我们感谢美国国立卫生研究院（1R21CA138373站起来，对这项工作的财政支持（SU2C）癌症。HY是感激，2009年亿利奖由美国癌症研究，从悉尼金梅尔基金会金梅尔学者奖协会癌症研究中心（SKF - 08 - 101），和美国国家科学基金会教师早期职业奖（NSF0954819）。

1。克隆的注意事项<ol><li> NheI和BamHI酶切位点和5&#39; -磷酸两侧的利益的战区导弹防御系统的商业化准备寡核苷酸可以连接到pTox7（我们实验室的一个碱基对的插入，修改后直接酶 BamHI酶切位点14）（图3）消化顺序用BamHI和NheI。寡核苷酸是一个例子所示： 5&#39;ctagcTMDSEQUENCEg3“ 3“gTMDSEQUENCEcctag5” </li></ol> TMD的顺序应该是12-24残基（短序列大概拉长矢量编码的疏水残基）。为了调查的接口，顺序残留的插入和随之而来的残留删除ToxR的战区导弹防御系统的相对旋转的结果 。15,16最后，阿拉伯糖的浓度应在0.001和0.01％之间不同，应建立战区导弹防御系统的设计的4个变种（W / V）来确定观察到的最大的差异是在不同的战区导弹防御系统的序列之间的β-半乳糖苷酶信号的浓度;测试不同表达水平的建议，以确定下可以区分不同的亲和力最好的条件。除了阿拉伯糖和抗生素，IPTG的0.4毫米，可用于增强亲和力的差异不同的TMDS。 ToxR测量应至少一式四份。整个过程应重复至少三次不同质粒转换。 2。生长的细菌培养<ol><li>轻轻解冻FHK12主管冰和转移到15 ml培养管细胞（200μL）。加入质粒DNA（200纳克）和细胞在冰上孵育30分钟。 </li><li>热休克细胞孵育90小号在42℃，孵育2分钟冰。 </li><li>添加SoC媒体（800μL），并在37样品°摇晃一小时（300转）彗星</li><li>氯霉素（30微克/毫升）和阿拉伯糖（0.0025％W / V）一式三份在15毫升培养管改造的混合物50μL接种5毫升LB培养基。孵育样品在37℃，摇晃20小时（300转） （或者5μL文化可以被用来在96孔板接种100μL介质处理大量样品时，这种方法非常有用，但错误会稍高。为了避免蒸发，这将导致错误，填补与媒体的最外层的水井，但不使用他们的样品，最后，双包装盖子和封口膜板之间的联合）。 </li></ol> 3。测量的β-半乳糖苷酶的活性<ol><li>板阅读器预热至28 ° C。 </li><li>转移到一个水库的Z -缓冲，具有较大的枪头，确保只占用上层（水层）。将一个96孔板的孔100μL新鲜准备Z-buffer/chloroform。转移到板一式四份井5μL每一种文化。忽略文化的四口井将作为空白。 </li><li>测量外径595 ​​的板，以确定细胞密度。 </li><li>加入50μL的Z-buffer/SDS板的所有油井。 10分析细胞板，摇板阅读器。确保细胞悬浮液是明确的裂解后，如果需要重复晃动一步。不完全溶解建议Z-buffer/chloroform是不新鲜配制。 </li><li>加入50μL新鲜配制Z-buffer/ONPG所有的井，并返回该板块的酶标仪和测量外径405每20分钟30秒。 </li><li>在使用下列公式（记住要减去空白）计算β-半乳糖苷酶的活性。外径405 /分的比例应该是使用所有数据点的OD 405范围0.0到1.0采用线性模型的拟合计算。 <img src="files/ftp_upload/2721/2721eq1.jpg" alt="公式1" /> 米勒的单位不同，有时在不同的日子记录。因此，这样的全球行动纲领“的参考构造应在每个测试测量。其值可用于ToxR值正常化。 </li></ol> 4。蛋白表达的控制<ol><li>执行免疫印迹验证甚至之间的结构蛋白的表达。