有很多人对这项工作作出了贡献和最卡弗里膜结构与功能的生物集团，过去和现在的成员。我们致以最热烈的感谢和赞赏。这项工作是支持的，一部分由爱尔兰科学基金会（07/IN.1/B1836），（GM75915）国家卫生研究院和利默里克大学的补助金。

在结构和功能的生物学领域的一个重要的重点是生物膜（图1）。膜，周围的细胞和亚细胞器出现时，只有两个脂质分子一个分子薄结构和与蛋白质云集。适用于脂质和蛋白质的结构和功能的利益。然而，本文的重点是限制膜蛋白。 寻求一个更好地了解如何在分子水平上的膜蛋白的功能是有两个原因。首先，是在知道他们是如何工作的智力满意。其次，通过了解蛋白质是如何工作的，总是能够解决应该发生故障或改善，甚至为特定的应用程序修改它的前景。药物设计是明显的这种类型的工作成果。辩别如何在分子水平上的一种膜蛋白的一种方法是，以确定其结构。这包括建立所有原子在三维空间的位置，或至少所有非氢原子，使蛋白质。我们为此目的使用的方法是大分子的X -射线结晶器（MX）。图2显示了一种膜蛋白的结构被确定使用MX的例子。衍射质量的蛋白质晶体是需要做的MX。 显然，使用大分子晶体结构的测定中涉及的许多步骤。图3说明了这一点。通常情况下，这些包括确定膜蛋白的目标，然后生产，提纯和结晶。使用一个家庭或一个同步辐射X射线源的晶体衍射测量上执行。高产与分子模型，然后将其安装的电子密度图的衍射数据处理。成品时，该模型，可以用于探索的蛋白质的作用机制和基于结构的药物设计。 本文的重点是显示我们如何产生所谓的中观方法使用脂质介孔的膜蛋白晶体衍射质量，的。最近进行的检讨的方法，其范围是在参考文献1（卡弗里，2009）。一步一步的协议，我们将按照这里的描述，在参考文献2（卡弗里和Cherezov，2009）。 总结参与设立一个中间膜蛋白结晶试验要求和时间步骤的流程图如图4所示。本文介绍了红色虚线线内的那些步骤。 第1部分:准备结晶板<ol><li>在工作​​台上的位置硅烷显微镜幻灯片。 </li><li>删除从一个带（市售，看到的特定试剂和设备下面的表）的穿孔双节棍间隔磁带表面的保护纸盖。 </li><li>胶带，粘面朝下放置，与表面的幻灯片接触。 </li><li>压力密封的磁带，幻灯片使用brayer或辊。可以放在铝箔之间的磁带和辊保护的水井的基础。 </li><li>从磁带中删除第二个纸盖，露出其上粘表面。 </li><li>的地方，个别硅烷盖玻片接近井快速高效的密封板，为尽快装载完成。 </li></ol>第2部分:准备脂质注射器<ol><li>脂质（常monoolein）放置在45 ° C 3分钟来渲染它熔融温度控制块。 </li><li>而脂质是融化准备在脂质蛋白混合步骤使用两个100μL气密汉密尔顿注射器。特氟隆卡环取下注射器将包含蛋白质溶液。将注射器将举行脂旁边（留在地方套圈）。 </li><li>耦合器的连接（请参阅表的特定试剂和设备下面，参考3（ 程等 。1998年））脂质注射器。用手拧紧耦合注射器，但不要过度拧紧。删除从每桶脂质注射器的柱塞。 </li><li>注意脂质必须熔化，然后再继续。设置移液器至30μL，慢慢约30μL熔融脂。慢慢地，你可以提供开放的照顾，不引入空气间隙的注射器结束熔融脂质。 </li><li>在与熔化的脂质接触，并慢慢地推进了每桶举行垂直视频显示注射器的柱塞，柱塞放入桶。空气可能无意中被困气泡上升的过程中被释放。 </li><li>仔细确定阅读的针筒上的标记，在注射器中的脂质量。 </li><li>滑动的FERRULE到从耦合器的扩展针。使用柱塞力了，慢慢地，轻轻地进入在耦合器的核心针桶和熔融脂质。 </li></ol>第3部分:准备蛋白质注射器<ol><li>纺14,000克蛋白质溶液应为5到10分钟，4 ° C到删除前设立的结晶试验的大聚合。从冰中取出的蛋白质溶液，并允许它在室温下平衡。 </li><li>计算蛋白质溶液的体积使用，形成了立方相或接近充分水化。对于monoolein在20℃，与水充分水化发生在5相图（图5），在接近40％的水（质量） 。 </li><li>随着一个25或50μL汉密尔顿注射器所需的蛋白质溶液体积。转移到聚四氟乙烯在每桶照顾，以避免气泡的蛋白质注射器的柱塞提示的解决方案。 </li><li>仔细撤回的针头和措施注射器中的蛋白质的解决方案的体积。它应该在上一步中提供的值相匹配。 </li><li>慢慢英寸了每桶蛋白质的解决方案，最终其开口端平齐。 </li><li>脂质注射器相连的耦合器的开口端拧入的蛋白质注射器。这是关键设备不能过分收紧。过度紧缩组装的搅拌装置，在这个阶段会损坏密封垫圈，造成泄漏。在紧缩也会导致漏水。 </li></ol>第4部分:混合蛋白质溶液和脂质:使中间相<ol><li>效果混合，混合机组组装到了极限的蛋白质方提前柱塞，用拇指或食指驾驶的蛋白质注射器的蛋白质溶液，通过耦合器和成脂质注射器。 </li><li>柱塞脂质方现在使用的驱动器通过耦合器和脂质注射器的蛋白质注射器的内容。 </li><li>这个过程是反复多次;偶尔，一百通道或以上须出示一个同质的中间相。在启动的混合材料通过耦合器来回运动可以是不平衡的，有时，，需要额外的力量是影响混合。通常伴随着最初的混合样品中的非均匀混浊的发展。由于同质化的不断进步，质地变得更均匀，粘性立方相的特征，作为新兴立方米中间相的视觉外观。 </li><li>如果条件合适和立方相形式，应该出现的色散光学透明的针筒。因此，针筒上的标记应清晰易读通过在桶的中间相。 </li><li>很轻微的混合冷却过程中的样品放置的时间很短，在冰上注射器混频器，可以加速同质化和透明度成就。然而，重要的是不overcool样品。 </li><li>应谨慎，以避免极其严厉的混合，因为这可能会导致样品的温度上升是由于摩擦加热 3 。 </li></ol>第5部分:载入饮水机<ol><li>删除从10μL汉密尔顿注射器的针头和柱塞。留在原地的特氟隆卡环。 </li><li>删除从一个小硬币的援助饮水机的固定螺母。 </li><li>随着棘轮手臂完全抽出，插入注射器 - 无柱塞和针 - 通过控股的饮水机环。 </li><li>更换的固定螺母，垫片面朝注射器，注射器开放紧紧拧。 应采取的注射器，以确保是正确控股环和中心的桶是平行的棘轮手臂的。都可以用肉眼来判断。这两个方面的要求是至关重要的，以避免泄漏。 </li><li>通过抓环柱塞与夹持螺母拧开了几圈，并引导到注射器的开口端柱塞。完全压下棘轮手臂。移动到每桶柱塞和检查，它的旅行自由和真正的桶。它可以帮助指导栏上松开螺丝，以方便自由移动的柱塞。确保它。压下活塞，直到其聚四氟乙烯提示μL桶零刻度线对齐。如果指导栏上的螺丝松动，拧紧它，并重新检查，柱塞可自由移动。 </li><li>组装的混合机组一侧移动，柱塞零μL刻度的中间相转移到其他注射器和耦合器。 </li><li>断开混合机组的空注射器（其在地方套圈），并立即装入注射器连接 - 耦合器一个ttached - 10μL配药注射器线程终止。松紧程度与耦合是非常关键，正如已经指出的。 </li><li>令人沮丧的100μL注射器的柱塞，使负载配药注射器通过耦合器的中间相转移。 </li><li>钢终止配药注射器安全短，平头的针，小心地拧紧到位。 </li><li>柱塞钳棘轮臂抓环拧紧螺母。应注意不要过分拧紧螺母，因为这可以得分和变形，柱塞，使其无法使用。重要的是提供棘轮臂夹紧条件的行程范围是有限的，以不超过一英寸，在视频显示。除此之外，活塞将有一种倾向，扣和交付失败。 </li><li>压下棘轮鼓几次提前在每桶柱塞，从而填补针的空隙体积和装载针。应重复此步骤（最多10次），直到一个连续字符串的中间相出现针尖。 </li><li>结晶试验应立即开始，一些蛋白质在不加沉淀立方相的不稳定。 </li></ol>第6部分:设置结晶板<ol><li>放置一个多井的结晶板和表面上的盖玻片提出为便于装载工作台以上几英寸。最佳的对比度和增强可视性是实现时，表面上是略暗。 </li><li>均匀沉淀的解决方案和摘帽的小瓶。设置为1μL沉淀配药吸管。 </li><li>一方面垂直举行的配药注射器，使用腾出手来在中心和基地以及数字1以上的针尖位置。按重复饮​​水机上的按钮驱逐到玻璃表面的中间相丸。推注量为200 NL时10μL注射器使用标准的重复饮水机。针尖应不超过几百微米以上的良好基础，以确保正确的交付。 </li><li>经过4个相邻井的中间相，1μL沉淀溶液使用2μL的吸管和标准一次性提示每个中间相丸上装载。 </li><li>尽快正视盖玻片放在充满井，覆盖均匀。要影响一个防水密封，用锅铲施加压力，以使暴露的间隔胶带粘物表面接触的盖玻片。 </li><li>中间相和沉淀配药过程可以反复进行，直至盘上所有井装载和密封。 </li><li>明确标示板，用于跟踪目的。感光蛋白质通常是由铝箔包装之前，他们在孵化室的板处理。 </li><li>放置在温度控制腔板，通常在20 ° C。 </li><li>定期的时间表，晶体生长的检查井，偏振光显微镜使用10或20倍的目标。在笔者的实验室中使用的时间表如下:0，1，2，3，5，7，14，21，30和60安装后。调整聚焦深度在0.14毫米厚的样品，仔细检查中间相丸。考试应在正常光线和交叉偏光片之间。 </li></ol>第7部分:代表结果导致晶体的外观将随膜蛋白的固有色，光的偏振用于检查（或缺乏）和照明的方法和质量。图6显示了几个可能的晶体出场。自然色的膜蛋白，在观日益正常光线查看时，在图6（b和d面板）所示的样子。无色透明的膜蛋白晶体，越来越多立方相正常光线查看时，可以在图6（小组E）所示的样子。最后，无色的膜蛋白晶体中观的偏振光观察时，可以像图6（面板A和C ）。 在结构测定的整个过程中的下一个步骤是收获和低温冷晶体，并记录和分析他们的X -射线衍射。这些主题都是在单独的朱庇特的文章。 <img src="/files/ftp_upload/1712/1712fig1.jpg" alt="图1" /> 图1显示了生物上的脂质双层膜的示意图，位于多种蛋白质。 <img src="/files/ftp_upload/1712/1712fig2.jpg" alt="图2" /> 图2的结构。维生素B 12运输蛋白，BtuB，解决使用MX和在此朱庇特的文章中 说明 6细观方法生长的晶体。 <img src="/files/ftp_upload/1712/1712fig3.jpg" alt="图3" /> 图3。结构与功能的周期，说明许多在完成有关的一种蛋白质的结构信息获取和利用所涉及的步骤。 <img src="/files/ftp_upload/1712/1712fig4.jpg" alt="图4" /> 图4。流程图总结细观方法在膜蛋白晶体的生产所涉及的步骤。只有那些虚线红线包围的步骤包括在此朱庇特的文章。从参考2。 <img src="/files/ftp_upload/1712/1712fig5.jpg" alt="图5" /> 图5。一个简化的温度成分为脂质（monoolein）/水体系相图。结晶试验，成立于20 ° C，其中40％水合脂与水饱和。详细的相图是在参考文献5。 <img src="/files/ftp_upload/1712/1712fig6.jpg" alt="图6" /> 图6。油脂中间相生长的膜蛋白质晶体。 （一）维生素B 12的运输蛋白质，BtuB 6，（二）捕光色素复合物Ⅱ7，（C）黏附/ invasin OPCA 8，（D）9，细菌视紫红质（E）从假单胞菌碳水化合物转运。拍摄的图像与正常光线（B，D，E）和交叉偏光片（A，C）之间。

