江实验室KvAP的研究已取得显着的帮助罗德里克麦金农博士在洛克菲勒大学的实验室。特别要感谢博士Kathlynn布朗和迈克尔McQuire的建议和帮助我们噬菌体屏实验。这项工作是由美国国立卫生研究院（GM088745 GM093271 Q-XJ）和AHA（12IRG9400019 Q-XJ）的补助支持。

<ol>
	<li>Alberts, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Biology of the Cell. , (2007).</li><li>Lee, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+lipids+and+proteins+interact+in+a+membrane:+a+molecular+approach.">How lipids and proteins interact in a membrane: a molecular approach.</a> Mol. Biosyst. 1, 203 (2005).</li><li>Lee, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+lipids+affect+the+activities+of+integral+membrane+proteins.">How lipids affect the activities of integral membrane proteins.</a> Biochim. Biophys. Acta. 1666, 62 (2004).</li><li>Anderson, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+caveolae+membrane+system.">The caveolae membrane system.</a> Annu. Rev. Biochem. 67, 199 (1998).</li><li>Simons, K., Vaz, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+systems,+lipid+rafts,+and+cell+membranes.">Model systems, lipid rafts, and cell membranes.</a> Annu. Rev. Biophys. Biomol. Struct. 33, 269 (2004).</li><li>Edidin, 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+state+of+lipid+rafts:+from+model+membranes+to+cells.">The state of lipid rafts: from model membranes to cells.</a> Annu. Rev. Biophys. Biomol. Struct. 32, 257 (2003).</li><li>Kapoor, R., Kim, J. H., Ingolfson, H., Andersen, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Artificial+Bilayers+for+Electrophysiology+Experiments.">Preparation of Artificial Bilayers for Electrophysiology Experiments.</a> J. Vis. Exp. (20), e1033 (2008).</li><li>Zheng, H., Liu, W., Anderson, L. Y., Jiang, Q. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid-dependent+gating+of+a+voltage-gated+potassium+channel.">Lipid-dependent gating of a voltage-gated potassium channel.</a> Nat. Commun. 2, 250 (2011).</li><li>Schmidt, D., Jiang, Q. X., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipids+and+the+origin+of+cationic+gating+charges+in+voltage+sensors.">Phospholipids and the origin of cationic gating charges in voltage sensors.</a> Nature. 444, 775 (2006).</li><li>Ruta, V., Jiang, Y., Lee, A., Chen, J., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+an+archaebacterial+voltage-dependent+K++channel.">Functional analysis of an archaebacterial voltage-dependent K+ channel.</a> Nature. 422, 180 (2003).</li><li>Jiang, Q. X., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+lipids+on+voltage-gated+ion+channels.">The influence of lipids on voltage-gated ion channels.</a> Curr. Opin. Struct. Biol. , 3408884 (2012).</li><li>Artimo, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ExPASy:+SIB+bioinformatics+resource+portal.">ExPASy: SIB bioinformatics resource portal.</a> Nucleic Acids Res. 40, W597 (2012).</li><li>Cladera, J., Rigaud, J. L., Villaverde, J., Dunach, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome+solubilization+and+membrane+protein+reconstitution+using+Chaps+and+Chapso.">Liposome solubilization and membrane protein reconstitution using Chaps and Chapso.</a> Eur. J. Biochem. 243, 798 (1997).</li><li>Levy, D., Bluzat, A., Seigneuret, M., Rigaud, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+study+of+liposome+and+proteoliposome+reconstitution+involving+Bio-Bead-mediated+Triton+X-100+removal.">A systematic study of liposome and proteoliposome reconstitution involving Bio-Bead-mediated Triton X-100 removal.</a> Biochim. Biophys. Acta. 1025, 179 (1990).</li><li>Young, H. S., Rigaud, J. L., Lacapere, J. J., Reddy, L. G., Stokes, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+tubular+crystals+by+reconstitution+of+detergent-solubilized+Ca2(+)-ATPase.">How to make tubular crystals by reconstitution of detergent-solubilized Ca2(+)-ATPase.</a> Biophys. J. 72, 2545 (1997).</li><li>Levy, D., Gulik, A., Bluzat, A., Rigaud, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+sarcoplasmic+reticulum+Ca(2+)-ATPase:+mechanisms+of+membrane+protein+insertion+into+liposomes+during+reconstitution+procedures+involving+the+use+of+detergents.">Reconstitution of the sarcoplasmic reticulum Ca(2+)-ATPase: mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents.</a> Biochim. Biophys. Acta. 1107, 283 (1992).</li><li>Cohen, F. S., Zimmerberg, J., Finkelstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+of+phospholipid+vesicles+with+planar+phospholipid+bilayer+membranes.+II.+Incorporation+of+a+vesicular+membrane+marker+into+the+planar+membrane.">Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. II. Incorporation of a vesicular membrane marker into the planar membrane.</a> The Journal of General Physiology. 75, 251 (1980).</li><li>Fuks, B., Homble, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+and+electrical+properties+of+planar+lipid+membranes+from+thylakoid+lipids.">Permeability and electrical properties of planar lipid membranes from thylakoid lipids.</a> Biophysical Journal. 66, 1404 (1994).</li><li>Hanke, W., Schlue, W. -. R., Sattelle, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+Lipid+Bilayers.+Methods+and+Applications.">Planar Lipid Bilayers. Methods and Applications.</a> Biological Techniques. , 133 (1993).</li><li>Tien, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bilayer lipid membranes (BLM). Theory and Practice. , 655-65 (1974).</li><li>Pagano, R. E., Ruysschaert, J. M., Miller, I. 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+molecular+composition+of+some+lipid+bilayer+membranes+in+aqueous+solution.">The molecular composition of some lipid bilayer membranes in aqueous solution.</a> The Journal of Membrane Biology. 10, 11 (1972).</li><li>Henn, F. A., Thompson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+lipid+bilayer+membranes+separating+two+aqueous+phases:+composition+studies.">Properties of lipid bilayer membranes separating two aqueous phases: composition studies.</a> Journal of Molecular Biology. 31, 227 (1968).</li><li>Tao, X., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+Kv1.2+and+paddle+chimera+Kv+channels+in+planar+lipid+bilayers.">Functional analysis of Kv1.2 and paddle chimera Kv channels in planar lipid bilayers.</a> J. Mol. Biol. 382, 24 (2008).</li><li>Cohen, F. S., Akabas, M. H., Zimmerberg, J., Finkelstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parameters+affecting+the+fusion+of+unilamellar+phospholipid+vesicles+with+planar+bilayer+membranes.">Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes.</a> The Journal of Cell Biology. 98, 1054 (1984).</li><li>Smith, G. P., Petrenko, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+Display.">Phage Display.</a> Chem. Rev. 97, 391-39 (1997).</li><li>McGuire, M. J., Li, S., Brown, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biopanning+of+phage+displayed+peptide+libraries+for+the+isolation+of+cell-specific+ligands.">Biopanning of phage displayed peptide libraries for the isolation of cell-specific ligands.</a> Methods Mol. Biol. 504, 291 (2009).</li><li>Rigaud, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+proteins:+functional+and+structural+studies+using+reconstituted+proteoliposomes+and+2-D+crystals.">Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals.</a> Braz. J. Med. Biol. Res. 35, 753 (2002).</li><li>Chami, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+octyl+beta-thioglucopyranoside+in+two-dimensional+crystallization+of+membrane+proteins.">Use of octyl beta-thioglucopyranoside in two-dimensional crystallization of membrane proteins.</a> J. Struct. Biol. 133, 64 (2001).</li><li>Kuhlbrandt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+crystallization+of+membrane+proteins.">Two-dimensional crystallization of membrane proteins.</a> Q. Rev. Biophys. 25, 1 (1992).</li><li>Rigaud, J. L., Levy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+membrane+proteins+into+liposomes.">Reconstitution of membrane proteins into liposomes.</a> Methods Enzymol. 372, 65 (2003).</li><li>Walz, T., Grigorieff, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+Crystallography+of+Two-Dimensional+Crystals+of+Membrane+Proteins.">Electron Crystallography of Two-Dimensional Crystals of Membrane Proteins.</a> J. Struct. Biol. 121 (2), 142 (1998).</li><li>Signorell, G. A., Kaufmann, T. C., Kukulski, W., Engel, A., Remigy, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+2D+crystallization+of+membrane+proteins+using+methyl-beta-cyclodextrin.">Controlled 2D crystallization of membrane proteins using methyl-beta-cyclodextrin.</a> J. Struct. Biol. 157, 321 (2007).</li><li>Vink, M., Derr, K., Love, J., Stokes, D. L., Ubarretxena-Belandia, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+strategy+to+screen+2D+crystallization+trials+of+membrane+proteins.">A high-throughput strategy to screen 2D crystallization trials of membrane proteins.</a> J. Struct. Biol. 160, 295 (2007).</li><li>Iacovache, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+2DX+robot:+a+membrane+protein+2D+crystallization+Swiss+Army+knife.">The 2DX robot: a membrane protein 2D crystallization Swiss Army knife.</a> J. Struct. Biol. 169, 370 (2010).</li></ol>