结合在离心50μL一式三份的文化和离心（2000转，4分钟）。吹打取出上清，重悬在2个样品上样缓冲液的残留颗粒。 </li><li>在一个标准的8％凝胶负载7.5μL进行电泳125 V为1小时5分钟。传输后，孵化，与抗- MBP HRP标记抗体和可视化;嵌合蛋白有时看到一些大约48 kDa的降解产物在约70 kDa的观察。内源性的MBP也观察到在45 kDa的（见图5）。 </li></ol>5。控制适当的膜插入麦芽糖结合蛋白缺失的细胞系在使用，以评估适当的膜插入嵌合的战区导弹防御系统的构造。当最小的媒体作为唯一碳源生长与麦芽糖，只有正确位于周质麦芽糖结合蛋白的表达不可或缺的一种膜表达产物能够成长。 <ol><li>变换PD28细胞（如FHK12细胞所述），接种2毫升LB培养基。细胞增长37 ° C的晃动（300转）过夜。 </li><li>在3500转离心沉淀细胞，10分钟，4 ° C和再悬浮在PBS（2毫升）洗轻柔吹打一个大型尖或温和的震荡。颗粒的细胞（如上述），第二次用PBS洗，颗粒，最后悬浮在PBS（1毫升）。 </li><li>使用25μL重悬细胞接种5毫升，一式三份，并培育基本培养基，在37 °（300转）用颤抖的彗星。 15-25小时，大约每2小时转移到96孔板和使用的板块，读者的阅读每个样品200μLOD 595读数。 </li></ol> 6。代表性的成果： ToxR转录报告基因检测分析如图4所示的跨膜结构域的寡聚化倾向使用的一个例子。以前，我们进行了调查跨膜齐聚的multispanning膜的组成蛋白质的潜伏膜蛋白1（LMP - 1）通过各种技术，包括ToxR 14个跨膜结构域五（TM5）表现出强烈的倾向oligomerize的;这是证明高米勒单位，与阳性对照，GPA，以及建立dimerizing序列。一个有害突变，D150A，TM5降低oligomerize序列的能力。 LMP - 1的TM1不显着oligomerize和展品一个非常低的米勒单位略高于空白，非FHK12细胞转化的信号，信号。 <img src="/files/ftp_upload/2721/2721fig1.jpg" alt="图1" /> 图1。卡通描绘ToxR记者分析。 ToxR和LacZ基因转录的激活二聚体跨膜结构域（TMD）的驱动齐聚结果。 LacZ基因产物，β-半乳糖苷酶可以量化作为衡量oligomerize一个战区导弹防御系统的倾向。 <img src="/files/ftp_upload/2721/2721fig2.jpg" alt="图2" /> 图2。生产的光吸收物种O - nitrophenolate（ONP）的β-半乳糖苷酶结果的ONPG水解断裂。 <img src="/files/ftp_upload/2721/2721fig3.jpg" alt="图3" /> 图3。pToxR7质粒图谱。 <img src="/files/ftp_upload/2721/2721fig4.jpg" alt="图4" /> 图4。代表ToxR转录报告基因检测分析潜伏膜蛋白1的跨膜结构域的寡聚化倾向。跨膜结构域5（TM5）oligomerizes强烈，而跨膜结构域1（TM1）展品只有弱相互作用。 TM5 D150A突变显着降低能力oligomerize。 GPA是作为一个强大的二聚体阳性对照序列。空白表示未转换FHK12细胞。 <img src="/files/ftp_upload/2721/2721fig5.jpg" alt="图5" /> 图5。蛋白表达的免疫印迹。 <img src="/files/ftp_upload/2721/2721fig6.jpg" alt="图6" /> 图6。PD28互补的检测，以控制正确的膜插入到周质。阴性对照代表缺乏麦芽糖结合蛋白的构造。

