结晶的微量接种技术,描述了接种容器的特点、硅化方法、实验技巧及其在蛋白质单晶培养中的应用。     

蛋白质的晶体生长及其进展

蛋白质晶体生长是用衍射法测定和研究蛋白质三维结构不可缺少的首要步骤,因而对于从分子水平了解生命过程和有效地开发蛋白质工程、理性药物设计等新的生物技术具有重要意义。这一结构测定步骤所处的落后状态,更使蛋白质晶体生长成为倍受重视的研究课题。蛋白质和核酸等生物大分子的结晶是一个受多个因素影响的过程。来自不同学科的研究人员从各个方面对蛋白质的晶体生长开展了研究,并取得了不同程度的进展。     

温度影响蛋白质结晶的研究现状

温度控制作为调控蛋白质结晶过程的手段,在结晶实验中被广泛采用。热历史效应作为蛋白质结晶实验中新的影响因素,已被越来越多的科学家所重视。控制温度可以改变蛋白质的溶解度,进一步改变溶液的过饱和度,从而影响结晶过程。我们简要总结了温度对蛋白质结晶的影响及应用温度技术控制蛋白质晶体生长的各种技术,为蛋白质结晶工作提供理论和实验依据。     

分子生物学在蛋白质结晶中的应用

获得具有高分辨率的蛋白质晶体是目前蛋白质结构测定的主要瓶颈 . 蛋白质结晶受很多因素影响,蛋白质自身是结晶时最重要的变量,可以说,蛋白质的内在特性在某种程度上决定了其能否结晶以及所得晶体分辨率的高低 . 近年来分子生物学尤其是蛋白质工程的应用有效地提高蛋白质的溶解度、均一性及可结晶性等内在特性,促进蛋白质的结晶,成为提高蛋白质结晶能力和蛋白质晶体分辨率的有效途径 .     

蛋白质结晶委托服务汇集不同领域的知识开发革新的结晶技术

在电工学领域研究结晶技术的大阪大学的研究者们向蛋白质发起挑战。他们与结构生物学的研究者们合作,创造出革新的结晶技术。2005年7月份,他们建立风险企业,开始提供结晶委托服务。[编者按]     

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。     

蛋白质晶体的优化生长

蛋白质晶体的优化生长是获得高质量蛋白质晶体, 进而得到高精度晶体结构的有效途径.针对不同的晶体生长方法,已尝试了不同的优化手段,这对改善某些蛋白质晶体的质量显示了明显的成效.然而,鉴于蛋白质晶体生长的多样性与复杂性,这些方面均未发展成为实用的技术.文章综述了这类研究进展,分析了各手段的利弊,并指出了应着重解决的问题.     

SOn-column Protein Refolding for Crystallization

One major bottleneck in protein production in Escherichia coli for structural genomics projects is the formation of insoluble protein aggregates (inclusion bodies). The efficient refolding of proteins from inclusion bodies is becoming an important tool that can provide soluble native proteins for structural and functional studies. Here we report an on-column refolding method established at the Berkeley Structural Genomics Center (BSGC). Our method is a combination of an ‘artificial chaperone-assisted refolding’ method previously proposed and affinity chromatography to take advantage of a chromatographic step: less time-consuming, no filtration or concentration, with the additional benefit of protein purification. It can be easily automated and formatted for high-throughput process.     

Protein Crystallization for X-ray Crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.Download video file.^{(59M, mov)}     

Statistical Analysis of Crystallization Database Links Protein Physico-Chemical Features with Crystallization Mechanisms

X-ray crystallography is the predominant method for obtaining atomic-scale information about biological macromolecules. Despite the success of the technique, obtaining well diffracting crystals still critically limits going from protein to structure. In practice, the crystallization process proceeds through knowledge-informed empiricism. Better physico-chemical understanding remains elusive because of the large number of variables involved, hence little guidance is available to systematically identify solution conditions that promote crystallization. To help determine relationships between macromolecular properties and their crystallization propensity, we have trained statistical models on samples for 182 proteins supplied by the Northeast Structural Genomics consortium. Gaussian processes, which capture trends beyond the reach of linear statistical models, distinguish between two main physico-chemical mechanisms driving crystallization. One is characterized by low levels of side chain entropy and has been extensively reported in the literature. The other identifies specific electrostatic interactions not previously described in the crystallization context. Because evidence for two distinct mechanisms can be gleaned both from crystal contacts and from solution conditions leading to successful crystallization, the model offers future avenues for optimizing crystallization screens based on partial structural information. The availability of crystallization data coupled with structural outcomes analyzed through state-of-the-art statistical models may thus guide macromolecular crystallization toward a more rational basis.     

Spectroscopic Imaging of Protein Crystals in Crystallization Drops

Automatic imaging and scoring of crystallization drops is an essential step in high-throughput crystallography. Presently, white-light images of crystallization drops are acquired robotically and the images are analyzed and scored using pattern recognition algorithms. However, the scoring part remains unreliable as crystals and microcrystals are not always recognized by existing feature-extraction and recognition algorithms. We propose a fundamental shift in crystal monitoring through spectroscopic imaging of crystallization drops. This method converts the problem of automatic crystal detection from one of pattern recognition into one of intensity (concentration) analysis. The latter can be more robust and reliable.     

The Facts and Fancy of Microgravity Protein Crystallization

In vivo molecular imaging enables non-invasive visualization of biological processes within living subjects, and holds great promise for diagnosis and monitoring of disease. The ability to create new agents that bind to molecular targets and deliver imaging probes to desired locations in the body is critically important to further advance this field. To address this need, phage display, an established technology for the discovery and development of novel binding agents, is increasingly becoming a key component of many molecular imaging research programs. This review discusses the expanding role played by phage display in the field of molecular imaging with a focus on in vivo applications. Furthermore, new methodological advances in phage display that can be directly applied to the discovery and development of molecular imaging agents are described. Various phage library selection strategies are summarized and compared, including selections against purified target, intact cells, and ex vivo tissue, plus in vivo homing strategies. An outline of the process for converting polypeptides obtained from phage display library selections into successful in vivo imaging agents is provided, including strategies to optimize in vivo performance. Additionally, the use selections are performed against pre-defined targets, the use of cell lines, tissue, and in vivo homing selections have also been valuable. These latter strategies avoid the need to identify a specific target at the outset, allow library selections under conditions potentially more relevant to a clinical setting, and can lead to the discovery of unanticipated and interesting targets. The full potential of phage display is far from being completely explored; many library formats and selection strategies have not been fully exploited for the production of molecular imaging agents. The successful and rapid translation of phage-derived molecular imaging agents into the clinic remains a challenge, but new methods and tools are becoming available for optimizing in vivo performance. In conclusion, phage display will continue to be a significant driving force and a key player in enabling in vivo molecular imaging to deliver on its promise for both basic science and clinical applications.