什么都没有透露。

8-10在几项研究中已被用于重建成不同的膜的KvAP频道。去污剂/脂质混合胶束和蛋白/去污剂/脂质的混合胶束之间的脂质确保分布的想法，我们能够达到几乎完全重构的KvAP制成的非常不同的脂质膜。每个四聚KvAP通道需要〜100脂质分子完全覆盖其跨膜结构域。的基本要求，是让足够的脂质分子融合蛋白/类脂/洗涤剂胶束前去除洗涤剂。确保我们的标准条件（0.5毫克的蛋白质和5.0毫克脂类）平均有〜1,000脂质分子，每个蛋白质分子。浮选实验和生化实验证实，重组几乎是完整的。电气录音通道插入黑脂膜，筛选重组通道一免受噬菌体展示肽库，而且增长的二维晶体膜的渠道都证明了成功的应用，用于各种用途的膜重建。 的KvAP频道和噬菌体展示肽库筛选对脂质的依赖构象的变化展示了新的途径，以通道阻滞剂的屏幕或通道开放剂通过生物 ​​化学的方法，而不是依赖于电，以保持通道的特定的构象8。在我们的屏幕的构象特异性结合的成功表明，同样的策略可以应用于以查找特定的粘结剂的活性构象。可以预见的是重组后的渠道在囊泡单链抗体库，厂库，同样可以用来对付其他膜蛋白可以运行通过这些操作，并确保他们的紧张可能是有用的各种用途的粘合剂。我们相信，这NEW法将在未来看到更多的一般应用。 再造膜系统将允许澄清的化学细节背后的脂质膜蛋白的影响11。脂质-蛋白质的相互作用已经知道许多膜蛋白是重要的，并已在过去3进行多个研究。在基于细胞的研究中，操作可以被实施，从而改变膜中的特定组件，然后在膜蛋白的功能变化与膜的结构和组成的变化。这样的连接是间接的，可能会导致多种因素没有得到很好的特点细胞膜。在重组的均匀的膜，它是在之间进行连接的膜蛋白的结构和功能改变和脂组合物和膜性能的变化更明确的。最终，以了解他背后的脂质 - 蛋白质相互作用的化学原理，我们需要划定的脂质分布各地的跨膜结构域，并了解这些脂质蛋白质的动态变化。改组后的系统出现朝着这样的理解是一个可靠的方式。 膜蛋白的重组需要控制去除清洁剂从蛋白质/脂质/洗涤剂混合胶束，混合胶束的融合，最终变成囊泡30路数。三种不同的方法被用来除去洗涤剂，透析，有孔玻璃珠，和环糊精14，31，32。但它仍然是难以实现良好的控制，逐步去除洗涤剂，体积小33，34。一个理想的方法去除清洁剂的清洁剂会采取出的水相均匀地在整个卷在一个可控的步伐，而不应施加强大的干扰Øn的重建双层膜。这样的方法可能会对能够改变的速度和重建的疗效，将有可能使在小体积的重组。缓慢稀释和任何三种常规方法去除洗涤剂的组合可能会接近这个目标。缓慢稀释到蛋白/去污剂/脂质混合物通过引入少量的水是一种可控的方式，均匀地减少洗涤剂浓度下其CMC。事后洗涤剂去除囊泡的形成是不太重要的，但仍然是重要的小囊泡融合的路数。其他途径来实现控制的洗涤剂除去，仍然需要被设计和开发。 我们的重建过程需要考虑脂质分布在混合胶束和囊泡以及混合胶束洗涤剂诱导的融合。它的成功铺平了道路，从而导致更广泛应用的Reconstituted囊泡，远远超过我们提出的三个方向。我们的程序，以适应其他膜蛋白应该不会遇到重大技术限制。尽管许多膜蛋白已被重组或其他方式，它一直难以达到近乎完整的重建，并从多个不同的角度来评价功能的蛋白质。在KvAP重建我们的努力表明，我们的方法可以让完整的重建，并将于适合这些用途。