<ol>
	<li>Parker, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligomerization+of+the+heptahelical+G+protein+coupling+receptors:+a+case+for+association+using+transmembrane+helices.">Oligomerization of the heptahelical G protein coupling receptors: a case for association using transmembrane helices.</a> Mini Rev Med Chem. 9, 329-339 (2009).</li><li>Mo, W., Zhang, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligomerization+of+human+ATP-binding+cassette+transporters+and+its+potential+significance+in+human+disease.">Oligomerization of human ATP-binding cassette transporters and its potential significance in human disease.</a> Expert Opin Drug Metab Toxicol. 5, 1049-1063 (2009).</li><li>von Heijne, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-protein+topology.">Membrane-protein topology.</a> Nat Rev Mol Cell Biol. 7, 909-918 (2006).</li><li>Van Horn, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+nuclear+magnetic+resonance+structure+of+membrane-integral+diacylglycerol+kinase.">Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase.</a> Science. 324, 1726-1729 (2009).</li><li>Hattori, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mg(2+)-dependent+gating+of+bacterial+MgtE+channel+underlies+Mg(2+)+homeostasis.">Mg(2+)-dependent gating of bacterial MgtE channel underlies Mg(2+) homeostasis.</a> Embo J. 28, 3602-3612 (2009).</li><li>Ulmschneider, M. B., Sansom, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acid+distributions+in+integral+membrane+protein+structures.">Amino acid distributions in integral membrane protein structures.</a> Biochim Biophys Acta. 1512, 1-14 (2001).</li><li>Deber, C. M., Goto, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+proteins+into+membranes.">Folding proteins into membranes.</a> Nat Struct Biol. 3, 815-818 (1996).</li><li>Gerber, D., Shai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+detection+of+hetero-association+of+glycophorin-A+and+its+mutants+within+the+membrane.">In vivo detection of hetero-association of glycophorin-A and its mutants within the membrane.</a> J Biol Chem. 276, 31229-31232 (2001).</li><li>Langosch, D., Brosig, B., Kolmar, H. p., Fritz, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimerisation+of+the+glycophorin+A+transmembrane+segment+in+membranes+probed+with+the+ToxR+transcription+activator.">Dimerisation of the glycophorin A transmembrane segment in membranes probed with the ToxR transcription activator.</a> J Mol Biol. 263, 525-530 (1996).</li><li>Russ, W. P., Engelman, 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=TOXCAT:+a+measure+of+transmembrane+helix+association+in+a+biological+membrane.">TOXCAT: a measure of transmembrane helix association in a biological membrane.</a> Proc Natl Acad Sci U S A. 96, 863-868 (1999).</li><li>Berger, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consensus+motif+for+integrin+transmembrane+helix+association.">Consensus motif for integrin transmembrane helix association.</a> Proc Natl Acad Sci U S A. 107, 703-708 (2010).</li><li>Oates, J., King, G., Dixon, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strong+oligomerization+behavior+of+PDGFbeta+receptor+transmembrane+domain+and+its+regulation+by+the+juxtamembrane+regions.">Strong oligomerization behavior of PDGFbeta receptor transmembrane domain and its regulation by the juxtamembrane regions.</a> Biochim Biophys Acta. 1798, 605-615 (2010).</li><li>Sal-Man, N., Gerber, D., Shai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hetero-assembly+between+all-L-+and+all-D-amino+acid+transmembrane+domains:+forces+involved+and+implication+for+inactivation+of+membrane+proteins.">Hetero-assembly between all-L- and all-D-amino acid transmembrane domains: forces involved and implication for inactivation of membrane proteins.</a> J Mol Biol. 344, 855-864 (2004).</li><li>Sammond, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmembrane+peptides+used+to+investigate+the+homo-oligomeric+interface+and+binding+hot-spot+of+the+oncogene,+latent+membrane+protein+1.">Transmembrane peptides used to investigate the homo-oligomeric interface and binding hot-spot of the oncogene, latent membrane protein 1.</a> , (2010).</li><li>Li, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimerization+of+the+transmembrane+domain+of+Integrin+alphaIIb+subunit+in+cell+membranes.">Dimerization of the transmembrane domain of Integrin alphaIIb subunit in cell membranes.</a> J Biol Chem. 279, 26666-26673 (2004).</li><li>Ruan, W., Becker, V., Klingmuller, U., Langosch, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interface+between+self-assembling+erythropoietin+receptor+transmembrane+segments+corresponds+to+a+membrane-spanning+leucine+zipper.">The interface between self-assembling erythropoietin receptor transmembrane segments corresponds to a membrane-spanning leucine zipper.</a> J Biol Chem. 279, 3273-3279 (2004).</li><li>Finger, C., Escher, C., Schneider, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+single+transmembrane+domains+of+human+receptor+tyrosine+kinases+encode+self-interactions.">The single transmembrane domains of human receptor tyrosine kinases encode self-interactions.</a> Sci Signal. 2, ra56-ra56 (2009).</li></ol>

