Name: determining membrane protein topology using fluorescence protease protection (fpp)
Uploaded: 2015-04-20T14:00:00.000+00:00
Duration: 8 min 14 s
Description: Watch this Scientific Journal Video about Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP at JoVE.com

We wish to thank the Calcium Imaging Core Facility at CMS for their help and guidance in image capture and analysis. This study was supported by the U.S. National Institutes of Health (NIH) HL102208 to N.A.B. This approach was originally pioneered by Holger Lorenz and Jennifer Lippincott-Schwartz at NIH.

1.代荧光蛋白嵌合体和验证<ol><li>附加绿色荧光蛋白（GFP）与感兴趣使用标准的重组DNA策略13中的蛋白质的氨基或羧基末端。 注意:虽然我们已经使用GFP的广泛应用，其他变型如青色荧光蛋白（CFP），黄色荧光蛋白（YFP），红色荧光蛋白和mCherry可以使用。正在不断取得荧光折叠效率，亮度和光稳定性的改进，和调查应利用最新一代的结构本身。 <ol><li>确认正确的DNA序列，并确保将GFP部分是在框架与感兴趣的蛋白质。使用标准的重组DNA策略21进行测序。 注:很多机构都核心DNA设施，研究者鼓励联系以获取更多信息。 </li><li>对于单程或双程膜蛋白，产生两个氨基和羧基末端的版本融合蛋白19的。 </li><li>对于多生成树蛋白插入推定膜外循环内的GFP，以及末端。产生跨膜嵌合体与附着于靶蛋白的氨基末端的荧光融合蛋白时作为信号序列被蛋白水解裂解通常一旦该蛋白已经通过对ER位子传递特别小心。 </li><li>在现存的信号序列的情况下，插入荧光融合蛋白远端切割位点，以便产生含有完整的氨基末端标记的荧光蛋白的成熟蛋白质。使用用于嵌合体21的生成标准的重组DNA的策略。 </li></ol></li><li>确定足以适当地本地化的荧光信号在感兴趣的细胞系的存在。 <ol><li>使用下appropri GFP的固有荧光观察下荧光显微镜的细胞吃了过滤器设置的条件。对于包括510-560纳米的GFP过滤器设置，确认正确的过滤器用于采用的荧光。 注:足够的信号达到评估时，用肉眼提供了足够强的信号，该信号驻留在其中的细胞结构很容易上述背景下解决。此外，绿色荧光蛋白的染色模式应该是与该标记相应的细胞器的一致，并优选相同的，天然的未标记的蛋白质。 </li></ol></li><li>确定是否基于所述蛋白被评估稳定或瞬时转染的方法将被使用。 注:稳定转染通常得到的表达水平低于瞬时系统。虽然瞬时表达产生的蛋白和增加信号强度（〜5-24小时转染后视蛋白）的高水平表达，这也可导致过度的工件，如蛋白质AGGRegation或蛋白靶向通路的饱和，从而导致错误的亚细胞定位。 </li></ol> 2.转染细胞和细胞培养的注:多种细胞是适合于FPP分析，包括HEK293，HeLa细胞，COS-7和CHO细胞14。下面的描述是基于表达膜蛋白在HEK293细胞。 <ol><li>生长的人胚胎肾细胞（的Hek293）以贴壁单层在6ml的Dulbecco氏改良的Eagles培养基（DMEM）中，在37℃补充有5％胎牛血清（FBS），1％丙酮酸钠，和250微克/毫升青霉素/链霉素下用5％的CO 2。执行在层流组织培养罩的所有工作用的细胞。生长的细胞在培养基中直到〜的T-75烧瓶中80％汇合。 </li><li>使用任何标准的高效率，低毒性的转染方法，包括脂质，viral-或电为基础的技术，以产生transien转染细胞吨或稳定表达。通常为2微克每1×10 6个细胞的质粒DNA给出了良好的重现性蛋白表达。保持在CO 2能够组织培养箱细胞为5-48小时在适当的组织培养基中，在37℃。 </li><li>监测细胞使用荧光显微镜的GFP融合蛋白的表达，采用的GFP的固有荧光信号，根据制造商的协议使用适当的过滤器集。荧光信号通常达到内24-72小时转染后一最大值。使用的细胞时，荧光信号达到最大电平。监测蛋白表达的免疫印迹分析使用针对感兴趣的任一​​蛋白或GFP的部分的抗体的水平。 </li><li>实验分析板细胞上涂盖玻片。查看或者下一个活细胞配置或以下的固定和安装在显微镜幻灯片。