的细胞交换与他们的的环境中的材料和信息通过特定的的膜蛋白1的职能，。在细胞膜上的膜蛋白的功能作为泵，渠道，受体，膜内内切酶器，连接器和跨膜的的的结构性支持者。影响的膜蛋白的的的基因突变，已一直与到许多的人类疾病的有关。事实上，有很多的的膜蛋白已经一直的首要的的的的药物靶标，，，因为他们顷重要的的，和很容易在单元格膜的访问的。因此，很重要的是了解膜不同的膜蛋白的结构和功能，并使得有可能设计新颖的方法，以减轻从突变蛋白在人类疾病中的不利影响。 脂质环绕所有集成在双层膜2，3的膜蛋白。在为真核细胞膜中，的各种的脂类的不同类型的顷公知，以被组织成的微区4，5。的许多的膜蛋白被示出将予分派产生这些的微区，以及作为了笨重的的的的膜心情，6的流体相为。该机制，背后的组织的的膜蛋白的的的的微区和的交付融入其中，的生理意义该等分派之的的是显然是重要的，，，，但仍然不佳理解的。的一个主要的在研究膜蛋白的的上的血脂特效时的的技术难度是生物化学上纯化的膜的成有良好对照的的的脂质组合物的膜的的蛋白质其他，以便，几乎所有的重新配制的蛋白质顷功能性的7的的可靠的复溶的。在的过去的的的几年里中，我们开发的方法的去重构一个从A.的"电压-门控性"原型钾离子通道pernix的 （KvAP）成各种膜系统的结构和功能研究8-10。其他人的数据，我们一起显示，脂类可能是一个决定因素中的构象变化的电压检测的一个电压门控离子信道和的网域可能会塑造的这些信道11中的的一些的的的结构。在未来，我们将提供我们的方法的详细说明，并提供关键技术的提示，可能会确保我们的研究结果，以及我们的方法扩展到其他膜蛋白的研究成功再现。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Tryptone</td>
 <td>RPI Corp.</td>
 <td>T60060</td>
 <td></td>
 </tr>
 <tr>
 <td>Yeast Extract</td>
 <td>RPI Corp.</td>
 <td>Y20020</td>
 <td></td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Fisher</td>
 <td>S271-3</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>RPI Corp.</td>
 <td>T60040</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium Chloride</td>
 <td>Fisher</td>
 <td>BP366-500</td>
 <td></td>
 </tr>
 <tr>
 <td>n-Dodecyl-&beta;-D-Maltoside</td>
 <td>Affymetrix</td>
 <td>D322S</td>
 <td>Sol-grade</td>
 </tr>
 <tr>
 <td>n-Octyl-&beta;-D-Glucoside </td>
 <td>Affymetrix</td>
 <td>O311</td>
 <td>Ana-grade</td>
 </tr>
 <tr>
 <td>Aprotinin</td>
 <td>RPI Corp.</td>
 <td>A20550-0.05</td>
 <td></td>
 </tr>
 <tr>
 <td>Leupeptin</td>
 <td>RPI Corp.</td>
 <td>L22035-0.025</td>
 <td></td>
 </tr>
 <tr>
 <td>Pepstatin A</td>
 <td>RPI Corp.</td>
 <td>P30100-0.025</td>
 <td></td>
 </tr>
 <tr>
 <td>PMSF</td>
 <td>SIGMA</td>
 <td>P7626</td>
 <td></td>
 </tr>
 <tr>
 <td>Dnase I</td>
 <td>Roche</td>
 <td>13407000</td>
 <td></td>
 </tr>
 <tr>
 <td>Bio-Bead SM-2</td>
 <td>Bio-Rad</td>
 <td>152-3920</td>
 <td></td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>RPI Corp.</td>
 <td>H75030</td>
 <td></td>
 </tr>
 <tr>
 <td>POPE</td>
 <td>Avanti Polar Lipids</td>
 <td>850757C</td>
 <td></td>
 </tr>
 <tr>
 <td>POPG</td>
 <td>Avanti Polar Lipids</td>
 <td>840457C</td>
 <td></td>
 </tr>
 <tr>
 <td>DOGS</td>
 <td>Avanti Polar Lipids</td>
 <td>870314C</td>
 <td></td>
 </tr>
 <tr>
 <td>DMPC </td>
 <td>Avanti Polar Lipids</td>
 <td>850345C</td>
 <td></td>
 </tr>
 <tr>
 <td>Biotin-DOPE </td>
 <td>Avanti Polar Lipids</td>
 <td>870282C</td>
 <td></td>
 </tr>
 <tr>
 <td>DOTAP</td>
 <td>Avanti Polar Lipids</td>
 <td>890890C</td>
 <td></td>
 </tr>
 <tr>
 <td>NeutrAvidin agarose beads </td>
 <td>Piercenet</td>
 <td>29200</td>
 <td></td>
 </tr>
 <tr>
 <td>Dialysis Tubing</td>
 <td>Spectrum Laboratories, Inc</td>
 <td>132-570</td>
 <td></td>
 </tr>
 <tr>
 <td>Pentane</td>
 <td>Fisher</td>
 <td>R399-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Decane</td>
 <td>TCI America</td>
 <td>D0011</td>
 <td></td>
 </tr>
 <tr>
 <td>MTS-PEG5000</td>
 <td>Toronto Research Cemicals</td>
 <td>M266501</td>
 <td></td>
 </tr>
 </tbody>
</table>

reconstitution of a kv channel into lipid membranes for structural and functional studies