ToxR转录报告基因检测是一个浅显的方式，以确定有oligomerize潜力的跨膜序列。由于内的细菌内膜发生的相互作用，这个实验绕过膜模仿的环境中学习系统的有效性相关的问题。这个实验，克隆到一个单一的质粒多个TMDS可以很容易地并行执行，并可以进行整个实验在96孔板格式，可用于高通量分析蛋白质序列的大量。一旦17已检测到的相互作用，可关键的功能残留审问突变分析，使所涉及的结构特征映射。在许多情况下，跨膜蛋白的晶体结构分析是有问题的的，需要替代的工具，如ToxR检测，建立功能的分子基础。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> BamHI位的限制酶</td><td> Invitrogen公司</td><td> 15201023 </td><td> Invitrogen公司的酶被发现比其他供应商更有效率</td></tr><tr><td> NheI限制酶</td><td> Invitrogen公司</td><td> 15444011 </td><td> Invitrogen公司的酶被发现比其他供应商更有效率</td></tr><tr><td> 15 mL培养管</td><td> Fisher Scientific则</td><td> 14 - 956 - 1J </td><td></td></tr><tr><td> SoC媒体</td><td> Teknova </td><td> S0225 </td><td>合适的音量和高压灭菌消毒。 </td></tr><tr><td> LB培养基</td><td> Sigma - Aldrich公司</td><td> L7275 </td><td>合适的音量和高压灭菌消毒。 </td></tr><tr><td>氯霉素</td><td> Sigma - Aldrich公司</td><td> CO 3的 78 </td><td>存放在冰箱的库存为30毫克/毫升的溶液中乙醇</td></tr><tr><td>阿拉伯糖</td><td> Fluka公司</td><td> 10839 </td><td>存放在冷冻水（W / V）的2.5％股份的解决方案</td></tr><tr><td> NA 2 HPO 4 </td><td> Sigma - Aldrich公司</td><td> S9390 </td><td></td></tr><tr><td>的NaH 2 PO 4 </td><td> Sigma - Aldrich公司</td><td> S9638 </td><td></td></tr><tr><td>氯化钾</td><td> Mallinckrodt化学品</td><td> 6858-06 </td><td></td></tr><tr><td>硫酸镁4 .7 H 2 O </td><td> Sigma - Aldrich公司</td><td> 63138 </td><td></td></tr><tr><td>十二烷基硫酸钠（SDS） </td><td> Sigma - Aldrich公司</td><td> L6026 </td><td></td></tr><tr><td> 2 - 硝基苯β- D -半乳糖苷（ONPG） </td><td> Sigma - Aldrich公司</td><td> 73660 </td><td></td></tr><tr><td> Z -缓冲</td><td></td><td></td><td> 16.1克钠2 HPO 4 5.5克的NaH 2 PO 4 0.75克氯化钾 0.246克硫酸镁4 化妆至1 L，pH值7.0 </td></tr><tr><td> Z-buffer/chloroform </td><td></td><td></td><td> 200毫升的β-巯基乙醇，2 mL氯仿，补至20 mL，与Z -缓冲。涡1分钟，1分钟800转的离心机。做出新的每块板。 </td></tr><tr><td> Z-buffer/SDS </td><td></td><td></td><td> 160毫克SDS溶解于10 mL Z -缓冲</td></tr><tr><td> Z-buffer/ONPG </td><td></td><td></td><td> 40毫克ONPG于10 mL Z -缓冲。做出新的每块板</td></tr><tr><td> β-巯基乙醇</td><td> Calbiochem </td><td> 444203 </td><td></td></tr><tr><td>抗- MBP单克隆抗体（HRP标记） </td><td> NEB </td><td> E8038S </td><td></td></tr><tr><td>最小媒体与麦芽糖</td><td></td><td></td><td> 1个M9的盐，0.4％麦芽糖，1毫克/毫升硫胺素，2毫米硫酸镁 </td></tr><tr><td> 96孔平底板</td><td> Sarstedt </td><td> 83.1835.300 </td><td></td></tr><tr><td>板阅读器</td><td> Beckman Coulter公司</td><td> DTX880模检测器</td><td></td></tr><tr><td>水浴</td><td> VWRI </td><td> 89032-204 </td><td></td></tr><tr><td>摇床</td><td> FormaScientific </td><td></td><td></td></tr></tbody></table>

