Name: from constructs to crystals – towards structure determination of β-barrel outer membrane proteins
Uploaded: 2016-07-04T13:00:00.000+00:00
Duration: 9 min 55 s
Description: Watch this Scientific Journal Video about From Constructs to Crystals â€“ Towards Structure Determination of Î²-barrel Outer Membrane Proteins at JoVE.com

We would like to thank Herve Celia of the CNRS for providing the UV images and Chris Dettmar and Garth Simpson in the Department of Chemistry at Purdue University for providing the SONICC images. We would like to acknowledge funding from the National Institute of Diabetes and Digestive and Kidney Diseases and the Intramural Research Program at the National Institutes of Health. Additionally, we would like to acknowledge additional funding from the National Institute of General Medical Sciences (A.M.S. and C.J.), National Institute of Allergy and Infectious Diseases (N.N. 1K22AI113078-01), and the Department of Biological Sciences at Purdue University (N.N.).

1.克隆与表达注意:为了使结构研究，高度纯化的蛋白质的足够的数量必须准备，这通常与目标β桶外膜蛋白在大肠杆菌中的克隆和表达（OMP）开始大肠杆菌 （ 图4）。迄今为止，所有的β桶的OMP结构，包括那些结构线粒体VDAC，已经从细菌表达的蛋白11的。这里，一般协议提出了克隆，并直接表达β桶的OMPs为（1）天然表达到细菌膜和（2）表达成包裹体在体外复性17。 <ol><li>设计表达构建<ol><li>获取或购买的密码子优化的目标OMP基因。 </li><li>购买或获得含有一个周质定位信号序列的T7表达载体（i）中，N末端6X组氨酸标签，并在体内表达于膜或（ii）在不进行表达，以包涵体在体外复性周质定位信号序列的TEV蛋白酶位点4,18,19。 注意:N-末端蛋白酶可切割的6倍或10倍，组氨酸标签将用于纯化的简单方法。与可溶性蛋白，变化亲和标签（组氨酸，链球菌，GST 等 ），亲和标记的位置，和蛋白酶位点的包容（TEV，肠激酶，凝血酶等 ）的选择用于后续标签去除。 </li><li> PCR扩增用合适的引物和亚克隆到表达载体中的靶序列。使用独立结扎的克隆（LIC）20,21或常规克隆技术（限制性内切酶/连接）进行亚克隆。然而，LIC可以促进高吞吐量，允许广大各种结构（截断，各种标签，多种启动子）的并行克隆更多容易。 </li></ol></li><li> 体内膜定位的表达<ol><li>使用序列分析来验证目标β桶的OMP的下游克隆并在帧与信号序列（ 即，规划环境地政局，OmpA的）中的表达构建体。 注意:信号序列指导新生链的二段易位子用于分泌到周质和随后组装成外膜。 </li><li>变换构造成细菌表达的表达菌株吹打1.0微升质粒入50μl的BL21（DE3）化学感受态细胞，并通过上下抽吸轻轻混匀。在冰上孵育30分钟。 </li><li>在42ºC加热脉冲用于使用30秒的水浴，然后放回冰上1分钟。 </li><li>加入1ml预热SOC培养基和使用台式摇床培养箱以1,000rpm于37ºC振摇1小时。 </li><li>板100微升细胞在LB琼脂平板含有适当抗菌素IC和孵育过夜在37ºC反转。 </li><li>通过接种5ml的LB +抗生素文化与单个菌落进行小规模试验的表达。重复5-10殖民地。 注意:由于β桶OMP的潜在毒性和对基础表达水平的依赖的，集落到集落变性有时观察到。因此，筛选多个殖民地，建议对每个结构进行测试。对于小规模的表达试验，而不是传统的5毫升培养，生长在125ml带挡板锥形瓶中25毫升培养建议。这些条件往往更好地反映了大规模增长会发生什么。此外，这是一个好主意，还筛选各类文化传媒（TB，LB，的2xYT，M9 等 ），因为不同水平的成功都依赖于靶蛋白上的报道。 <ol><li>在37ºC增长振荡至〜0.6的OD 600。 </li><li>通过广告诱导靶OMP的表达丁5微升的1M异丙基β-D-1-硫代半乳糖苷（IPTG）到每个培养管中，并允许生长额外1-2小时。 </li><li>通过使用离心在15000 XG离心1毫升各种文化（中〜0.6 OD 600）1分钟比较为所有殖民地的表达水平。 </li><li>除去上清液和重悬细胞于200μl1×SDS-PAGE上样缓冲液中。热，在100ºC为5分钟，然后离心再次以15,000×g离心5分钟。 </li><li>吹打20微升到10％凝胶的各孔分析使用SDS-PAGE的样品。在恒定200V。运行凝胶35分钟 注意:这将是在这一点上有利，以便能够确定该蛋白靶标被正确折叠的或没有。这通常可以通过这样做的小规模纯化或通过测定热可修改来实现。 </li></ol></li><li>选择出最能表达殖民地和执行使用12-24 L培养基的大规模表达。 注意:常规方法典型地生长在37ºC培养振摇至0.6〜的OD 600，然后用适当的诱导物诱导（ 即，0.1-1毫为IPTG或〜为阿拉伯糖0.2％）额外2-4小时。如果需要的话，前向感应，温度降低到低至20℃，延长诱导时间。 <ol><li>对于泄露表达方法，同比增长25毫升，37接种文化 ℃下在LB培养基含有适当抗生素（S），直至OD 600达到〜0.6。然后，加入1ml接种到TB培养基加抗生素（多个）十二1升烧瓶中并在20℃下生长至饱和（〜3天）。 </li></ol></li><li>收获离心将细胞在6000×g离心10分钟。 </li><li>继续进行纯化步骤2.1节或在-80ºC冻结在用于长期贮存液氮细胞沉淀。 </li></ol></li><li>表达包涵体体外 Refol丁<ol><li>使用序列分析来验证表达构建体不包含信号序列。没有信号序列的直接将靶蛋白在胞质溶胶中作为包涵体累积。 </li><li>变换表达构建成细菌表达的表达菌株。对于包涵体表达，最好是控制感应（ 即 IPTG，阿拉伯糖等 ）的表达，以尽量减少可能的毒性作用。 </li><li>板100微升转化细胞在含有合适的抗生素的LB琼脂平板上并在37过夜ºC反转。 </li><li>通过接种5ml的LB +抗生素文化与单个菌落进行小规模试验的表达。 </li><li>重复步骤1.3.4 2-4额外的殖民地。 </li><li>在37ºC增长振荡至〜0.6的OD 600。 </li><li>用1-2小时的适当诱导剂诱导。 </li><li>比较表达水平对所有t他菌落使用SDS-PAGE分析（参见1.2.6步）。选择出最能表达殖民地和执行使用12-24 L培养基大规模的表达。 <ol><li>生长在37℃下将细胞以0.6-0.8的OD 600，并在37℃下诱导3-5小时。而表达包涵体是比其他类型的表达的更稳健，与所有的蛋白表达的实验中，表达可以通过改变生长培养基，诱导时间，诱导温度，诱导剂的浓度来提高。 </li></ol></li><li>收获离心将细胞在6000×g离心10分钟。 </li><li>继续进行纯化步骤2.2节或在-80ºC冻结在用于长期贮存液氮细胞沉淀。 </li></ol></li></ol> 2.净化<ol><li>从膜组分分离 注意:相对于可溶性蛋白，整合膜蛋白嵌入到脂质双层，因此需要洗涤剂提取它们用于进一步纯化和分析（ 图5）。下面的表达式，在β桶OMP的纯化的第一步是从膜级分萃取。 <ol><li>以5毫升/克细胞糊的比例重悬细胞在裂解缓冲液（50mM的Tris-HCl，pH值7.4，200mM的氯化钠，10mM的MgCl 2的50微克/毫升AEBSF，5微克/毫升DNA酶I）。 </li><li>裂解用法国压机或细胞匀浆细胞。旋裂解的细胞以15,000 xg离心30分钟，在4℃到除去未裂解的细胞和细胞碎片。 </li><li>在4ºC高速（200,000 XG）再次转移上清至干净的试管离心1小时。所得粒料的膜部分含有感兴趣的蛋白质。 </li><li>使用Dounce匀浆，重悬膜部分，第一转印膜，然后加入增溶缓冲液（50mM KH 2 PO 4 pH为7.5，200 mM氯化钠，20mM咪唑的，pH值8.0）（每个20 G细胞50ml）中以2倍浓度，不用洗衣粉。 </li><li>倾将再悬浮的膜入一个小烧杯，并添加洗涤剂（ 即，正十二烷基β-D-麦芽糖苷（DDM），月桂基二甲基-N-氧化物（LDAO），正辛基β-D-吡喃葡糖苷（OG），曲通X-100 等 ）慢慢倍至10倍的临界胶束浓度（CMC）的〜终浓度。 </li><li>加水，直至1×增溶缓冲液的终浓度。搅拌0.5-16小时，在4ºC，这取决于如何容易目标蛋白质从膜中提取。 注:洗涤剂用来慢慢溶解的膜，以提取目标蛋白质。有3种类型的洗涤剂，可以用在这里:离子（SDS，脱氧胆酸），非离子（Elugent，TWEEN，曲拉通X-100，OG，DDM），和两性离子（LDAO，CHAPS）。蛋白质 - 去污剂复合物，在纯化步骤是稳定的，单分散性将膜蛋白crystallizat的成功极大地帮助离子。关于洗涤剂，其属性，CMC的信息，以及它们在膜蛋白和其他应用的纯化使用的更多信息可以在商业供应商的网站上找到。 </li><li>在4ºC离心溶解的样品30万XG 1小时。上清现在包含洗涤剂溶解的目标β桶OMP。 </li><li>第2.3节进行概述如下。 </li></ol></li><li>从包涵体复性 注意:对于体外复性，目标β桶OMP是直接表达成包涵体。