JoVE Science Education
Biochemistry
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JoVE Science Education Biochemistry
Protein Crystallization
  • 00:00Overview
  • 00:50Principles of Protein Crystallization
  • 03:16Protocol for Protein Expression, Crystallization, and X-Ray Diffraction
  • 05:15Applications
  • 07:20Summary

蛋白质结晶

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Overview

蛋白质结晶,获得一个坚实的格子的生物分子,阐明了蛋白质的结构,并使蛋白功能的研究。结晶涉及干燥组合的许多因素,包括 ph 值、 温度、 离子强度、 蛋白质浓度下的纯化的蛋白。一旦获得晶体,可以通过 x 射线衍射和电子密度模型计算阐明蛋白质结构。

本视频介绍了蛋白质结晶和显示的一般程序。的过程中,涵盖了蛋白的表达和纯化、 结晶和 x-射线衍射。蛋白质结晶的应用包括在硅片药物设计、 绑定现场测定和膜蛋白结构分析。

蛋白质结晶是获得格构式固体形式的一种蛋白质的过程。这些水晶是结构生物学家,协助蛋白功能的研究尤为重要。其他技术,如质谱法或 SDS-PAGE,只能提供信息的一维结构的蛋白质。蛋白质结晶被辅的重组蛋白的表达和 x 射线衍射技术。本视频将显示在生化领域的蛋白质结晶,一般实验室程序,和几个及其应用原则。

在过程中所需的第一步是蛋白的获得毫克量的很纯,通常使用重组蛋白的表达。对应于感兴趣的蛋白质的基因表达载体,克隆并表达的蛋白融合到亲和标记,例如聚-组氨酸,协助亲和层析纯化。要了解更多信息,见此集合的视频上亲和层析。

纯化蛋白形成晶体是依赖于许多因素,包括 ph 值、 离子强度、 沉淀剂浓度和蛋白质、 温度和速率平衡的适当结合。使用的最常见方法是蒸气扩散,其中有两种类别: 挂滴和坐在下降。液滴含纯蛋白、 缓冲区和沉淀剂、 离子固体,束缚水分子,减少蛋白质的水供应和模仿蛋白质浓度越高,处于封闭微孔与储层更高浓度的相同的缓冲区和沉淀剂混合物。在开始时,蛋白质和沉淀剂的浓度是太低,不足以引起结晶。在实验过程中,水从液滴蒸发和收集在储层中;水在滴减少会导致系统变得饱和,和成核,其次是结晶,可以发生。水从液滴的净转移处于平衡状态,和系统维护过程完成之前。

若要可视化的三维结构,使用了 x 射线衍射。要获得晶体的 x 射线数据,它被放在单色 x 射线,在那里它被暴露在各个角度梁。每次曝光提供的图像,在每个地方是衍射 x 射线,从晶体产生、 由探测器注册。数据相结合,制作一个模型在晶体内部原子的排列。由此产生的晶体结构演示原子,典型分辨率为 2 埃的 3 维位置。

既然我们已经涵盖了蛋白质结晶的原则,让我们看看一个广义的协议。

开始执行程序将包含感兴趣的基因表达载体转化为细胞。孵育细胞和阶段中期日志,表达式由添加诱导剂,如 IPTG,触发基因的 mRNA 转录启动。后蛋白表达,原油材料悬浮在裂解缓冲液中,然后用离心法澄清。

阐明了裂解液然后装到镍列和应运而生标记蛋白将绑定到列,而所有其他生物分子都冲走了。

一旦获得了几毫克的纯蛋白质,它是准备由蒸气扩散的结晶。24 井挂/坐滴托盘充满不同浓度的氯化钠和钠醋酸缓冲溶液。坐滴方法,相同体积的蛋白质和储层的解决方案吸取到每口井,上方的架子上,然后托盘布满了透明胶带。托盘然后放在孵化室,并在井监视增长第二天,然后每隔几天。

一旦它获得了适当的水晶是准备 x 射线衍射分析。水晶被安装在测角仪定位在所选的方向位置的晶体。水晶被照明用的 x 射线在所有的角度,产生衍射图案单色光束。该软件将转换的二维图像,在不同的方向,对三维模型的测定原子在晶体中的位置晶体中的电子密度。

我们已经讨论了一种程序,让我们回顾一些有用的应用程序的蛋白质结晶和另一种结晶技术。

可用于蛋白质结晶硅药物设计中。流感病毒聚合酶碱性蛋白 2,其中已链接到病毒感染在哺乳动物中,三维结构测定结晶和 x 射线衍射。潜在的蛋白结合位点的可视化,并使用对接程序,设计了一个三维分子,会插入在蛋白质中的裂缝。

合作的结晶的蛋白质-DNA 复合物也是一个有用的技术。DNA 结合蛋白调节种类繁多的生物功能,如转录和 DNA 聚合和 DNA 修复;和这些配合物的晶体结构可以洞察蛋白质的功能、 机制及特异性相互作用的性质。大肠杆菌蛋白 SeqA,负性调节因子的 DNA 复制,是与半甲基化 DNA 位共同结晶。

一体式膜蛋白质如 G 蛋白偶联受体或 GCPRs,很难结晶由于其数量有限的极性表面积供形成晶体的晶格接触,导致融合蛋白辅助蛋白质结晶的发展。编码 β 2 肾上腺素能受体、 GCPR 和溶菌酶基因被插入表达载体。Β2AR 溶菌酶融合蛋白质结晶过程被达到由于增加胞外亲水表面自然疏水 β2AR,提供的溶菌酶,成型包装的相互作用在晶体点阵的必要条件。

你刚看了朱庇特的视频对蛋白质结晶。这段视频描述其原则、 广义的议定书 》,和一些及其在生物医学领域的应用。谢谢观赏 !