transmembrane domain oligomerization propensity determined by toxr assay

蛋白质跨膜结构域的过于简单化的看法只是在磷脂双层锚早已disproven。跨膜蛋白在许多情况下，已经发展了高度复杂的行动机制。1-3的一种方式在膜蛋白可以调节自己的结构和功能是疏水螺旋的直接和具体的联系，形成结构化的跨膜低聚物4,5近来工作的重点分布在膜环境优先氨基酸在水溶液和不同的分子间作用力，驱动蛋白协会。6,7然而，在跨膜结构域的蛋白质的分子识别研究仍落后于那些滞后相比水溶性地区。一个主要的障碍依然存在：尽管跨膜齐聚可以实现显着的特异性和亲和力，他们协会的8直接测量是具有挑战性的。可应用的整合膜蛋白的功能研究的传统方法阻碍了固有的不溶性考试序列。生物物理研究代表跨膜结构域的合成肽获得的见解可以提供有用的结构的洞察力。然而，洗涤剂胶束或在这些研究中用来模仿细胞膜的脂质体系统的生物相关性往往是质疑;做肽在这些条件下采取类似母语的结构和其功能的行为，真正体现内的原生膜的行动模式？为了研究天然磷脂双层的跨膜序列之间的相互作用，Langosch实验室开发ToxR转录记者分析。利益的跨膜结构域嵌合蛋白与麦芽糖结合蛋白的位置，周质和ToxR表示提供的一份报告齐聚水平（图1）。 在过去的十年中，几个其他团体（如Engelman，DeGrado，夏嘉曦）进一步优化和应用这ToxR记者分析。10-13各种ToxR检测已成为金标准，以测试在细胞膜蛋白质相互作用。我们在此展示一个典型的实验操作，在我们的实验室，主要如下Langosch开发协议中进行。这个普遍适用的方法是非常有用的跨膜结构域自我协会在大肠杆菌中的分析大肠杆菌 ，其中β-半乳糖苷酶的生产是用来评估TMD齐聚倾向。战区导弹防御系统，诱导二聚化后，ToxR结合造成的β-半乳糖苷酶的LacZ基因调节的 CTX发起人。 ONPG除了lyzed细胞是通过比色读数。生产的光吸收物种O - nitrophenolate（ONP）（图2）中的β-半乳糖苷酶结果的ONPG水解断裂。

Watch this Scientific Journal Video about Transmembrane Domain Oligomerization Propensity determined by ToxR Assay at JoVE.com

Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

一个高效的程序，以评估单通跨膜结构域的寡聚化倾向（TMDS）描述。融合到ToxR的战区导弹防御系统组成的嵌合蛋白在大肠杆菌记者应变中表达。 TMD诱导齐聚的原因ToxR，记者​​蛋白的转录和生产，半乳糖苷酶的激活二聚体。

跨膜结构域齐聚倾向ToxR含量决定

The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven.  In many cases ...