这里的一个优点是，这些蛋白质可以以高的水平来制备。然而缺点是复性往往是低效率的，并从一个折叠试验到下一个而变化。不过，也有已经成功重折叠用于结构研究蛋白质的许多例子。下面的表达式成包涵体，人们现在必须隔离包涵体重折叠实验秒。 <ol><li>悬浮细胞在裂解缓冲液（50mM的Tris-HCl，pH值7.4，200mM的氯化钠，10mM的MgCl 2的50微克/毫升AEBSF，5微克/毫升DNA酶I，4毫摩尔2-巯基乙醇（BME））（5毫升，每G细胞粘贴）。裂解使用一个法国记者，超声处理，或细胞匀浆。 </li><li>沉淀通过低速旋包涵体以6000 xg离心10分钟，4ºC。 </li><li>通过使用Dounce匀浆在25毫升的1.0M尿素再悬浮洗涤包涵体。再由低速旋转在6000 xg离心在4ºC沉淀10分钟。 </li><li>重复步骤必要2.2.3。 </li><li>悬浮洗涤包涵体至10毫克的终浓度/ ml的用含8.0 M尿素缓冲液（或6.0米胍盐酸盐（GdCl））以2倍的CMC或更高加洗涤剂（ 即，DCM或LDAO）。 注:如果需要的话，对于含有一个组氨酸标签膜蛋白靶，固定化金属亲和层析（IMAC）纯化可使用变性共进行nditions（在8.0 M尿素或6.0米GdCl），为类似于第2.3节概述如下的初始步骤。 </li><li>执行由缓慢（过夜）通过透析缓冲液中除去变性剂缺乏变性剂的重折叠反应。 注意:这可能需要透析缓冲液的一些变化做到这一点。或者，如果包涵体首先结合到IMAC柱，可以执行重折叠反应，同时仍通过缓慢交换缓冲剂以除去变性剂结合到柱上。在这两种情况下，请记住，这是一种膜蛋白，因此，洗涤剂必须存在以稳定靶。此外，如果所用的去污剂是可透析，它必须被添加到透析缓冲液（S），以及。 </li><li>旋复性的样品为20万XG 1小时，4ºC。上清包含复性洗涤剂溶解的目标β桶OMP。 </li><li>第2.3节进行概述如下。 </li></ol></li><li>纯化，用我MAC，离子交换和凝胶过滤 注意:假定蛋白已被工程改造用组氨酸标签，无论是本地表达或复性使用体外方法，则固定化金属亲和层析（IMAC）的纯化进行很像组氨酸标签的可溶性蛋白，但洗涤剂必须保持在所有后续的缓冲器，以防止溶解和稳定的目标β桶OMP。 <ol><li>准备1-5毫升IMAC柱或使用预装列。在4℃下执行后续层析步骤。用清水冲洗，以去除防腐剂的任何痕迹。如果使用自动净化系统，根据制造商的说明安装柱。 </li><li>制成500毫升之IMAC缓冲液A（50mM的K 2 HPO 4，pH为7.5，200 mM氯化钠，0.1％DDM）和250毫升IMAC缓冲液B（50mM的K 2 HPO 4，pH为7.5，200 mM氯化钠，0.1％的DDM，和1.0M咪唑）。 注意:可以使用的我的其它洗涤剂nclude Cymal-6（0.05％），曲拉通X-100（0.03％），和LDAO（0.05％）。 </li><li>平衡用IMAC缓冲液A的10倍柱体积的IMAC柱</li><li>到蛋白质样品添加咪唑至25mM的终浓度并充分混合。以2毫升/分钟加载样品到平衡的IMAC柱。收集通过的流动。 </li><li>与每个增加使用缓冲液B的咪唑浓度的5倍柱体积洗涤IMAC柱（ 即 25毫米，50毫米，100毫米）。收集在2ml级分的洗涤。 </li><li>洗脱用250毫为5倍柱体积的最终浓度的样品。收集在2ml级分洗脱的样品。 </li><li>通过分析的流动，洗涤级分，并使用SDS-PAGE分析洗脱的级分的基础上在280nm其吸光度（参考步骤1.2.6）。池含有靶β桶的OMP如通过SDS-PAGE分析证实的级分。 </li><li> （根据除去通过加入TEV蛋白酶的合并样本的6×组氨酸标签制造商的协议），并在4ºC温和摇动温育过夜。 </li><li>再次装入样品/蛋白酶溶液到一个IMAC柱从切割的标签和任何未切割的样品中分离目标β桶OMP（流过）。流通过将包含缺乏标记的切割样品。 注意:这也将消除任何蛋白酶像TEV-His的，其中也有一个非切割的6×组氨酸标签本身。否则，将需要其他方法消化后，除去蛋白酶。 </li><li>任选地，进行离子交换色谱进一步纯化所述样本。按照制造商的说明使用的列，请务必提供在所有的缓冲区洗涤剂。 </li><li>到结晶制备，将样品上的凝胶过滤柱加载到包含所述洗涤剂的缓冲液可用于结晶，例如25毫的Tris-HCl，pH值7.5，200mM的氯化钠，1％OG。 </li><li>收集1- ml的组分分析USING SDS-PAGE分析（见 基于在280nm的吸光度步骤1.2.6）。用在280nm处的吸光度池馏分，因为它们含有的目标蛋白质。浓缩至〜10毫克/毫升。 请注意:如果需要的话，适当的折叠可使用热修改性测定如下所述进行评估。 </li></ol></li><li>热火修改性分析 注意:一种方法来监控纯化的β桶的正确折叠外膜蛋白是执行使用半天然SDS-PAGE 22热修改性测定。这里，被正确折叠的未加热的样品一般将迁移不同于那些热变性。这家酒店是唯一的β桶外膜蛋白，并广泛用于研究该家族的膜蛋白。 <ol><li>在此之前的样品制备，组装胶设备。