若要研究的脂质在一个还原论的的时尚的 - 蛋白相互作用，它是必要的，，以纳入成定义良好-的的脂质组合物的膜的的的膜蛋白。我们正在研究在了一个原型电压-门控性钾（Kv值）信道中的脂质-依赖的的的门控效果，，，，并已摸索出到不同的膜系统中的的详细程序，以重新组建的渠道。我们的的复溶程序也须花上代价为两个的囊泡的以的洗涤剂-诱导的的的融合菜式，和的融合菜式的与的脂质/界面活性剂的混合胶束的的蛋白质/去污剂微胶束中，以及作为在的重要性的的脂类达成一项平衡态分布产生的蛋白质/清洁剂/脂质和的洗涤剂/脂质的混合的胶束。我们的的数据所表示的，在的油脂泡囊中中的信道的插入的是相对地在取向中随机抽出的，，，并，没有可检测到的蛋白质聚集体被在分馏实验中看到的，了复溶效率是如此之高的。我们已动用了重新组建，，以确定在不同的脂类中的的渠道的构象状态的d个频道，请记录一个小的数目间在平面脂双层膜的中注册成立之的信道，屏幕为从一个噬菌体-显示的的的的肽库的构象-特定的的配体的电活动的，，并支持的增长的二维晶体的:在膜的中的信道。可适于在脂质双层中，特别是对于真核电压门控离子通道的脂质的影响的调查研究其他膜蛋白的重组此处描述的步骤。

