Name: high-throughput crystallization of membrane proteins using the lipidic bicelle method
Uploaded: 2012-01-09T13:00:00
Description: Watch this Scientific Journal Video about High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method at JoVE.com

我们要感谢博士。詹姆斯Bowie和萨利姆Faham全线Bicelle方法和有益的讨论特拉维夫拉巴斯博士提供的技术专长和指导。我们承认实验支持乐杜。 Rachna Ujwal MemX生物科技有限责任公司，但是，不支持这项工作的财务利益。这项工作是支持部分由美国国立卫生研究院（RO1 GM078844）资助。

全线Bicelle基础的结晶，是由四个基本步骤（图2）：我）准备一个全线Bicelle形成脂质：两亲分子的混合物;二）进入全线Bicelle介质的纯化蛋白质的结合;三）结晶试验（手动或自动机械）;及iv）可视化，晶体的提取和冻结。下面详细介绍这些步骤 1。准备的Bicelles Bicelles可以形成：两亲组合在各种脂质的浓度范围。因此，基于以往的成功条件，初步构成建议?...

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	<li>Landau, E. M., Rosenbusch, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipidic+cubic+phases:+A+novel+concept+for+the+crystallization+of+membrane+proteins.">Lipidic cubic phases: A novel concept for the crystallization of membrane proteins.</a> Proceedings of the National Academy of Sciences. 93, 14532-14535 (1996).</li><li>Caffrey, M., Lyons, J., Smyth, T., Hart, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+4+Monoacylglycerols:+The+Workhorse+Lipids+for+Crystallizing+Membrane+Proteins+in+Mesophases.">Chapter 4 Monoacylglycerols: The Workhorse Lipids for Crystallizing Membrane Proteins in Mesophases.</a> Current Topics in Membranes. 63, 83-108 (2009).</li><li>Nollert, P., Landau, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymic+release+of+crystals+from+lipidic+cubic+phases.">Enzymic release of crystals from lipidic cubic phases.</a> Biochem. Soc. Trans. 26, 709-713 (1998).</li><li>Cheng, A., Hummel, B., Qiu, H., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+mechanical+mixer+for+small+viscous+lipid-containing+samples.">A simple mechanical mixer for small viscous lipid-containing samples.</a> Chem. Phys. Lipids. 95, 11-21 (1998).</li><li>Faham, S., Bowie, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bicelle+crystallization:+a+new+method+for+crystallizing+membrane+proteins+yields+a+monomeric+bacteriorhodopsin+structure.">Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure.</a> J. Mol. Biol. 316, 1-6 (2002).</li><li>Faham, S., Ujwal, R., Abramson, J., Bowie, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+5+Practical+Aspects+of+Membrane+Proteins+Crystallization+in+Bicelles.">Chapter 5 Practical Aspects of Membrane Proteins Crystallization in Bicelles.</a> Current Topics in Membranes. 63, 109-125 (2009).</li><li>Faham, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+of+bacteriorhodopsin+from+bicelle+formulations+at+room+temperature.">Crystallization of bacteriorhodopsin from bicelle formulations at room temperature.</a> Protein Science. 14, 836-840 (2005).</li><li>Luecke, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallographic+structure+of+xanthorhodopsin,+the+light-driven+proton+pump+with+a+dual+chromophore.">Crystallographic structure of xanthorhodopsin, the light-driven proton pump with a dual chromophore.</a> Proceedings of the National Academy of Sciences. 105, 16561-16565 (2008).</li><li>Ujwal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crystal+structure+of+mouse+VDAC1+at+2.3+&#197;+resolution+reveals+mechanistic+insights+into+metabolite+gating.">The crystal structure of mouse VDAC1 at 2.3 &#197; resolution reveals mechanistic insights into metabolite gating.</a> Proceedings of the National Academy of Sciences. 105, 17742-17747 (2008).</li><li>Vinothkumar, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+rhomboid+protease+in+a+lipid+environment.">Structure of rhomboid protease in a lipid environment.</a> J. Mol. Biol. 407, 232-247 (2011).</li><li>Rasmussen, S. G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+human+[bgr]2+adrenergic+G-protein-coupled+receptor.">Crystal structure of the human [bgr]2 adrenergic G-protein-coupled receptor.</a> Nature. 450, 383-387 (2007).</li><li>Prosser, R. S., Hwang, J. S., Vold, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetically+aligned+phospholipid+bilayers+with+positive+ordering:+a+new+model+membrane+system.">Magnetically aligned phospholipid bilayers with positive ordering: a new model membrane system.</a> Biophys. J. 74, 2405-2418 (1998).</li></ol>