首先，将缓冲罐成一个冰桶和冰完全包围。插入一个内部的铸造或商业天然梯度凝胶插入支架和放置到罐中。填写ŧ他完全罐用冷的1×MES运行缓冲液（50mM 2- [N-吗啉代]乙磺酸，50毫摩尔Tris碱，1mM EDTA中，0.1％SDS，pH7.3）中。 </li><li>吸管0.25微升样品（10毫克/毫升）的成2个1.5 ml离心管中。标记一个作为"煮沸"，另一个为"RT"。 </li><li>添加9.75微升样品缓冲液为每吹打混合。到两个样品，加10微升2×SDS上样缓冲液（100mM的Tris-HCl，pH值6.8，2％SDS，0.2％溴酚蓝和20％甘油）中，并通过移液轻轻混匀。 </li><li>熬&#39;水煮&#39;样品在95°C下5分钟，同时保持在室温的"RT"样本。旋&#39;水煮&#39;样本简要介绍。 </li><li>负载20微升两个样本的上预装的天然凝胶。在恒定150 V. 60分钟运行中取出凝胶，并在染色溶液中浸泡，以可视化的结果。 </li></ol></li></ol> 3.结晶注意:对于两者的结晶可溶性和膜蛋白的目标，这是标准的协议，以最大限度地提高样品纯度和稳定性（ 即 ，最好洗涤剂，配体，辅因子等 ）。对于一般结晶膜蛋白靶现行方法包括满足双层包埋蛋白质的两亲要求三个主要的方法:（1）洗涤剂，（2）bicelle，和（3）脂质立方相（LCP）（ 图6）23。强烈建议一个纳升结晶机器人的使用时可能以增加的可筛选对于给定的样品体积的条件在旨在帮助结构测定（ 图7）的工具的数量，以及，利用最新进展。 <ol><li>使用清洁剂结晶<ol><li>得到的洗涤剂增溶的蛋白质样品是在〜10毫克/毫升浓度。任选地，直接添加添加剂1,2,3- heptanetriol到样品的最终浓度为3％，以减少deterg耳鼻喉科胶束大小。 </li><li>使用0.22微米的离心过滤器以除去颗粒和沉淀过滤样品。 </li><li>使用结晶机器人，执行广泛的基质结晶使用市售的96孔的屏幕无论是通过悬挂或坐滴汽化法〜200 NL蛋白质样品和〜200 NL结晶缓冲组成滴筛选。 </li><li>在〜21℃培养结晶板。检查每周用于使用立体显微镜晶体生长平板。 </li><li>通过改变在一个系统的方式在结晶条件部件优化结晶引线（增加/减少的盐或沉淀剂浓度，缓冲液pH值，培养温度，和/或蛋白质浓度）。 </li></ol></li><li>结晶使用Bicelles 注:bicelle技术的更详细论证先前已经由Ujwal和艾布拉姆森24描述。 <ol><li>准备或购买35％bicelle混合物由DMPC的:CHAPSO以2.8比:根据公布的协议25 1。其他浓度也可以被检测，以及，其它脂质和去污剂，如必要的。 </li><li>得到的洗涤剂增溶的蛋白质样品是在〜10毫克/毫升浓度。 </li><li>添加bicelle混合物，在冰上，以4为起始比:1体积/体积（蛋白质:bicelle）（ 例如，加10微升bicelle混合物至40微升的蛋白质，并通过移液快速混合）。 </li><li>在冰上孵育30-60分钟。 注:蛋白质:bicelle的解决方案应该主要留清楚，但往往会略有不透明。但是，如果该蛋白:bicelle溶液变为乳白色，表明沉淀，然后其他变量应该测定（ 即，洗涤剂，缓冲液等 ）。对于新的样品，做小规模的测试（8微升蛋白质和2微升bicelles）建议，以验证稳定扩大，以防止不必要的样品损失之前。</li><li>使用结晶机器人，执行广泛的基质结晶使用市售的96孔的屏幕无论是通过悬挂或坐滴汽化法〜200 NL蛋白质样品和〜200 NL结晶缓冲组成滴筛选。 </li><li>在〜21℃培养结晶板。检查每周用于使用立体显微镜晶体生长平板。 </li><li>通过改变在一个系统的方式在结晶条件部件优化结晶引线（增加/减少的盐或沉淀剂浓度，缓冲液pH值，培养温度，和/或蛋白质浓度）。 </li></ol></li><li>结晶使用脂质立方相（LCP） 注:LCP技术的更详细论证先前已经由刘和Cherezov 26,27描述。 <ol><li>得到的洗涤剂增溶的蛋白质样品是在约20毫克/毫升浓度。 </li><li>预暖油酸酯到〜40ºC。加载60微升油酸单甘油酯成一个气密100微升注射器和40微升蛋白质样品到另一个。 </li><li>使用混合耦合器，连接注射器，小心不要引入空气。 </li><li>通过在注射器柱塞从一个针筒推混合物到另一个完全直到样品变成半透明且均匀施加交替压力轻轻混匀。 </li><li>使用结晶机器人专为LCP方法，进行广泛的基质结晶使用市售的96孔屏幕，三明治式结晶板（0.1毫米的厚度也）用100 NL蛋白样品和750 NL结晶缓冲组成滴筛选。 注:如果需要的话删除比率可被调节。此外，固体覆盖（ 即，玻璃或厚塑料）建议支持液滴很好，而不是薄膜，这往往使滴移动有关以及将板处理。 </li><li>孵育结晶p在鲈〜21℃。检查每周用于使用立体显微镜晶体生长平板。 </li><li>通过改变在一个系统的方式在结晶条件部件优化结晶引线（增加/减少的盐或沉淀剂浓度，缓冲液pH值，培养温度，和/或蛋白质浓度）。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