transmembrane-domain-oligomerization-propensity-determined-toxr

Universidad San Sebastian

Research

JoVE Journal

Biology

15.2K 次观看.  University of Colorado at Boulder. 一个高效的程序，以评估单通跨膜结构域的寡聚化倾向（TMDS）描述。融合到ToxR的战区导弹防御系统组成的嵌合蛋白在大肠杆菌记者应变中表达。 TMD诱导齐聚的原因ToxR，记者​​蛋白的转录和生产，半乳糖苷酶的激活二聚体。

视频: Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin. In recent years, small molecules have been heavily analyzed as potential LC inhibitors based on their increased cellular permeability relative to larger therapeutics (peptides, aptamers, etc.). Lead identification often involves high-throughput screening (HTS), where large libraries of small molecules are screened based on their ability to modulate therapeutic target function. Here we describe a FRET-based assay with a commercial BoNT/A LC substrate and recombinant LC that can be automated for HTS of potential BoNT inhibitors. Moreover, we describe a manual technique that can be used for follow-up secondary screening, or for comparing the potency of several candidate compounds.

The botulinum neurotoxin type A light chain (BoNT/A LC) is a metalloprotease that enters motor neurons, cleaves its substrate SNAP-25, and disrupts neurotransmission, thereby resulting in flaccid paralysis. Utilizing a high-throughput-compatible FRET-based assay, large libraries of small molecules can be screened for their impact on BoNT/A LC enzymatic activity.

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and ...

科研

化学

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

生物化学

Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1. These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol2-4. In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target5. The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER6-12.

To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)13-15. The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.

In this report, we describe how surface plasmon resonance is used to detect toxin entry into the host cytosol. This highly sensitive method can provide quantitative data on the amount of cytosolic toxin, and it can be applied to a range of toxins.

表面等离子体共振检测到宿主细胞质的毒素易位

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1.  These toxins are secreted into the extracellular milieu, but they act upon ...

免疫与感染

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

遗传筛选隔离弓形虫宿主细胞的出口突变

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

Demonstration of the Sequence Alignment to Predict Across Species Susceptibility Tool for Rapid Assessment of Protein Conservation

The US Environmental Protection Agency Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool is a fast, freely available, online screening application that allows researchers and regulators to extrapolate toxicity information across species. For biological targets in model systems such as human cells, mice, rats, and zebrafish, toxicity data are available for a variety of chemicals. Through the evaluation of protein target conservation, this tool can be used to extrapolate data generated from such model systems to thousands of other species lacking toxicity data, yielding predictions of relative intrinsic chemical susceptibility. The latest releases of the tool (versions 2.0-6.1) have incorporated new features that allow for the rapid synthesis, interpretation, and use of the data for publication plus presentation-quality graphics.
Among these features are customizable data visualizations and a comprehensive summary report designed to summarize SeqAPASS data for ease of interpretation. This paper describes the protocol to guide users through submitting jobs, navigating the various levels of protein sequence comparisons, and interpreting and displaying the resulting data. New features of SeqAPASS v2.0-6.0 are highlighted. Furthermore, two use-cases focused on transthyretin and opioid receptor protein conservation using this tool are described. Finally, SeqAPASS&#39; strengths and limitations are discussed to define the domain of applicability for the tool and highlight different applications for cross-species extrapolation.

Here, we present a protocol to utilize the latest version of the US Environmental Protection Agency Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool. This protocol demonstrates the application of the online tool to rapidly analyze protein conservation and provide customizable and easily interpretable predictions of chemical susceptibility across species.

演示序列比对以预测跨物种敏感性的工具，用于快速评估蛋白质保存

The US Environmental Protection Agency Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool is a fast, freely available, online ...

遗传学

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their&#160;cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050H60A H307A variant.

X射线晶体学研究热木体马里蒂玛M42氨基肽TmPep1050的寡聚态过渡

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion ...