Bicelles是一个独特的，提供原生双层式的环境，而表现犹如洗涤剂溶解油脂媒体。此属性，给出了bicelles一个明显的优势超过其他脂质体的结晶方法，因为没有学习曲线，这种技术所需的专用设备。一旦bicelles，无论是商业或准备在实验室中，他们可以直接与纯化的蛋白质，从这个角度混合结晶试验几乎完全与标准洗衣粉的协议进行的。此外，bicelles提供了包括与其他技术相比，延长贮存期，蛋白?...

high-throughput crystallization of membrane proteins using the lipidic bicelle method

在许多生理过程，如抽水整个否则周围所有的细胞和细胞器的防渗膜双层的具体分子膜蛋白（MPS）发挥了关键作用。 MPS导致许多人类的疾病和失调的功能发生变化，因此，其结构复杂的认识，仍然是生物学研究的重要目标。然而，国会议员的决心结构仍然是一个重大的挑战往往来自其疏水性所引起的。 国会议员的双层内嵌入大量的疏水区域。经常使用的洗涤剂这些蛋白质溶解产生一种蛋白质，然后可以在一个类似的方式为可溶性蛋白操纵洗涤剂胶束双层。传统上，进行结晶试验使用一种蛋白质清洁剂的混合物，但他们往往抵制结晶或生产质量较差的晶体。出现这些问题，因洗涤剂无法充分模仿双层导致稳定性差和异质性。此外，洗涤剂盾牌疏水表面的MP减少晶体接触的表面积。来规避这些弊端国会议员可结晶油脂的媒体，更加紧密地模拟其内源性环境，并在最近成为了MP的结晶新技术。 油脂立方米阶段（LCP）是一个立体的脂质双层渗透 1水通道的互联系统。虽然monoolein是选择脂质，如monopalmitolein和monovaccenin相关的血脂也被用来做LCP 的 2 。国会议员被纳入LCP的，他们在三个层面和饲料晶核弥漫。 LCP的一个巨大优势是，该蛋白在更自然的环境中仍然存在，但该方法具有技术的弊端，包括高VISCosity（需要专门的仪器），并在晶体中的可视化和操纵3,4的困难。由于这些技术上的困难，我们利用另一个结晶bicelles 5,6（ 图1）油脂中。 Bicelles是与两亲（CHAPSO）或短链的脂质（DHPC）一个混合的磷脂酰胆碱脂质（DMPC）所形成的脂质/两亲分子的混合物。在每个全线Bicelle光盘，脂质分子生成双层而行两亲分子的非极性边缘的双层和洗涤剂提供有益的特性。重要的是，其转变温度以下，蛋白全线Bicelle混合物粘度降低和控制在一个类似的方式溶解洗涤剂，国会议员，bicelles兼容结晶机器人。 Bicelles已成功地用于结晶几个膜蛋白5,7-11（见表1）。这种增长的集合蛋白质演示的bicelles结晶从原核和真核来源的α-螺旋和β表国会议员的多功能性。由于这些成功和高通量实施简单，bicelles应该是每一个膜蛋白晶体学的武器库中的一部分。在这个视频中，我们描述全线Bicelle方法，并提供一个一步一步的协议，使用标准的机器人纯化国会议员成立的高通量结晶试验。

Watch this Scientific Journal Video about High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method at JoVE.com

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Bicelles脂质/两亲分子的混合物保持在脂质双层膜蛋白（MPS），但有独特的相行为，有利于高通量筛选的结晶机器人。这项技术已成功地生产从原核和真核来源的高分辨率结构。本视频介绍了生成油脂全线Bicelle混合物，纳入全线Bicelle混合物的国会议员，成立从中长期的结晶试验（手动以及作为机器人）和收割晶体的协议。

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Research

JoVE Journal

Biology

A high-throughput method to globally study the organelle morphology in S. cerevisiae

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

科研

生物学

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

评估电子晶体结构生物学研究的小膜蛋白的二维结晶试验

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Crystallization of Membrane Proteins in Lipidic Mesophases

在油脂介孔膜蛋白的结晶

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

使用机器人在油脂中间相高通量膜蛋白的结晶

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

在油脂中间相结构的测定生物大分子晶体生长的膜蛋白晶体收获和低温冷

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

使用高通量形状的RNA二级结构预测

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

检测miRNA靶在高通量的使用3&#39;LIFE分析

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...