β桶的OMPs服务于革兰氏阴性菌，线粒体和叶绿体重要作用，并且用于结构分析，提供了大量关于在这些相应的细胞器的外膜基本分子机制信息的重要目标。然而，对于结构分析产生足够样品并不总是直截了当，因此，一般的管道提出了生产足够数量的目标β桶的OMPs结构测定的，详细说明从构建体晶体的过程。虽然这些协议已被测试，发现从革兰氏阴性细菌最外膜蛋白工作，有其局限性，有一些目...

β桶的OMPs只能在线粒体，叶绿体外膜中找到，和革兰氏阴性菌1-3。而他们服务相似的角色为α螺旋的蛋白质，它们有非常不同的褶皱包括中央膜包埋β桶结构域范围从8-26反平行β链与各链被紧密连接到两个相邻的股线的（ 图1和2）。的β桶结构域的第一个和最后一个链，然后与另一个在反平行的方式进行交互，几乎完全（除线粒体VDAC），以关闭和从周围膜密封β桶结构域。所有β桶的OMPs具有不同序列和长度的胞外环从而起到配体的相互作用和/或蛋白质 - 蛋白质接触中起重要作用，与这些环有时是75个残基，如在奈瑟氏菌转发现结合亲大蛋白A（TBPA）4。 β桶外膜蛋白也可以有哪些作为其他域的蛋白质的功能性用途的N端或C端周质的扩展（ 例如，巴马5-7，FimD 8,9，法德勒10）。而存在11许多类型的β桶的OMPs的，两种较常见的类型在下面描述为用于那些不太熟悉的领域中，（1）的TonB依赖性转运和（2）autotransporters例子。 的TonB依赖性转运（ 例如，FEPA，TBPA，BtuB，CIR， 等 ）是用于营养进口基本与包含一个N-末端插头域选自由被发现夹着内侧的22链β-C-末端〜150个残基的桶域嵌入到外膜12（ 图3）。虽然这种插头域防止基板从自由穿过枪管域，底物结合诱导插头域第内的构象变化在导致孔形成（通过插头重排或插头的部分/完整喷射）然后可以促进跨外膜衬底输送到周质。的TonB依赖性转运是革兰氏阴性细菌的一些致病菌株的存活特别重要，如已经进化出劫持营养素如来自人类宿主蛋白质4,13,14铁直接专门转运脑膜炎奈瑟氏球菌 。 Autotransporters属于革兰氏阴性菌的V型分泌系统，是指带有一个β桶结构域（通常为12-链与ESTA和ESPP）的是任一分泌或提出了在β-桶外膜蛋白和一位乘客域单元15,16（ 图3）的表面上。这些β桶的OMPs往往成为在细胞存活和毒力的重要作用与服务于乘客域或者作为蛋白酶，粘附，和/或其它EFfector介导的发病机制。 结构的方法，如X射线晶体学，核磁共振光谱，和电子显微镜（EM）允许我们确定在原子分辨率可反过来用于破译它们外膜内究竟如何发挥作用的外膜蛋白的模型。然后可用于药物和疫苗的开发，如果适用本宝贵信息。例如，转铁蛋白结合蛋白A（TBPA）被奈瑟氏球菌的表面上发现的和所需的发病机制，因为它直接结合人转铁蛋白，然后提取并导入铁为自身的生存。无TBPA， 奈瑟不能从人类宿主清除铁和呈现非致病。势必TBPA 4人转铁的晶体结构得到解决之后，它成为更清晰的两种蛋白质如何相关，哪些区域TBPA介导的相互作用，什么残基为铁提取重要由TBPA，和怎么一会开发针对脑膜炎奈瑟氏疗法针对跨界保护区。因此，给定的β桶的OMPs的革兰氏阴性菌的生存和发病机制中的重要性，以及在线粒体和叶绿体功能，以及需要了解这个唯一的类膜蛋白的额外的结构信息，并且其中它们的功能的系统，一般的协议都带有表达和结构方法在高位纯化的目标外膜蛋白为特征的总体目标。