Procedure

Disclosures

No conflicts of interest declared.

Transcript

Protein crystallization is the process of obtaining a latticed solid form of a protein. These crystals are especially valuable to structural biologists, assisting in the study of protein function. Other techniques, such as mass spec or SDS-PAGE, can only provide information on the one-dimensional structure of proteins. Protein crystallization is complemented by the techniques of recombinant protein expression and x-ray diffraction. This video will show the principles of protein crystallization, a general laboratory procedure, and several of its applications in the biochemical field.

The first step required in the process is to obtain milligram quantities of very pure protein, typically using recombinant protein expression. The gene corresponding to the protein of interest is cloned into an expression vector, and the expressed protein is fused to an affinity tag, such as poly-histidine, to assist in the purification by affinity chromatography. To learn more, see this collection’s video on affinity chromatography.

Formation of the purified protein into crystals is dependent on the proper combination of many factors, including pH, ionic strength, concentrations of precipitant and protein, temperature, and rate of equilibration. The most common method used is vapor diffusion, of which there are two categories: hanging drop and sitting drop. A droplet containing pure protein, buffer, and precipitant, which is an ionic solid that binds water molecules, reducing water availability for the protein and mimicking higher protein concentration, is in an enclosed microwell with a reservoir with a more highly concentrated mixture of the same buffer and precipitant. At the beginning, the concentrations of protein and precipitant are too low to cause crystallization. During the course of the experiment, water vaporizes from the droplet and collects in the reservoir; a decrease in the amount of water in the droplet causes the system to become supersaturated, and nucleation, followed by crystallization, can occur. The net transfer of water from the droplet is in equilibrium, and the system is maintained until the process is complete.

To visualize the 3D structure, x-ray diffraction is used. To obtain x-ray data from a crystal, it is placed in a monochromatic x-ray beam, where it is exposed to the beam at all angles. Each exposure provides an image, where each spot is a diffracted x-ray, which emerges from the crystal and is registered by a detector. The data are combined to produce a model of the arrangement of atoms within the crystal. The resulting crystal structure demonstrates the 3-dimensional placement of the atoms, with a typical resolution of 2 angstroms.

Now that we have covered the principles of protein crystallization, let us look at a generalized protocol.

To begin the procedure an expression vector containing the gene of interest is transformed into cells. The cells are incubated and at mid-log phase, expression is initiated by adding an inducer, such as IPTG, which triggers transcription of the gene’s mRNA. After protein expression, the crude material is suspended in lysis buffer, and then clarified by centrifugation.

The clarified lysate is then loaded onto a nickel column, and the polyhistidine-tagged protein binds to the column while all other biomolecules are washed away.

Once several milligrams of pure protein have been obtained, it is ready for crystallization by vapor diffusion. A 24-well hanging/sitting drop tray is filled with varying concentrations of sodium chloride and sodium acetate buffer solutions. For the sitting drop method, equal volumes of protein and reservoir solution are pipetted onto the shelf above each well, and then the tray is covered with transparent tape. The tray is then placed in an incubation chamber, and the wells are monitored for growth the following day, then every few days.

Once a proper crystal has been obtained it is ready for x-ray diffraction analysis. The crystal is mounted on a goniometer to position the crystal at selected orientations. The crystal is illuminated with a monochromatic beam of x-rays at all angles, producing a diffraction pattern. The software converts the two-dimensional images, taken at different orientations, to a three-dimensional model of the density of electrons within the crystal by determining the positions of the atoms in the crystal.

Now that we have reviewed a procedure, let’s review some useful applications of protein crystallization, and another crystallization technique.

Protein crystallization may be used for in silico drug design. The three-dimensional structure of Influenza virus’s polymerase basic protein 2, which has been linked to viral infection in mammals, was determined by crystallization and x-ray diffraction. Potential binding sites in the protein are visualized, and with the use of a docking program, a three-dimensional molecule was designed that would insert into a cleft in the protein.

Co-crystallization of protein-DNA complexes is also a useful technique. DNA-binding proteins modulate a wide variety of biological functions such as transcription and DNA polymerization and DNA repair; and crystal structures of these complexes can provide insight into protein function, mechanism, and the nature of the specific interaction. The E. coli protein SeqA, a negative regulator of DNA replication, was co-crystallized with hemimethylated DNA.

Integral membrane proteins such as G-protein coupled receptors, or GCPRs, are difficult to crystallize due to their limited amount of polar surface area available for forming crystal lattice contacts, which has led to the development of fusion-protein-assisted protein crystallization. Genes encoding β2 adrenergic receptor, a GCPR, and a lysozyme were inserted into an expression vector. The crystallization of the β2AR- lysozyme fusion protein was achieved due to the increased extracellular hydrophilic surface over the naturally hydrophobic β2AR, provided by the lysozyme, necessary for forming packing interactions in the crystal lattice.

You’ve just watched JoVE’s video on protein crystallization. This video described its principles, a generalized protocol, and some its uses in the biomedical field. Thanks for watching!

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JoVE Science Education Database. JoVE Science Education. Protein Crystallization. JoVE, Cambridge, MA, (2023).

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