的实验净化KvAP的通道进入生化同质性的一般流程，在图1A中所述。结果显示在图1B中的SDS-PAGE凝胶中的蛋白质的表达和纯化过程中的典型的样品。 IMAC净化后的蛋白质比较纯粹。的KvAP通道的产率是约1.0毫克/升培养。 用清洁剂溶脂囊泡需要制定出每对脂质与洗涤剂。由DM /教皇教皇的小单室脂质体的增溶图2A，小泡浮选的结果通常是相当简单。典型的分数梯度SDS-PAGE分析结果显示在图2B。在蔗糖密度梯度中，含通道的POPE / POPG囊泡通常都集中在10％的层之间的界面d为35％的层。的含KvAP的DOTAP或DOGS囊泡的重量更轻，通常是在接口之间的5％和10％（轻微渗透到10％的层）。如果有一个显着的部分的多层囊泡，它们通常是低于10〜35％接口发白并浓缩。我们发现，这通常发生在蛋白质洗涤剂脂质混合物没有培养足够长的时间，以达到良好的血脂分布。 图3所示的电气录音从KvAP在平面脂双层组成频道。典型的电流跟踪显示，在-80 mV，渠道很安静。切换到80 mV提供满意的电容峰（尖锐的电压切换时）。一个缓慢的上升阶段，目前的渠道变得活跃，并能够进行外向钾电流。的峰值的上升阶段后，电流开始下降，名为失活的步骤。 inactivati需要几百毫秒的时间来完成。一旦电压切换回至-80 mV，向下电容峰值后是返回阶段，这反映了公开渠道的关闭，被称为失活（ 图3C）。 噬菌体屏幕的中间，我们测试了放大的噬菌体上KvAP的通道中的双层的POPE / POPG的活性。 12个选择周期后，扩增噬菌体抑制信道活动（ 图4B中 ，选择的噬菌体）。但起动噬菌体展示库的通道（ 图4B，控制噬菌体），没有发现任何影响。尽管所选择的噬菌体的活动不是很高，因为只有低浓度的阳性噬菌体周围，明确的，可重复的效应，结果表明有活性的阳性克隆表现出高结合亲和力，，应选择性地富集在剩余筛选CYCLES一旦丰富，膜通道上可能有较强的抑制活性。 在筛选的二维晶体的早期阶段，我们看到了小格，在许多小囊泡。有了优化，一些大张显示了很多周围的小囊泡（ 图5A）。这些结晶的冷冻蚀刻电镜图像总是显示的特定的局部缺陷（ 图5B），这表明需要进一步的优化，以取得更好的晶体。一旦晶体被很好的优化，他们表现出锋利的边缘，并出现单张纸。在高倍率，个别单位显然要更好地下令（ 图5C）。 <table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>蛋白胨</td><td> RPI公司</td><td> T60060</td><td></td></tr><tr><td>酵母提取物</td><td> RPI公司</td><td> Y20020 </td><td></td></tr><tr><td>氯化钠</td><td>费舍尔</td><td> S271-3 </td><td></td></tr><tr><td> Tris碱</td><td> RPI公司</td><td> T60040 </td><td></td></tr><tr><td>氯化钾</td><td>费舍尔</td><td> BP366-500 </td><td></td></tr><tr><td> n-十二烷基-β-D-麦芽糖苷</td><td> Affymetrix公司</td><td> D322S </td><td>溶胶级</td></tr><tr><td> N-辛基-β-D-葡萄糖苷</td><td> Affymetrix公司</td><td> O311 </td><td>安娜级</td></tr><tr><td>抑肽酶</td><td> RPI公司</td><td> A20550-0.05 </td><td></td></tr><tr><td>亮肽素</td><td> RPI公司</td><td> L22035-0.025 </td><td></td></tr><tr><td>胃酶抑素</td><td> RPI公司</td><td> P30100-0.025 </td><td></td></tr><tr><td> PMSF </td><tD&gt; SIGMA </td><td> P7626 </td><td></td></tr><tr><td> DNA酶I </td><td>罗氏</td><td> 13407000 </td><td></td></tr><tr><td>生物珠SM-2 </td><td> Bio-Rad公司</td><td> 152-3920 </td><td></td></tr><tr><td> HEPES </td><td> RPI公司</td><td> H75030 </td><td></td></tr><tr><td>教皇</td><td>阿凡提极地血脂</td><td> 850757C </td><td></td></tr><tr><td> POPG </td><td>阿凡提极地血脂</td><td> 840457C </td><td></td></tr><tr><td>爱狗</td><td>阿凡提极地血脂</td><td> 870314C </td><td></td></tr><tr><td> DMPC </td><td>阿凡提极地血脂</td><td> 850345C </td><td></td></tr><tr><td>生物素掺杂</td><td>阿凡提极地血脂</td><td> 870282C </td><td></td></tr><tr><td> DOTAP </td><td>阿凡提极地血脂</td><td> 890890C </td><td></td></tr><tr><td> NeutrAvidin AGA玫瑰珠</td><td> Piercenet </td><td> 29200 </td><td></td></tr><tr><td>透析管</td><td>光谱实验室，公司</td><td> 132-570 </td><td></td></tr><tr><td>戊烷</td><td>费舍尔</td><td> R399-1 </td><td></td></tr><tr><td>癸</td><td> TCI美国</td><td> D0011 </td><td></td></tr><tr><td> MTS-PEG5000 </td><td>多伦多研究化学品</td><td> M266501 </td><td></td></tr></tbody></table> 表1中。