<ol>
	<li>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=Crystallizing+membrane+proteins+for+structure+determination.+Use+of+lipidic+mesophases.">Crystallizing membrane proteins for structure determination. Use of lipidic mesophases.</a> Annu. Rev. Biophys. 38, 29-51 (2009).</li><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=Crystallising+membrane+proteins+in+lipidic+mesophases.">Crystallising membrane proteins in lipidic mesophases.</a> Nat. Protoc. 4, 706-731 (2009).</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=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>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=The+phase+diagram+of+the+monoolein/water+system:+metastability+and+equilibrium+aspects.">The phase diagram of the monoolein/water system: metastability and equilibrium aspects.</a> Biomaterials. 21, 223-234 (2000).</li><li>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=In+meso+structure+of+the+cobalamin+transporter,+BtuB,+at+1.95+A+resolution.">In meso structure of the cobalamin transporter, BtuB, at 1.95 A resolution.</a> J. Mol. Biol. 364, 716-734 (2006).</li><li>Cherezov, V., Clogston, J., Papiz, M. Z., 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=Room+to+move:+crystallizing+membrane+proteins+in+swollen+lipidic+mesophases.">Room to move: crystallizing membrane proteins in swollen lipidic mesophases.</a> J. Mol. Biol. 357, 1605-1618 (2006).</li><li>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=In+meso+crystal+structure+and+docking+simulations+suggest+an+alternative+proteoglycan+binding+site+in+the+OpcA+outer+membrane+adhesin.">In meso crystal structure and docking simulations suggest an alternative proteoglycan binding site in the OpcA outer membrane adhesin.</a> Proteins. 71, 24-34 (2008).</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 Biol. Crystallogr. 60, 1795-1807 (2004).</li></ol>

