这项工作是在部分资助，由美国国立卫生研究院拨款GM073197和RR025336。

一个典型的轮廓观结晶实验是在图1,2所示。预结晶LCP - FRAP实验是可选的;但是，它们可以显着加快搜索初始结晶条件的过程中特别是在困难的膜蛋白3情况下，。 1。在LCP蛋白质重构<ol><li>净化膜蛋白的洗涤剂溶液中的兴趣和集中的蛋白质/去污剂复合物〜10 - 20毫克/毫升，注意不要过度浓缩洗衣粉 1,4 。 </li><li>转移一个LCP主机脂质（通常monoolein）或脂质混合物为1.5毫升塑料管和孵育40〜25毫克° C直到几分钟脂融化。 </li><li>将100μL气密注射器注射器耦合。 </li><li>将熔融使用音量可调移液器脂质注射器。记录在注射器中的脂质量。 </li><li>一种蛋白质的解决方案，以脂质比2 / 3 V /蛋白质溶液装入另外100μL注射器诉</li><li>通过注射器耦合连接两个注射器。 </li><li>推注射器活塞交替脂质和蛋白质，通过耦合器内针来回移动，直到脂质中间相变得均匀。 LCP的形式自发地机械搅拌后，在LCP的脂质双层重组蛋白质。 LCP的形成，可核实birefringency缺乏透明和凝胶体状的一致性和跨偏振片配备显微镜下观察时，或者，如果可能的话，用小角X - 射线衍射1。 </li></ol> 2。 LCP - FRAP预结晶实验 LCP - FRAP分析是用来衡量膜蛋白的扩散性质在LCP重组的各种筛选条件3。在LCP膜蛋白的远距离扩散成功的结晶，是必不可少的;然而，LCP的微观结构，制约了大量的蛋白质或低聚蛋白质总量的扩散。 观结晶实验失败的一个常见的原因是一个快速的蛋白质聚集，导致亏损扩散。它已被证明的一种蛋白质的聚集行为取决于特定的蛋白质的构造，主机脂质和 3的筛选解决方案的组成。 <ol><li>标签用荧光染料（Cy3标记或类似）的100 / 1〜的蛋白/染料比率的蛋白质，除去未反应的染料和集中的蛋白质〜1毫克/毫升。标签无论是免费胺或免费硫醇。当标签自由胺，pH值7和7.5之间为主标签免费的N -末端。请注意氨基酸的标签也可以标签纯化蛋白质 2,3血脂。 </li><li>重组在LCP在第1条所述的标记蛋白）。 </li><li>设置检测板，在第3节所述）使用LCP - FRAP筛选解决方案，而不是结晶屏幕2。 </li><li>孵育板块在20 ° C黑暗的至少12小时，以达到平衡状态环境中。 </li><li> LCP的FRAP站和第一次使用10倍的目标，重点放在板块之一。 </li><li>掌握5​​荧光图像，捕捉到最初的预漂白状态。 </li><li>触发激光。激光功率和脉冲数应调整漂白〜30 - 70％在漂白当场中的标记蛋白质。 </li><li>激光触发后，立即开始录制〜200幅图像序列快速漂白后以最快的速率。 </li><li>跟随一个缓慢的漂白后〜50张图片，选择1-20小号的图像之间的延迟，这取决于对蛋白质的扩散速率的序列记录。 </li><li>内部集成所有帧中的漂白剂点的强度和正确的漂白和光强度的波动，收购期间内漂白现货除以参考现货以外的激光漂白面积的平均强度，强度。 </li><li>规范化纠正的力度，使预漂白的强度等于1，初始漂白强度等于0。 </li><li>适合的归一化强度与时间，F（T）曲线，使用下列公式5： F（T）= M X EXP（- 2T / T）×（我0（2T / T）+ 1（2T / T）），（Eq.1） 其中M是扩散分子的移动部分，T为特征的扩散时间 ，t是每个记录帧的实时，0 和 1 0 日和1日为了修正贝塞尔函数。 </li><li>计算的扩散系数，D为： D = R 2 / 4T（Eq.2） 其中，R为半径的漂白现货。 </li><li>移动吨Ø未来以及重复步骤2.5） - 2.13）。 </li><li>比较移动的分数和不同的筛选条件获得的扩散系数。屏幕的基础上，促进蛋白质的扩散和不包括未观察到的蛋白扩散条件的组件设计新的结晶。如果蛋白质没有在任何筛选条件弥漫，考虑扩大筛选空间，或试图建构一个新的蛋白质。 </li></ol> 3。设置LCP的结晶试验<ol><li>重组蛋白在LCP中所述第1条）。 </li><li>转移到10μL气密注射器连接到一个重复的注射器饮水机蛋白质载货的LCP。 </li><li>将一个可拆卸的短针（表26，10毫米长）10μL注射器。 </li><li>免除表面上形成一个2x2的正方形的四个相邻井200 NL LCP的推注。 </li><li>覆盖每个LCP的推注1μL相应的结晶屏解决方案。 </li><li>第一个18平方毫米的玻璃盖玻片加载井。应用盖玻片温和的压力，密封井。 </li><li>与下一组的4口井重复步骤3.4）-3.6），直到充满整个板块。 </li><li>孵育板在一个恒定的温度，定期检查晶体的形成和生长。 </li></ol> 4。收获来自LCP的晶体<ol><li>将一盘线性旋转偏光器和分析仪的配备，可变变焦体视显微镜下的蛋白质晶体。 </li><li>重点以及使用一个低功率放大，使整个以及视野内放置的利益。 </li><li>分数在四个方内部使用的陶瓷毛细管切割石材的尖角以及边界招的玻璃盖玻片。 </li><li>具有很强的尖锐点镊子取得周边各地新闻传播盖玻片玻璃的厚度划痕。 </li><li>冲床的得分广场的对面角落的两个小孔。 </li><li>通过一个孔注入数微升沉淀的解决方案，在随后的步骤，以减少脱水。 </li><li>使用角度尖锐的针探头打破玻璃沿着一个或双方自由切出的平方。 </li><li>小心地抬起立方相丸玻璃广场观看。如果丸是坚持以盖玻片，然后翻转井底超过玻璃广场和地点。 </li><li>沉淀的解决方案中添加额外的几微升，辅以一个冷冻保护剂，如果必要的话，暴露在良好的立方相丸上。 </li><li>增加显微镜的放大倍率，并专注于一个晶体。 </li><li>调整角度之间的偏光器和分析仪增加双折射晶体和背景之间的对比度，同时保持足够的光线，看到收获的循环。 </li><li>选择匹配晶体的大小与一个直径MiTeGen MicroMount，然后收获从LCP的晶体直接舀MicroMount。 </li><li>闪存冻结在液态氮的收获水晶MicroMount，并运到同步辐射光源光束线为X射线数据收集 6 。 </li></ol> 5。代表性的成果： 一个精心设计的人β2肾上腺素G蛋白偶联受体（β2 AR - T4L）表示在杆状病毒感染Sf9细胞和dodecylmaltoside（DDM）/胆固醇琥珀酸单酯（CHS）的洗衣粉溶液局部反相激动剂carazolol 7纯化。用Cy3 NHS酯蛋白标记和使用LCP - FRAP预结晶实验（图2）。粗格栅基于LCP - FRAP分析结果选定的几个条件的屏幕产生了最初的水晶般的点击（图3）。沉淀条件的进一步优化产生衍射的晶体（图4）。 <img src="/files/ftp_upload/2501/2501fig1.jpg" alt="图1" /> 图1一个典型的LCP结晶实验流程图。在灰色的方块中的步骤不是在当前的协议。 <img src="/files/ftp_upload/2501/2501fig2.jpg" alt="图2" /> 图2。LCP - FRAP测定β2 monoolein基于LCP的AR-T4L/carazolol。一）一个LCP - FRAP实验结果进行自动高通量模式，在每个样品的96孔板漂白和荧光恢复后30分钟潜伏期测量。所获得的荧光回收率，代表每个样本中的移动部分，绘制所有96个样品。筛选解决方案中包含的0.1 M Tris pH值8，30％V / V聚乙二醇400结合两个不同浓度的48个不同的盐。二）数代表的荧光恢复配置文件resentative条件。实线曲线代表由方程的拟合。 1，移动分数和扩散系数的确定使用式。 1和2。不到10％的样品含有钠氯的快速恢复是由于荧光标记的脂质与蛋白质共同纯化。 <img src="/files/ftp_upload/2501/2501fig3.jpg" alt="图3" /> 图3β2 AR-T4L/carazolol初步晶体点击获得一个粗网格筛选围绕确定的最有前途的条件的LCP - FRAP，含有钠硫酸盐（A组）和Na甲酸（B组）。用Cy3 NHS酯蛋白标记和荧光图像是使用激发波长543纳米和605纳米的排放。 <img src="/files/ftp_upload/2501/2501fig4.jpg" alt="图4" /> 图4β2 AR-T4L/carazolol优化晶体。晶体的图像成长中存在的钠硫酸盐（小组A和B）和K甲酸（板C和D）在明模式（面板A和C），并采用交叉偏光片（板B和D）。