试剂和材料清单。 <table border="1"><tbody><tr><td><ul><li>细菌培养摇床</li><li>分光光度计</li><li>微探头超声发生器的</li><li>跷</li><li>落地式离心机用于收集细菌培养和浓缩样品</li><li>空列</li><li> superdex 200柱连接到FPLC系统</li></ul></td&gt; </tr></tbody></table> 表2中。所需设备。 <table border="1"><tbody><tr><td> 缓冲区的名字 </td><td> 内容 </td></tr><tr><td> IMAC裂解缓冲液</td><td>的50mM Tris（pH为8.0），100mM的氯化钾</td></tr><tr><td> IMAC洗涤液</td><td>的50mM Tris（pH为8.0），100mM的氯化钾，5.0 mM的DM </td></tr><tr><td> IMAC洗脱缓冲液</td><td>的50mM Tris（pH为8.0），100mM的氯化钾，5.0 mM的DM，300 mM咪唑</td></tr><tr><td> SDS-上样缓冲液（5X）（非还原） </td><td> 60毫摩尔Tris-HCl（pH为6.8）中，25％甘油，2.0％SDS，0.10％溴酚蓝。 （1.0％2 - 巯基乙醇的加入使减少缓冲液）。 </td></tr><tr><td>浓缩胶缓冲SDS-PAGE（4X） </td><td> 0.50米，0.40％SDS的Tris-HCl（pH值6.8） </td></tr><tr><td>分辨率凝胶缓冲液SDS-PAGE（4X） </td><td> 1.5 M的Tris-HCl（pH为8.8），0.40％SDS </td></tr><tr><td> FPLC平衡态配给</td><td> 20毫米的Tris pH值8.0，氯化钾100毫米，5.0毫米DM </td></tr><tr><td>脂质体透析</td><td> 10毫米HEPES，氯化钾100毫米</td></tr></tbody></table> 表3中。缓冲区的名称和内容。 <img alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/50436/50436fig1highres.jpg" src="/files/ftp_upload/50436/50436fig1.jpg" /> 图1。制备重组KvAP。一般工作流程的蛋白表达，纯化和重建。B.生化纯化的蛋白质。诱导培养的大肠杆菌大肠杆菌 XL1-蓝色表示KvAP，处理和纯化的KvAP。 20微升的细胞培养物（泳道1）之前或之后（泳道2）诱导的洗涤剂提取物（泳道3），流通过IMAC色谱法（泳道4），洗涤步骤（5.6），和5.0μg总后的蛋白300 mM咪唑洗脱（泳道7）和5.0微克的KvAP四聚体的尺寸排阻FPLC（泳道8），进行非还原性12％SDS-PAGE和考马斯亮蓝染色。 <img alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/50436/50436fig2highres.jpg" src="/files/ftp_upload/50436/50436fig2.jpg" /> 图2。重组在教皇/ POPG的囊泡KvAP。 。囊泡融合和溶解洗涤剂浓度的函数。用于监视的囊泡的光散射（减去基线没有洗涤剂分数），在410 nm处的吸光度。脂质（POPE / POPG 3:1）分别为10毫克/毫升。小单室脂质体后，超声强，单分散的，由于它们的大小，直径30-50纳米的弱散射。进行了介绍，当洗涤剂溶液相之间的分布和脂质膜阶段。由于在小强曲率<html>单层脂质体，引入的洗涤剂触发囊泡融合和曲率的释放（从而低的表面电势的能量）。的稠囊泡尺寸更大，并表现出较强的散射波长为410 nm。 （灰色区）峰值的上升阶段，因此反映了洗涤剂引起的融合，一个好的制度蛋白胶束囊泡融合。为我们的重整过程中的洗涤剂的浓度（DM，例如，在这里），上面的吸收峰确实选择。B. 50微升再造KvAP囊泡（KvAP 0.5毫克/毫升），用蔗糖密度梯度分离。样品100μl的馏分从顶部到底部（1〜9）蔗糖梯度中的12％减少的SDS-PAGE测定。凝胶考马斯亮蓝染色。 3巷包含〜2微克KvAP。 / files/ftp_upload/50436/50436fig3.jpg的"/&gt; 图3。电气活动的通道双层KvAP的重组。 A.录制里面设置了一个法拉第笼，1;两个电极，2;反侧，3;顺侧，4;盐桥。B.放大部分的减薄部分的一侧示出的孔5的双层杯这是用于膜。C.电气活性的融合成黑色的脂质膜的KvAP通道被记录下来。虽然被关押在-80 mV，膜电位为​​150毫秒脉冲为80 mV，再变回控股的潜力。离子电流在电压钳模式下使用Axopatch 200B放大器。 <img alt="图4" fo:content-width="6in" fo:src="/files/ftp_upload/50436/50436fig4highres.jpg" src="/files/ftp_upload/50436/50436fig4.jpg" /> 图4。