板的细胞来实现在24-48小时的60％-80％汇合。 <ol><li>外套盖玻片用聚赖氨酸溶液（0.1毫克/毫升），这样整个表面被覆盖。孵育下紫外光在室温搅拌30分钟，随后在细胞培养孵化器60分钟，在37℃。去除多余的多聚赖氨酸和冲洗的PBS 3倍的盖玻片。 </li><li>替代地，盘上的腔盖玻璃组成的细胞培养板用光学玻璃底的细胞，以允许直接成像油浸目标。在实际的显微镜设置取决于可用的研究者在设备上，但应能迅速解决的变化。根据细胞类型和细胞形状，细胞应为约60-90％汇合的FPP分析的时间。 </li></ol></li></ol> 3.荧光显微镜安装<ol><li>使用的最大信号采集和空间分辨率，如60X，1.42 NA油浸物镜高数值孔径（NA）油浸物镜。使用日Ë以下激光器激发荧光团:氩气488纳米（GFP和YFP）和561二极管（RFP，mCherry）。 </li><li>确定图像的每个结构的最佳曝光，增益和捕获率。调节激光的强度和照射时间，以减少光漂白（见步骤7.1.2）。 </li><li>用于使用多个荧光团（ 例如 ，可溶性标记在音乐会的膜蛋白标志物膜通透性），选择适当的过滤器的研究设置以最小化信号的串扰和提高信号噪声比。 </li></ol> 4.建立最佳条件等离子膜通透性<ol><li>除去从细胞中只表达可溶性的荧光蛋白质的细胞培养基（ 例如绿色荧光蛋白，红色荧光蛋白）。在KHM缓冲洗细胞3次（每次1分钟）（110毫醋酸钾，3mM的MgCl 2的，20毫米的HEPES-氢氧化钠，pH为7.2）。在室温下执行与细胞的实验和所有试剂。 </li><li>地方细胞ØÑ​​盖玻片（参见步骤2.4）上的荧光显微镜阶段KHM缓冲器，并收集第一图像，其表示该控制"非透化"阶段。 </li><li>以选择性地透质膜，在KHM缓冲器添加毛地黄皂苷（30μM）细胞中。灌注细胞用缓冲液（缓冲液+毛地黄皂苷）中/速率为5毫升分钟为10-60秒。 </li><li>通过逐渐增加毛地黄皂苷的浓度确定最佳的毛地黄皂苷浓度。当有荧光信号的内10-60秒毛地黄皂苷申请完全丧失从可溶性EGFP最优毛地黄皂苷浓度达到。对于许多细胞应用的10-50微米毛地黄皂苷是足够通透的细胞膜。 <ol><li>对于所有的应用程序都使用毛地黄皂苷的尽可能低的浓度。毛地黄皂苷透了透化背景信号后，采集图像。 </li></ol></li><li>确认感兴趣的GFP蛋白嵌合体是膜在通过确认蜂窝荧光信号的保留以下质膜16的毛地黄皂苷透化tegrated。 <ol><li>表达一个特定的细胞器荧光蛋白23。线粒体蛋白质在这方面16， 例如 ，pDsRed2-三刀载体很好地工作。决定按照步骤2.1中所述的最佳表达条件。 </li><li>确认毛地黄皂苷浓度是适合于通过确保所使用的细胞类型的细胞内细胞器的形态保持不变通过长时间（最多60分钟）暴露于毛地黄皂苷。 注意:使用抗体对各种细胞区室用标准免疫荧光显微镜的协议，如溶酶体（ 例如 ，LAMP1），高尔基（ 例如 ，TGN38），内质网（ 例如 ，钙联接蛋白），内体（ 例如 ，Rab5的），线粒体（ 例如 ，细胞色素C）。如果细胞内的细胞器形态/完整性急剧变化奥雅纳R 60分钟，它是可能的毛地黄皂苷浓度过高，并且需要在毛地黄皂苷浓度和/或曝光时间的减少。 注:毛地黄皂苷是吸入和皮肤接触有毒。穿戴合适的个人防护装备（PPE）。 </li></ol></li></ol>荧光蛋白5.蛋白酶处理<ol><li>洗细胞中KHM缓冲，然后添加蛋白酶（蛋白酶K;在KHM缓冲器50微克/毫升）直接到细胞上。灌注细胞以5ml使用重力给料的灌注系统连续浴中的制剂/分钟的流速。 <ol><li>在50毫升的注射器商店的解决方案和聚乙烯管连接独立储到歧管与在浴（5毫升/分钟流速）的单一出口。定位的吸入吸管以保持恒定的（0.5毫升）浴体积和通过灌注容器之间手动切换实现溶液交换。 </li></ol></li><li>收集图像，以确定是否所述氟escent信号持续或降级。 &gt; 90％，比30-60秒的初始信号强度的损失是与GFP蛋白嵌合体的蛋白水解降解相一致（见代表结果）。 </li><li>如果没有信号衰减是用蛋白酶K观察到的，当预计这种损失，取代蛋白酶K溶液用胰蛋白酶（4-8毫米）中KHM缓冲器。如果需要的话，利用蛋白酶K和胰蛋白酶的混合物。没有必要以淬灭蛋白酶活性。 </li></ol>对于质膜蛋白质与外部域6.替代方法<ol><li>对于等离子的膜蛋白，进行检测的，以洋地黄通透前外部荧光蛋白嵌合体。洗在KHM缓冲器盖玻片的细胞（5毫升/分钟×3分钟），并采取预蛋白酶处理的图像和量化的预蛋白酶荧光信号，如步骤7。 </li><li>细胞暴露于蛋白酶（蛋白酶K; 50微克/毫升胰蛋白酶或4-8毫米KHM缓冲液中）在叔他通过灌注细胞KHM含蛋白酶的媒体（流速5毫升/分钟）没有毛地黄皂苷的。收集荧光蛋白的图像，以确定如何快速的信号消失要不了多久，信号仍然存在。 </li><li>毛地黄皂苷透化前，淬火所有蛋白酶活性通过在含有10％血清（FBS）的细胞培养基洗涤细胞三次（每次1分钟）。 </li><li>通过用KHM缓冲液（5毫升/分钟流速）进行2分钟灌注洗细胞。获得的预通透图像。透化细胞膜如步骤4中所述。 </li><li>如在步骤5中所述收集下列蛋白酶加成图像。 </li></ol> 7.图像分析<ol><li>使用倒置显微镜能够"活细胞"的记录，其中，荧光信号的损耗可以实时监测。连续监测，采用相同的参数（曝光时间，增益，激光强度等 ）。 <ol><li>确定EXPERimental参数，通过观察从控制条件（ 即 KBH单独的缓冲液）下感兴趣的GFP蛋白的荧光信号。调整在计算机驱动的显微镜的增益，激光强度和曝光时间设置，以确保有信号强度没有明显的损失超过10-20分钟的时间。 注意:信号强度不是一个绝对值，而是基于显微镜系统和设置可用于每个PI上。监测变化在相对荧光强度。 </li></ol></li><li>此外，治疗的细胞在设定的时间点洋地黄其次是蛋白酶，并修复细胞安装在盖玻片成像之前。确定时间点凭经验按照步骤4和5。初步调查程序，从0开始，30，60，120和300秒后添加洋地黄或蛋白酶。 <ol><li>固定用的固定剂的足量（在PBS中3.7％的多聚甲醛），以充分淹没的Th细胞（20分钟室温）E细胞。 </li><li>通过用含有20mM甘氨酸（3×5分钟）的PBS漂洗细胞淬灭残余的多聚甲醛。 </li><li>摩盖玻片通过从盖玻片除去多余的液体和反相盖玻片上50微升安装媒体上的玻璃显微镜载玻片进行成像。允许幻灯片前成像隔夜干燥。 </li></ol></li><li>收集荧光显微镜图像（根据适当的过滤器设置与所使用的荧光团兼容）通过使用计算机控制的荧光显微镜视频图像捕捉功能。 </li><li>定量荧光信号强度，作为孵化条件的功能。获得的感兴趣区域（ROI）的区域内包围单个小区中的平均像素强度。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+protease+protection+reveals+the+topology+of+the+integral+membrane+protein+Lemur+Tyrosine+Kinase+2+(LMTK2).">Fluorescence protease protection reveals the topology of the integral membrane protein Lemur Tyrosine Kinase 2 (LMTK2).</a> Am J Physiol. 304 (2), C164-C169 (2012).</li><li>Tagawa, A., 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=Assembly+and+trafficking+of+caveolar+domains+in+the+cell:+caveolae+as+stable,+cargo-triggered,+vesicular+transporters.">Assembly and trafficking of caveolar domains in the cell: caveolae as stable, cargo-triggered, vesicular transporters.</a> J Cell Biol. 170, 769-779 (2005).</li><li>Rizzuto, R., 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+gene+specifying+subunit+VIII+of+human+cytochrome+c+oxidase+is+localized+to+chromosome+11+and+is+expressed+in+both+muscle+and+non-muscle+tissues.">A gene specifying subunit VIII of human cytochrome c oxidase is localized to chromosome 11 and is expressed in both muscle and non-muscle tissues.</a> J Biol Chem. 264, 10595-10600 (1989).</li><li>Rizzuto, R., Brini, M., Pizzo, P., Murgia, M., Pozzan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+green+fluorescent+protein+as+a+tool+for+visualizing+subcellular+organelles+in+living+cells.">Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells.</a> Curr Biol. 5, 635-642 (1995).</li><li>Lorenz, H., Windl, O., Kretzschmar, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+phenotyping+of+secretory+and+nuclear+prion+proteins+associated+with+inherited+prion+diseases.">Cellular phenotyping of secretory and nuclear prion proteins associated with inherited prion diseases.</a> J Biol Chem. 277, 8508-8516 (2002).</li><li>Ben-Gedalya, T., 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=Cyclosporin-A-induced+prion+protein+aggresomes+are+dynamic+quality-control+cellular+compartments.">Cyclosporin-A-induced prion protein aggresomes are dynamic quality-control cellular compartments.</a> J Cell Sci. 124, 1891-1902 (2011).</li><li>Rogers, 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=Epitope+mapping+of+the+Syrian+hamster+prion+protein+utilizing+chimeric+and+mutant+genes+in+a+vaccinia+virus+expression+system.">Epitope mapping of the Syrian hamster prion protein utilizing chimeric and mutant genes in a vaccinia virus expression system.</a> J Immunol. 147, 3568-3574 (1991).</li><li>Lisenbee, C. S., Karnik, S. K., Trelease, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+and+mislocalization+of+a+tail-anchored+GFP+redefines+the+identity+of+peroxisomal+ER.">Overexpression and mislocalization of a tail-anchored GFP redefines the identity of peroxisomal ER.</a> Traffic. 4, 491-501 (2003).</li><li>Brandee, A. 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=Mislocalization+and+degradation+of+human+P23H-Rhodopsin-GFP+in+knockin+mouse+model+of+retinitis+pigmentosa.">Mislocalization and degradation of human P23H-Rhodopsin-GFP in knockin mouse model of retinitis pigmentosa.</a> Invest Ophthalmol Vis Sci. 52, 9728-9736 (2011).</li><li>Kuznetsov, A. V., 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=Analysis+of+mitochondrial+function+in+situ+in+permeabilized+muscle+fibers,+tissues+and+cells.">Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells.</a> Nat Protoc. 3, 965-976 (2008).</li></ol>