<ol>
	<li>Caffrey, M., Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallizing+membrane+proteins+using+lipidic+mesophases.">Crystallizing membrane proteins using lipidic mesophases.</a> Nat. Protoc. 4, 706-731 (2009).</li><li>Cherezov, V., Abola, E., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+the+structure+determination+of+GPCRs,+a+membrane+protein+family+with+high+potential+as+pharmaceutical+targets.">Recent progress in the structure determination of GPCRs, a membrane protein family with high potential as pharmaceutical targets.</a> Methods Mol. Biol. 654, 141-170 (2010).</li><li>Cherezov, V., Liu, J., Hanson, M. A., Griffith, M. T., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LCP-FRAP+assay+for+pre-screening+membrane+proteins+for+in+meso+crystallization.">LCP-FRAP assay for pre-screening membrane proteins for in meso crystallization.</a> J. Cryst. Growth Design. 8, 4307-4315 (2008).</li><li>Misquitta, Y., 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=Detergents+destabilize+the+cubic+phase+of+monoolein:+implications+for+membrane+protein+crystallization.">Detergents destabilize the cubic phase of monoolein: implications for membrane protein crystallization.</a> Biophys. J. 79, 394-405 (2003).</li><li>Soumpasis, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+analysis+of+fluorescence+photobleaching+recovery+experiments.">Theoretical analysis of fluorescence photobleaching recovery experiments.</a> Biophys. J. 41, 95-97 (1983).</li><li>Cherezov, V., Hanson, M. A., Griffith, M. T., Hilgart, M. C., Sanishvili, R., Nagarajan, V., Stepanov, S., Fischetti, R. F., Kuhn, P., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rastering+strategy+for+screening+and+centering+of+microcrystal+samples+of+human+membrane+proteins+with+a+sub-10+micrometer+size+X-ray+synchrotron+beam.">Rastering strategy for screening and centering of microcrystal samples of human membrane proteins with a sub-10 micrometer size X-ray synchrotron beam.</a> J. R. Soc. Interface. 6, S587-S597 (2009).</li><li>Cherezov, V., Rosenbaum, D. M., Hanson, M. A., Rasmussen, S. G., Thian, F. S., Kobilka, T. S., Choi, H. J., Kuhn, P., Weis, W. I., Kobilka, B. K., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+crystal+structure+of+an+engineered+human+beta2-adrenergic+G+protein-coupled+receptor.">High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor.</a> Science. 318, 1258-1265 (2007).</li><li>Cherezov, V., Peddi, A., Muthusubramaniam, L., Zheng, Y. F., 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+robotic+system+for+crystallizing+membrane+and+soluble+proteins+in+lipidic+mesophases.">A robotic system for crystallizing membrane and soluble proteins in lipidic mesophases.</a> Acta Crystallogr. D. 60, 1795-1807 (2004).</li><li>Kissick, D. J., Gualtieri, E. J., Simpson, G. J., Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+optical+imaging+of+integral+membrane+protein+crystals+in+lipidic+mesophases.">Nonlinear optical imaging of integral membrane protein crystals in lipidic mesophases.</a> Anal. Chem. 82, 491-497 (2010).</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>Cherezov, V., 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+and+inexpensive+nanoliter-volume+dispenser+for+highly+viscous+materials+used+in+membrane+protein+crystallization.">A simple and inexpensive nanoliter-volume dispenser for highly viscous materials used in membrane protein crystallization.</a> J. Appl. Cryst. 38, 398-400 (2005).</li><li>Cherezov, V., 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=Nano-volume+plates+with+excellent+optical+properties+for+fast,+inexpensive+crystallization+screening+of+membrane+proteins.">Nano-volume plates with excellent optical properties for fast, inexpensive crystallization screening of membrane proteins.</a> J. Appl. Cryst. 36, 1372-1377 (2003).</li><li>Cherezov, V., Fersi, 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=Crystallization+screens:+compatibility+with+the+lipidic+cubic+phase+for+in+meso+crystallization+of+membrane+proteins.">Crystallization screens: compatibility with the lipidic cubic phase for in meso crystallization of membrane proteins.</a> Biophys J. 81, 225-242 (2001).</li></ol>