<table><tbody><tr><td>Crystallization Robot</td><td>TTP Labtech, Art Robbins</td><td>-</td><td>Any should work here, except for LCP crystallization</td></tr><tr><td>PCR thermocycler</td><td>Eppendorf, BioRad</td><td>-</td><td></td></tr><tr><td>Media Shaker</td><td>New Brunswick, Infors HT</td><td>-</td><td></td></tr><tr><td>UV-vis spectrometer</td><td>Eppendorf</td><td>-</td><td></td></tr><tr><td>SDS-PAGE apparatus</td><td>BioRad</td><td>1645050, 1658005</td><td></td></tr><tr><td>SDS-PAGE and native gels</td><td>BioRad, Life Technologies</td><td>4561084, EC6035BOX (BN1002BOX)</td><td></td></tr><tr><td>AkTA Prime</td><td>GE Healthcare</td><td>-</td><td></td></tr><tr><td>AkTA Purifier</td><td>GE Healthcare</td><td>-</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>-</td><td></td></tr><tr><td>Centrifuge (low-medium speed)</td><td>Beckman-Coulter</td><td>-</td><td></td></tr><tr><td>Ultracentrifuge (high speed)</td><td>Beckman-Coulter</td><td>-</td><td></td></tr><tr><td>SS34 rotor</td><td>Sorvall</td><td>-</td><td></td></tr><tr><td>Type 45 Ti rotor</td><td>Beckman-Coulter</td><td>-</td><td></td></tr><tr><td>Type 70 Ti rotor</td><td>Beckman-Coulter</td><td>-</td><td></td></tr><tr><td>Dounce homogenizer</td><td>Fisher Scientific</td><td>06 435C</td><td></td></tr><tr><td>Emulsiflex</td><td>Avestin</td><td>-</td><td></td></tr><tr><td>Dialysis tubing</td><td>Sigma</td><td>D9652</td><td></td></tr><tr><td>LCP tools</td><td>Hamilton, TTP Labtech</td><td>-</td><td></td></tr><tr><td>VDX 24 well plates</td><td>Hampton Research</td><td>HR3-172</td><td></td></tr><tr><td>Sandwich plates</td><td>Hampton Research, Molecular Dimensions</td><td>HR3-151, MD11-50 (MD11-53)</td><td></td></tr><tr><td>Grace Crystallization sheets</td><td>Grace Bio-Labs</td><td>875238</td><td></td></tr><tr><td>HiPrep S300 HR column</td><td>GE Healthcare</td><td>17-1167-01</td><td></td></tr><tr><td>Q-Sepharose column</td><td>GE Healthcare</td><td>17-0510-01</td><td></td></tr><tr><td>Crystallization screens</td><td>Hampton Research, Qiagen, Molecular Dimensions</td><td>-</td><td></td></tr><tr><td>Gas-tight syringe (100 ml)</td><td>Hamilton&amp;nbsp;</td><td></td><td></td></tr></tbody></table>