The authors declare no conflict of interest.

膜蛋白的正确方向和拓扑是为他们的正常功能是必不可少的。尽管认识膜蛋白拓扑的重要性，有许多蛋白质​​的量这样的数据被完全缺乏。 FPP的提供确定膜蛋白拓扑的一个简单而有效的方式，并且是可以由大多数实验室中进行。在FPP方法比以前的方法测定蛋白质拓扑能提供显著优势。例如，没有必要有定向感兴趣31的蛋白质的再次各种域上的多个抗体的面板。在许多情况下，抗体的这?...

质膜，以及众多的细胞内膜，作为分离两个隔室的含水的障碍。在质膜的情况下，该分离是外部之间和小区内;对于细胞内细胞器是细胞质和细胞器内腔之间。例如，内质网（ER）膜分离的ER从胞质还原环境1管腔内氧化环境。膜蛋白合成的雌激素受体 ​​相关的核糖体，并实现其内质网膜2内做出最终的拓扑结构。收购合适的膜方向和拓扑的蛋白质是其正常功能的关键。正确拓扑允许膜蛋白有关的结构域与其结合配偶相互作用，它允许发生临界翻译后修饰，并在质膜蛋白质的情况下，允许细胞进行交互和响应吨o它的环境。充分理解的膜蛋白的功能，它显然是必须知道这样的蛋白质被定向相对于在其中驻留的膜， 也就是说 ，它的膜拓扑结构。除了 ​​获得的基础科学知识，理解的膜蛋白和其中蛋白质表面的各方面暴露于不同环境的拓扑已标记的临床意义，因为膜蛋白包含多数药理靶3。直到最近，接近已确定需要时间和金钱的大量投资或已经所需的试剂，是很难得的膜蛋白的拓扑结构。 既，在硅片的实验和方法已被用于确定驻留在质膜内蛋白的膜拓扑结构。因为基于INDIVI的评价疏水性跨膜结构域的第一个预测双氨基酸3，众多的预测算法，现已在互联网上，而只要求蛋白质的氨基酸序列的知识。然而，假设常常是中央到这样的建模程序，假设，可导致拓扑4,5的不正确的分配。此外，虽然这些基于计算机的预测可以试探性地指派跨膜区域，它们不总是确定的氨基或蛋白质的羧基末端是否在细胞质中，细胞器腔或细胞外。即使具有增加的计算能力，以及利用机器学习算法6中，这样的数据仍然是一个模型，并且必须用实验获得的数据进行验证。直接的实验测定膜拓扑结构采用了与整个蛋白质，其中已经取得了他们的免疫反应的评估之前和细胞通透性后散发称为抗原决定簇的单克隆抗体的面板已经开展。此方法要求一组抗体，其可能不提供对感兴趣的蛋白质。 另一种策略是工程表位的标记，如myc基因或血凝素（HA）到各个位置的蛋白质，再其次是确定免疫前和膜通透性后。除了 ​​免疫标记，酶促标记（包括碱性磷酸酶，β半乳糖苷酶，或β内酰胺酶）和化学修饰，如半胱氨酸扫描都被用来确定膜蛋白拓扑7,8。用于质膜蛋白的拓扑映射的附加方法依赖于一个稍微不同的方式来表位标签。在该方法的标签序列是N联糖基化的共有序列NXS / T。由于糖基化作用，只有当这样的序列是存在于所述生物合成途径中，标签在一管腔相对于存在的内腔发生一个胞质区室是很容易观察到在SDS-PAGE凝胶质量偏移。这样的方法已被应用到multispanning离子通道CFTR 9。虽然所有这些方法都在使用，但很明显，它们都需要在分子生物学相当大的投资，以产生和测序所述歧管结构。 以确定关于位于细胞内细胞器内的跨膜蛋白拓扑信息已被证明是更具挑战性。基于荧光技术的应用然而，已经取得了膜拓扑简单了很多的决心。的双分子荧光互补（附设）的技术依赖于一种荧光蛋白的两个非荧光片段之间的相互作用，恢复该蛋白10的荧光性质。虽然用于确定在体内 10的蛋白质-蛋白质相互作用的最初描述，这种方法也被用于以确定膜蛋白拓扑在植物细胞11。然而，该方法也耗费时间，因为它需要的产生不仅融合蛋白与感兴趣的蛋白质，但也有多种靶向胞质溶胶或细胞器管腔融合蛋白的，并且需要知道哪些细胞内膜蛋白的兴趣在于。 另一种更简单的方法来判断膜蛋白的拓扑结构已被描述12。在测定中，荧光蛋白酶保护（FPP）要求GFP和目的基因之间的融合蛋白的产生。该方法是基于非特异性蛋白酶的GFP部分的相对无障碍;根据是否GFP是由驻留在细胞内的细胞器的管腔免受蛋白水解或通过存在于胞质溶胶暴露于蛋白水解。因此，如果在感兴趣的蛋白质的GFP部分朝向细胞质中，它会被暴露于蛋白酶活性和荧光信号丢失。相反，如果在感兴趣的蛋白质的GFP部分面临来自蛋白酶的环境"保护的"（如高尔基管腔），则荧光信号将持续存在。 以允许蛋白酶进入细胞，但不进入细胞内的膜隔室的胆固醇结合药毛地黄皂苷被使用。胆固醇是在脊椎动物中主要的甾醇，并在质膜相对于细胞内区室是特别丰富。糖苷毒素，毛地黄皂苷，是从植物洋地黄萃取。毛地黄皂苷具有丰富胆固醇的膜具有亲和性，在那里它会导致选择性隔膜permeabiliztion 14,16（ 图1）。除了允许小胞质成分出口，毛地黄皂苷透化还允许外源性分子，例如蛋白酶K或胰蛋白酶的条目。在FPP协议取FAC优势吨的质膜含有高达80％的细胞内胆固醇17的，而其它的细胞器，如ER，高尔基体，内体，线粒体，它们具有非常低的胆固醇含量，保持完整14。胆甾醇的选择性掺入质膜在许多真核细胞中被观察到，允许使用毛地黄皂苷依赖性质膜透化等不同的真核物种如S.酵母对人体14,18。对FPP分析提供了确定（a）一种蛋白质是否是膜结合的/关联的或在胞质溶胶和（b），该膜蛋白的结构域自由扩散朝向胞质溶胶或细胞器内腔的一个简单，快速，相当坚固的装置。应的膜蛋白有多个方向，信号将产生于主要形式和次要形式将不会被检测到。而可以有一些担心，另外一个GFP的部分与上感兴趣的蛋白的可能影响其功能和/或亚细胞定位，这实际上比实际更多的理论。实际上，许多研究已经清楚地表明，将GFP标记不改变蛋白质19,20的属性。