筛选conformati的从噬菌体展示库中的特定的配位体。对离子通道的筛选。计划背后囊泡重组。用生物素-DOPE掺杂囊泡，它们可以被拉出NeutrAvidin有孔玻璃珠的溶液。控制的特定脂质的KvAP通道的构象。负选择之前完成对渠道在不同的构象珠，空囊泡和囊泡噬菌体培养目标通道的构象状态（在这里作为一个例子DOTAP或狗）。选择的噬菌体可以被放大，并测试对双分子层中的信道，在屏幕中间的选择的噬菌体B.测试。电气双层KvAP活动增加了选择的噬菌体:黑迹后，在不同时间点-除了前黄色轨迹- 2分钟后，红色轨迹- 10分钟后再加。:噬菌体抗体库（总〜10 10噬菌体附加的双层）上测试双分子层中的信道，被发现有没有检测到活性，因为每个噬菌体克隆具有只有约100份， 下图:12个选择周期后，噬菌体仍然不同无性系的混合物。在双层铅明显抑制通道活性的渠道，增加约10噬菌体提示有阳性克隆，应该有高亲和力。 <a href="https://www.jove.com/files/ftp_upload/50436/50436fig4large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/50436/50436fig5highres.jpg" src="/files/ftp_upload/50436/50436fig5.jpg" /> 图5。二维的KvAP结晶膜。 A.负染的单层的二维晶体在晶体优化的中间图像。的结晶，用6.0％的硫酸铵染色钼酸盐，pH值6.4加0.50％的海藻糖。由于糖，它们有时会相互重叠起来。的黑色方框指定的区域，使人们产生要〜20埃的衍射点的右侧所示的衍射图案。B.冷冻蚀刻电镜图像的二维晶体从（A）中使用的相同的样品中的一项。混合试样，用3％的海藻糖和冻结直接飞泻，下冷冻蚀刻电镜成像。在50000倍，得到的图像。很显然，在晶体包装的局部缺陷。C.冷冻蚀刻电镜图像的进一步优化晶体。的检体被嵌入在0.75％的单宁酸和10％的海藻糖和拍摄图像时的直线和紧密地堆积在50000 X.建议，在这种类型的晶体排列的通道。两个黑色的箭头标示的正方形格子。 盒: 专栏1。 IMAC柱的制备 <ol><li>彻底重悬IMAC树脂。 </li><li>紧接在悬浮液中所需的树脂量转移到50毫升管。我们使用2毫升树脂床体积的净化KvAP从2 L培养。 </li><li>两分钟以沉淀下来的树脂，以700×g离心管。 </li><li>取出并弃上清。 </li><li> MQH 2 O 10床体积的添加，并简要混合冲洗树脂。 </li><li>以700×g，2分钟沉淀下来的树脂Recentrifuge。弃上清。 </li><li>添加MQH 2 O 10床体积的，倒入空列。 </li><li>等待，直到树脂落户，关闭列流变压器低压液相色谱。 </li><li>通过列MQH 2 O和5床体积的平衡缓冲运行5个床体积。 </li></ol> 专栏2。纯化抗体 抗体转型-第1天 <ol><li>改造100ng的质粒含有抗体为6〜60微升JM83感受态细胞，板4含有100微克/毫升氨苄青霉素，和在37℃培养箱中孵育过夜的LB琼脂平板上的细菌。 </li><li>准备6×1 L LB进行细菌培养。 </li></ol> 抗体的表达-第2天 <ol start="3"><li>废钢板LB培养基的殖民地。加入该悬浮液中的细菌，以6×1升LB氨苄青霉素（100毫克/升）的存在下温育，直到OD600达到0.5。 </li><li>外径达到0.5时，温度降低到20°C和RPM为115人。当温度下降到〜20℃下，加无水四环素（100微升的DMF为1mg/ml至1 L）发起的诱导蛋白的表达，并孵育5小时，在20°C与正常晃动。 </li><li>在4000×g离心15分钟，在1升的离心瓶中收获细菌。倾出上清液尽可能地。在冰上保持收获的细菌4℃冷室中过夜。 </li></ol> 释放的Fv分子从细菌-第3天 <ol start="6"><li>在150毫升的Fv释放缓冲区（的50mM Tris pH值8.0，20％蔗糖，1.0mM的EDTA）重悬细菌。 </li><li>用磁性搅拌棒搅拌的同时，加入溶菌酶至0.1毫克/毫升，并保持细菌在冰上放置30分钟。此后，添加氯化镁 2.0毫米，并保持在冰上10-15分钟。 </li><li>离心处理过的细菌在4℃下以20,000 xg离心30分钟细菌spheroblasts都应该去沉淀，在上清液中释放的Fv分子。 </li><li>透析的上清液对4.0升的洗涤缓冲液（20毫摩尔Tris，100mM NaCl的pH为8.0）在冷室中进行至少4小时。在年底的一天，改变新鲜透析液，透析过夜。由于在溶液中的蔗糖，透析液的体积将增加约50％。多余的透析管应该被使用。 </li></ol> IMAC纯化和FPLC - 4天 <ol start="10"><li>在50毫升管与透析缓冲液中平衡10毫升床体积的IMAC纯化树脂（见专栏1）。 </li><li>从油管，将透析液转移的10mM MgCl 2的添加，增加至200毫升的体积。混合预平衡树脂和孵育30分钟，在4°C </li><li>包与培养树脂的空柱，并用5倍柱床体积的洗涤缓冲液中，然后5倍柱​​床体积的洗涤缓冲液，10 mM咪唑，30 mM咪唑每个洗涤树脂。 </li><li>用20毫升的洗涤缓冲液加300 mM咪唑洗脱结合的蛋白。 </li><li>运行浓缩抗体通过Superdex 200柱用洗涤缓冲液预平衡的。游泳池含有FV（典型峰显示保留量17.6毫升）一起集中。确定样本浓度，使用的抗体在280nm处的消光系数。 </li></ol>