from constructs to crystals &#8211; towards structure determination of &#946;-barrel outer membrane proteins

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, &#945;-helical and &#946;-barrel. While &#945;-helical membrane proteins can be found in nearly all cellular membranes, &#946;-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite &#946;-barrel outer membrane protein (OMP), general protocols are presented for the production of target &#946;-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

YiuR是一个依赖的TonB铁转运即对鼠疫耶尔森菌一个假定的疫苗的目标。使用微阵列分析它最初鉴定。这里，被送往确定利用X射线晶体YiuR的结构的步骤概述（ 图9）。用于克隆，YiuR的（去掉N端信号序列）的DNA序列进行PCR从基因组DNA扩增并亚克隆到含N-末端PELB信号序列和10×组氨酸亲和标签后跟一个TEV蛋白酶位点的载体。对于表达，含YiuR质粒...

Watch this Scientific Journal Video about From Constructs to Crystals â€“ Towards Structure Determination of Î²-barrel Outer Membrane Proteins at JoVE.com

从构建体到晶体β-桶到β-桶外膜蛋白的结构测定|化学|视频

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

&#946;-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of &#946;-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

从构造到晶体 - 迈向β桶外膜蛋白的结构解析

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% ...

from-constructs-to-crystals-towards-structure-determination-barrel

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13.4K 次观看.  Purdue University. β-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of β-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

视频: From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins

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 ...

科研

生物化学

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

监视分泌细菌毒力因子的组装使用站点特定的交联

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

免疫与感染

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

枯草芽孢杆菌中细菌体和内膜的可视化

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

生物工程

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to assess the purity of the bilayers. The OM has an increased overall density compared to the inner membrane, largely due to the presence of lipooligosaccharides (LOS) and lipopolysaccharides (LPS) within the outer leaflet. LOS and LPS molecules are amphipathic glycolipids that have a similar structure, which consists of a lipid-A disaccharolipid and core-oligosaccharide substituent. However, only LPS molecules are decorated with a third subunit known as the O-polysaccharide, or O-antigen. The type and amount of glycolipids present will impact an organism&#8217;s OM density. Therefore, we tested whether the membranes of bacteria with varied glycolipid content could be similarly isolated using our technique. For the LPS-producing organisms, Salmonella enterica serovar Typhimurium and Escherichia coli, the membranes were easily isolated and the LPS O-antigen moiety did not impact bilayer partitioning. Acinetobacter baumannii produces LOS molecules, which have a similar mass to O-antigen deficient LPS molecules; however, the membranes of these microbes could not initially be separated. We reasoned that the OM of A. baumannii was less dense than that of Enterobacteriaceae, so the sucrose gradient was adjusted and the membranes were isolated. The technique can therefore be adapted and modified for use with other organisms.

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

Gram-negative bacteria produce two spatially segregated membranes. The outer membrane is partitioned from the inner membrane by a periplasm and a peptidoglycan layer. The ability to isolate the dual bilayers of these microbes has been critical for understanding their physiology and pathogenesis.

将葛兰阴性细菌的细胞包络分离到内膜和外膜分数，对阿辛托巴基的培养素进行技术调整

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to ...

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous &beta;2-Microglobulin

The major histocompatibility complex (MHC) plays a pivotal role in antigen peptide presentation and T cell immune responses against infectious disease and tumor development. The hybrid MHC I complexed with heterologous &#946;2-microglobulin (&#946;2m) substitution from different species can be stabilized in vitro. This is a feasible means to study MHC I of mammals, when the homologous &#946;2m is not available. Meanwhile, it is indicated that mammalian &#946;2m substitution does not significantly affect peptide presentation. However, there is limited summarization regarding the methodology and the technology for the hybrid MHC I complexed with heterologous &#946;2-microglobulin (&#946;2m). Herein, methods to evaluate the feasibility of heterologous &#946;2m substitution in MHC I study are presented. These methods include preparation of expression constructs; purification of inclusion bodies and refolding of the MHC complex; determination of protein thermostability; crystal screening and structure determination. This study provides a recommendation for understanding function and structure of MHC I, and is also significant for T cell response evaluation during infectious disease and tumor immunotherapy.