<table><tbody><tr><td>Digitonin</td><td>Calbiochem</td><td>300410</td><td></td></tr><tr><td>Proteinase K</td><td>Sigma</td><td>P2308</td><td></td></tr><tr><td>Trypsin</td><td>Sigma</td><td>T3924</td><td></td></tr><tr><td>Cav1-GFP</td><td>Addgene</td><td>44433</td><td></td></tr><tr><td>pAcGFP-1 Golgi</td><td>Clontech</td><td>632464</td><td></td></tr><tr><td>Polylysine</td><td>Sigma</td><td>P4707</td><td></td></tr><tr><td>Mounting Media</td><td>Dako</td><td>cS704</td><td></td></tr></tbody></table>

determining membrane protein topology using fluorescence protease protection (fpp)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

等离子膜通透性 高效质膜透化是通过使用可溶的荧光蛋白质（ 例如 ，绿色荧光蛋白，红色荧光蛋白）（步骤4）的测定。这些蛋白质，当在细胞中表达，可自由在细胞质中扩散，并且当质膜用毛地黄皂苷（ 图2）被透化都将丢失。荧光信号的完全消失，应该发生在10〜60秒毛地黄皂苷应用。所感兴趣的蛋白与细胞内细胞器相关联的确认是通过注意荧?...

Watch this Scientific Journal Video about Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP at JoVE.com

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

确定膜蛋白拓扑使用荧光蛋白酶保护（FPP）

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...

determining-membrane-protein-topology-using-fluorescence-protease

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

17.7K 次观看.  Chicago Medical School. Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP. 