该协议提供了一个基本的视觉指导，参与表演观的结晶实验的主要步骤。深度有关这些协议的细节，强调可能存在的缺陷，缺陷或替代路线可在别处 1,2 。可选LCP - FRAP分析可以帮助在早期阶段，选择最有前途的蛋白质构造，LCP主机脂质和脂质添加剂，以及限制可能的沉淀和缓冲条件 3范围。一旦发现一个初步的结晶命中，应该优化，以​​获得更好的质量的晶体。 观结晶条件的优化是增加 1 LCP的组成相关的额外的参数优化与可溶性蛋白的条件基本类似。膜蛋白油脂中间相生长的晶体，通常是规模较小比洗衣粉溶液中获得的晶体，但更有序，因此，强烈受益从使用微聚焦光束线在现代同步加速器源6。 观的结晶，包括设立结晶或检测板，进行LCP - FRAP分析和检测晶体， 相关的程序，有许多已半自动或全自动化1,2,8,9，允许大范围的条件筛选而消耗少量的蛋白质和脂质。另一方面，在LCP和蛋白质晶体收获reconstitutions保持手册和操作比较繁琐，因此，有需要改进的地方。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>100 &mu;L gas-tight syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>7656-01</td>
<td>2 syringes are required</td>
</tr>
<tr>
<td>10 &mu;L gas-tight syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>7653-01</td>
<td>&nbsp;</td>
<tr>
<td>Syringe coupler</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>7770-02 30902</td>
<td>The coupler can be made using available parts as described in refs. 10</td>
</tr>
<tr>
<td>Repetitive syringe dispenser</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>83700</td>
<td>The repetitive syringe dispenser can be modified to reduce dispensing volume by ~3 times11</td>
</tr>
<tr>
<td>Short (0.375&#x201D;) flat-tipped removable needle (point style 3, gauge 26)</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>7804-03</td>
<td>&nbsp;</td>
<tr>
<td>96-well glass sandwich plate</td>
<td>&nbsp;</td>
<td>Marienfeld</td>
<td>08 900 03</td>
<td>For manual operations it is more convenient to assemble the glass sandwich plate using a standard microscope glass slides and a double sticky tape with punched holes1,2,12.</td>
</tr>
<tr>
<td>Glass cover slip</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>63787-01</td>
<td>&nbsp;</td>
<tr>
<td>monoolein</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M7765</td>
<td>&nbsp;</td>
<tr>
<td>Crystallization screens</td>
<td>&nbsp;</td>
<td>Hampton Research, Molecular Dimensions, Emerald Biosystems, Jena Bioscience</td>
<td>&nbsp;</td>
<td>Most of available commercial screens can be used for initial screening. Conditions that consistently disrupt LCP can be diluted 2x for better compatibility13.</td>
</tr>
<tr>
<td>Capillary cutting stone</td>
<td>&nbsp;</td>
<td>Hampton Research</td>
<td>HR4-334</td>
<td>&nbsp;</td>
<tr>
<td>Fine point tweezers</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>510</td>
<td>&nbsp;</td>
<tr>
<td>Angled sharp probe</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>13650</td>
<td>&nbsp;</td>
<tr>
<td>MicroMounts</td>
<td>&nbsp;</td>
<td>MiTeGen</td>
<td>M1-Lxx-xx</td>
<td>Select MicroMount diameter to match the crystal size</td>
</tr>		</tbody>
		</table>
		</div>