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous &#946;2-Microglobulin

The protocol describes experimental methods to obtain stable major histocompatibility complex (MHC) class I through potential &#946;2-microglobulin (&#946;2m) substitutions from different species. The structural comparison of MHC I stabilized by homologous and heterologous &#946;2m were investigated.

蝙蝠主要组织兼容性复杂类 I 的稳定性和结构与异质β2- 微球蛋白

The major histocompatibility complex (MHC) plays a pivotal role in antigen peptide presentation and T cell immune responses against infectious disease and ...

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.

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 ...

生物学

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

从巨 Unilamellar 泡中提取膜纳米管

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

Multi-pass Transmembrane Proteins and &#946;-barrels

In multi-pass transmembrane proteins, the polypeptide chain crosses the membrane more than once. The transmembrane polypeptide chain either forms an &#945;-helix or &#946;-strand structure. &#945;-Helix containing multi-pass transmembrane proteins are ubiquitous, whereas &#946;-strand&#160;containing ones are mainly found in gram-negative bacteria, mitochondria, and chloroplasts.
&#945;-Helix containing multi-pass transmembrane proteins 
Multi-pass transmembrane proteins such as G-protein-linked receptors (GPCRs) and bacteriorhodopsin contain multiple transmembrane domains. All GPCRs have seven transmembrane &#945;-helices, but each receptor has its specific extracellular domain and G-protein-binding site. GPCRs bind a ligand and activate membrane protein called G-protein. The activated G-protein then interacts with either an ion channel or an enzyme in the membrane.
Bacteriorhodopsin found in certain photosynthetic bacteria has seven transmembrane &#945;-helices. It is a light-driven proton pump that helps in ATP production by generating a proton gradient across the membrane.
&#946;-Strands containing multi-pass transmembrane proteins 
Some bacterial membrane proteins contain transmembrane &#946;-strands, which are arranged into a ring-like structure called the &#946;-barrel. A minimum of eight &#946;-strands are required to form the barrel-like structure where the edges of the two closely spaced &#946;-strands form hydrogen bonds together. The porin protein observed in the membrane of some gram-negative bacteria contains a transmembrane &#946;-barrel. Apart from bacteria, porin proteins are also found in mitochondrial and chloroplast membranes, which support the hypothesis that both mitochondria and chloroplast are evolved from prokaryotes.
Some part of this text is adapted from Openstax, Biology 2e, <a href="https://openstax.org/books/biology-2e/pages/9-1-signaling-molecules-and-cellular-receptors">Section 9.1: Signaling Molecules and Cellular Receptors</a>.

Multi-pass Transmembrane Proteins and &amp;#946;-barrels

多通道跨膜蛋白和 β 桶

In multi-pass transmembrane proteins, the polypeptide chain crosses the membrane more than once. The transmembrane polypeptide chain either forms an ...

教育

细胞生物学

Unit 1

Membrane Structure and Components

关键术语与概念

Structure of Porins

Mitochondria, chloroplasts, and gram-negative bacteria have transmembrane, beta-barrel proteins called porins to mediate the free diffusion of ions and metabolites across the membrane. Mitochondrial porin precursors contain conserved amino acid sequences called beta signals at their C-terminal. Beta signals have a&#160; motif of PoXGXXHyXHy (Po-Polar, X-Any amino acid, G-Glycine, Hy-LargeHydrophobic), which are crucial for precursor recognition to initiate precursor assembly. Beta-barrel precursors move through the TOM complex on the outer mitochondrial membrane into the intermembrane space. The precursors interact with TIM chaperones inside the intermembrane space and are guided to a trimeric complex called the Sorting and Assembly machinery complex or SAM complex.&#160;
SAM complex has three components: a beta-barrel core called the SAM50 and accessory proteins SAM35 and SAM37. SAM50 comprises 16-beta-strands that span the outer membrane and expose an N-terminal polypeptide transport-associated domain or POTRA domain in the intermembrane space. The POTRA domain functions as a receptor for beta-barrel precursors and facilitates beta-strand assembly. In contrast, SAM50 interacts closely with accessory proteins SAM35/SAM37 towards the cytosolic side to help fold the beta-barrel precursors entering the intermembrane space.

孔蛋白的结构

Mitochondria, chloroplasts, and gram-negative bacteria have transmembrane, beta-barrel proteins called porins to mediate the free diffusion of ions and ...