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence (TIRF) Microscopy

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient association of pore-forming and auxiliary subunits, lateral diffusion, and clustering with other proteins. However, the relationship between lateral diffusion and function is poorly understood. To approach this problem, we describe how lateral mobility and activity of individual channels in supported lipid membranes can be monitored and correlated using total internal reflection fluorescence (TIRF) microscopy. Membranes are fabricated on an ultrathin hydrogel substrate using the droplet interface bilayer (DIB) technique. Compared to other types of model membranes, these membranes have the advantage of being mechanically robust and suitable for highly sensitive analytical techniques. This protocol measures Ca2+ ion flux through single channels by observing the fluorescence emission of a Ca2+-sensitive dye in close proximity to the membrane. In contrast to classical single-molecule tracking approaches, no fluorescent fusion proteins or labels, which can interfere with lateral movement and function in the membrane, are required. Possible changes in ion flux associated with conformational changes of the protein are only due to protein lateral motion in the membrane. Representative results are shown using the mitochondrial protein translocation channel TOM-CC and the bacterial channel OmpF. In contrast to OmpF, the gating of TOM-CC is very sensitive to molecular confinement and the nature of lateral diffusion. Hence, supported droplet-interface bilayers are a powerful tool to characterize the link between lateral diffusion and the function of ion channels.

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence TIRF Microscopy

This protocol describes how to use TIRF microscopy to track individual ion channels and determine their activity in supported lipid membranes, thereby defining the interplay between lateral membrane movement and channel function. It describes how to prepare the membranes, record the data, and analyze the results.

使用全内反射荧光（TIRF）显微镜对脂质双层中的横向迁移率和离子通道活性进行单分子成像

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient ...

使用生物物理测定表征 PROTAC 中三元复合物的形成

The Development and Application of Biophysical Assays for Evaluating Ternary Complex Formation Induced by Proteolysis Targeting Chimeras (PROTACS)

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics and thermodynamics of the interactions involved contribute to efficiency of ubiquitination and resulting degradation of the protein. Biophysical techniques such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC) provide valuable information that can be used in the optimization of those interactions. Using two model systems, a biophysical assay tool kit for understanding the cooperativity of ternary complex formation and the impact of the &#39;hook effect&#39; on binding kinetics was established. In one case, a proteolysis targeting chimera (PROTAC) molecule that induced ternary complex formation between Brd4BD2 and VHL was evaluated. The heterobifunctional molecule, MZ1, has nM affinities for both the Brd4BD2 protein (SPR KD = 1 nM, ITC KD = 4 nM) and the VHL complex (SPR KD = 29 nM, ITC KD = 66 nM). For this system, robust SPR, BLI, and ITC assays were developed that reproduced published results demonstrating the cooperativity of ternary complex formation. In the other case, a molecule that induced ternary complexes between a 46.0 kDa protein, PPM1D, and cereblon [CRBN (319-442)] was studied. The heterobifunctional molecule, BRD-5110, has an SPR KD = 1 nM for PPM1D but much weaker binding against the truncated CRBN (319-442) complex (SPR KD= ~ 3 &#181;M). In that case, the binding for CRBN in SPR was not saturable, resulting in a &#34;hook-effect&#34;. Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.

作者聚焦：评估用于表征 PROTACS 三元复合物的生物物理检测 

Here we describe protocols for the biophysical characterization of ternary complex formation induced by proteolysis targeting chimeras (PROTACS) that involve the ubiquitin ligases Von Hippel-Lindau E3 ligase (VHL) and Cereblon (CRBN). Biophysical methods illustrated herein include surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC).

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics ...

跨膜结构域齐聚倾向ToxR含量决定

摘要

探索更多视频

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

表面等离子体共振检测到宿主细胞质的毒素易位

遗传筛选隔离弓形虫宿主细胞的出口突变

演示序列比对以预测跨物种敏感性的工具，用于快速评估蛋白质保存

X射线晶体学研究热木体马里蒂玛M42氨基肽TmPep1050的寡聚态过渡

使用全内反射荧光（TIRF）显微镜对脂质双层中的横向迁移率和离子通道活性进行单分子成像

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用