视频: Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity

When novel proteins are identified through affinity-based isolation and bioinformatics analysis, they are often largely uncharacterized. Antibodies against specific peptides within the predicted sequence allow some localization experiments. However, other possible interactions with the antibodies often cannot be excluded. This situation provided an opportunity to develop a set of assays dependent on the protein sequence. Specifically, a construct containing the gene sequence coupled to the GFP coding sequence at the C-terminal end of the protein was obtained and employed for these purposes. Experiments to characterize localization, ligand affinity, and gain of function were originally designed and carried out to confirm the identification of TMEM184A as a heparin receptor1. In addition, the construct can be employed for studies addressing membrane topology questions and detailed protein-ligand interactions. The present report presents a range of experimental protocols based on the GFP-TMEM184A construct expressed in vascular cells that could easily be adapted for other novel proteins.

A construct encoding TMEM184A with a GFP tag at the carboxy-terminus designed for eukaryotic expression, was employed in assays designed to confirm the identification of TMEM184A as a heparin receptor in vascular cells.

用GFP标记TMEM184A构建肝素受体身份确认

When novel proteins are identified through affinity-based isolation and bioinformatics analysis, they are often largely uncharacterized. Antibodies ...

科研

遗传学

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

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

膜脊柱：一个生化工具来确定膜蛋白的蛋白质 - 蛋白质相互作用<em&gt;在体内</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

生物工程

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes.
In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

Here, we present the detailed procedure of a recently developed protease assay platform utilizing N-terminal hexahistidine/maltose-binding protein and fluorescent protein-fused recombinant substrates attached to the surface of nickel-nitrilotriacetic acid magnetic agarose beads. A subsequent in-gel analysis of the assay samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is also presented.

重组融合蛋白在荧光蛋白酶检测平台中的应用及其凝胶内再生

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different tags, truncations, deletions, fusion partner insertions, and stabilizing mutations to find one that is not aggregated after extraction from the membrane. Furthermore, buffer screening to determine the detergent, additive, ligand, or polymer that provides the most stabilizing condition for the membrane protein is an important practice. The early characterization of membrane protein quality by fluorescent size exclusion chromatography provides a powerful tool to assess and rank different constructs or conditions without the requirement for protein purification, and this tool also minimizes the sample requirement. The membrane proteins must be fluorescently tagged, commonly by expressing them with a GFP tag or similar. The protein can be solubilized directly from whole cells and then crudely clarified by centrifugation; subsequently, the protein is passed down a size exclusion column, and a fluorescent trace is collected. Here, a method for running FSEC and representative FSEC data on the GPCR targets sphingosine-1-phosphate receptor (S1PR1) and serotonin receptor (5HT2AR) are presented.

The present protocol describes a procedure to perform fluorescent size exclusion chromatography (FSEC) on membrane proteins to assess their quality for downstream functional and structural analysis. Representative FSEC results collected for several G-protein coupled receptors (GPCRs) under detergent-solubilized and detergent-free conditions are presented.

通过荧光体积排阻色谱快速评估膜蛋白质量

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different ...

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

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

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

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

研究膜蛋白的构象动力学原位网站的荧光标记

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

生物学

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

蛋白膜覆盖含量：一个协议测试可溶性和不溶性蛋白质之间的相互作用在体外</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

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 ...

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.

Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker.

外周相关蛋白血浆膜分型的测定

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

Fluorescence-Detection Size-Exclusion Chromatography: A Technique to Identify the Integrity of Fluorescent Membrane Proteins upon Detergent Solubilization

Source: Bird, L. E., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/52357">Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli</a>. J. Vis. Exp. (2015)
In this video, we demonstrate a fluorescence-detection size-exclusion chromatography technique to screen for green fluorescent protein (GFP)-fused membrane protein stability in Escherichia coli following detergent solubilization. Proteins exhibiting a symmetrical peak in the size-exclusion profile with little or no free GFP indicate purified proteins with minimum degradation and aggregation, which can subsequently be selected for structure-function analysis.

荧光检测尺寸排阻色谱法:一种在去污剂溶解后鉴定荧光膜蛋白完整性的技术

Source: Bird, L. E., et al. Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli. J. Vis. Exp. (2015)
In this ...

确定膜蛋白拓扑使用荧光蛋白酶保护（FPP）

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