立方相是一个微妙的和动态的环境可以改变一个变量的数目急剧变化。这是不可能的，给予说明在手动模式下，描述了所有潜在的隐患的中间结晶试验的设置。然而，许多困难，可避免通过练习前把它应用到昂贵的蛋白质溶液的技术和应用在混合注射器的压力放缓。做得正确， 在中观方法可以产生各种各样的蛋白质晶体，其数量是不断增加。 这里设置在中间的结晶试验的描述主要集中在手动模式。这个过程就可以了，经常被修改，以促进在这些情况下，需要大规模的结晶条件筛选的结晶板自动设置。

*您自己的窄径耦合加工的全部细节，提供参考文献3。

crystallizing membrane proteins for structure determination using lipidic mesophases

使用油脂介孔膜蛋白结晶的一个详细的协议描述。这种方法有不同的被称为油脂立方相， 或在中观方法。该方法已被证明是相当通用的，因为它已被用于解决X射线晶体结构的原核和真核细胞的蛋白质，蛋白质是单体，同源异多聚体，生色含和生色，和alpha螺旋和β-管蛋白。最近取得的成功观的结晶，是人类设计的β2-肾上腺素和腺苷A2A G蛋白偶联受体。协议提出的重组膜蛋白的monoolein为基础的中间相，并设立在手动模式下的结晶。额外的步骤，在整个过程中，如水晶收获，要在未来的视频文章处理。所需的时间准备蛋白质装载的中间相，并设立结晶板，手工大约一个小时。

Watch this Scientific Journal Video about Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases at JoVE.com

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

此处所描述的卡弗里膜的结构与功能的生物集团成立手动油脂介孔膜蛋白的结晶试验实施的过程。

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 ...

crystallizing-membrane-proteins-for-structure-determination-using

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

29.9K 次观看.  Trinity College Dublin. 此处所描述的卡弗里膜的结构与功能的生物集团成立手动油脂介孔膜蛋白的结晶试验实施的过程。

视频: Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

正确的蛋白浓度的动力学结构与功能分析和亲和力测定的重要性

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

科研

生物学

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

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 ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

使用微量的生物规格的纳米分光光度计核酸和蛋白的浓度测定

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

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 ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

在斑马鱼发育的细胞追踪使用Photoconvertible蛋白

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

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 ...

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, ...

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 ...