Watch this Scientific Journal Video about Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies at JoVE.com

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

完整的原型脂膜的电压门控钾离子通道进入重组程序中描述的。重组的渠道都适合生化检测，电气录音，配体筛选和电子晶体学研究。这些方法可以有其它膜蛋白的结构和功能研究的一般应用。

重组脂膜的结构和功能研究的钾离子通道

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

reconstitution-kv-channel-into-lipid-membranes-for-structural

Universidad San Sebastian

Research

JoVE Journal

Biology

19.3K 次观看.  University of Texas Southwestern Medical Center at Dallas. 完整的原型脂膜的电压门控钾离子通道进入重组程序中描述的。重组的渠道都适合生化检测，电气录音，配体筛选和电子晶体学研究。这些方法可以有其它膜蛋白的结构和功能研究的一般应用。

视频: Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

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 ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination1-7. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism8-13. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.
Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA14. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotin-containing messages.
The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,14-16. To examine the function of these isolated ribosome complexes, peptidyl-transferase assays can be performed in the presence of the antibiotic puromycin1. To study structural changes in translational complexes, well established procedures can be used, such as i) crosslinking to specific amino acids14 and/or ii) alkylation protection assays1,14,17.

A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.

分离翻译含肽- tRNA的核糖体的功能和结构分析

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK2 complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted.

Nanodiscs are small discoid particles that incorporate membrane proteins into a small patch of phospholipid bilayer. We provide a visual protocol that shows the step-by-step incorporation of the MalFGK2 transporter into a disc.

一个循序渐进的步骤方法的到Nanodisc脂质颗粒的ABC转运蛋白的重组

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

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 ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

在小鼠的心房和心室肌细胞的分离及钾离子通道的录音

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

跨膜蛋白的重组，电压门控离子通道，KvAP，进入巨人单层囊的显微镜和膜片钳研究

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...