crystallization of membrane proteins in lipidic mesophases

膜蛋白相关的信号传导，运输和能源的转换，正因为如此，在众多的故障和疾病有牵连的活细胞执行的关键功能。然而，强烈的膜蛋白的结构和功能的认识是落后可溶性伙伴，主要是由于与衍射晶体的溶解和代困难。油脂介孔结晶（又称观或LCP的结晶）是一种很有前途的技术成功地应用于获得微生物视紫质，光合蛋白，外膜的beta桶和G蛋白偶联受体的高分辨率结构。在中观的结晶，需要一个类似母语的膜环境的优势，通常会产生较低的溶剂含量和更好的排序相比，洗涤剂解决方案的传统结晶的晶体。该方法并不难，但需要了解脂质的相行为和处理粘性中间相材料的做法。在这里，我们展示了一个简单有效的方法，使LCP和改组使用注射器搅拌机，配药到一个试验或结晶板LCP的纳升的部分，进行预结晶实验，并从收割晶体的LCP脂质双层膜蛋白LCP的矩阵。这些协议提供了一个在中间的结晶试验临近的基本指导，但是，任何结晶实验，广泛的筛选和优化，并取得成功的结果并不一定保证。

Watch this Scientific Journal Video about Crystallization of Membrane Proteins in Lipidic Mesophases at JoVE.com

Crystallization of Membrane Proteins in Lipidic Mesophases

协议描述获得一个膜蛋白，重组蛋白在油脂立方米阶段（LCP）起的晶体衍射的质量必不可少的步骤，寻找LCP - FRAP预结晶实验的初始条件，设立LCP的结晶试验和收获晶体。

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 ...

crystallization-of-membrane-proteins-in-lipidic-mesophases

Research

JoVE Journal

Biology

31.2K 次观看.  The Scripps Research Institute. 协议描述获得一个膜蛋白，重组蛋白在油脂立方米阶段（LCP）起的晶体衍射的质量必不可少的步骤，寻找LCP - FRAP预结晶实验的初始条件，设立LCP的结晶试验和收获晶体。

视频: Crystallization of Membrane Proteins in Lipidic Mesophases

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in 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 ...

科研

生物学

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

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

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 ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

研究膜蛋白的构象动力学原位网站的荧光标记

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

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

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

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 ...

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

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

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

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray 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 ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

外层膜蛋白的分离线粒体的流式细胞仪免疫检测

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

微灌技术在调查微血管渗透性的大鼠肠系膜条例

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...