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>

<ol>
	<li>Walther, D. M., Rapaport, D., Tommassen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+of+beta-barrel+membrane+proteins+in+bacteria+and+eukaryotes:+evolutionary+conservation+and+divergence.">Biogenesis of beta-barrel membrane proteins in bacteria and eukaryotes: evolutionary conservation and divergence.</a> Cell Mol Life Sci. 66, 2789-2804 (2009).</li><li>Ricci, D. P., Silhavy, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bam+machine:+A+molecular+cooper.">The Bam machine: A molecular cooper.</a> Biochim Biophys Acta. 1818, 1067-1084 (2012).</li><li>Hagan, C. L., Silhavy, T. J., Kahne, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Barrel+membrane+protein+assembly+by+the+Bam+complex.">beta-Barrel membrane protein assembly by the Bam complex.</a> Ann Rev of Biochem. 80, 189-210 (2011).</li><li>Noinaj, N., 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=Structural+basis+for+iron+piracy+by+pathogenic+Neisseria.">Structural basis for iron piracy by pathogenic Neisseria.</a> Nature. 483, 53-58 (2012).</li><li>Noinaj, N., 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=Structural+insight+into+the+biogenesis+of+beta-barrel+membrane+proteins.">Structural insight into the biogenesis of beta-barrel membrane proteins.</a> Nature. 501, 385-390 (2013).</li><li>Gatzeva-Topalova, P. Z., Walton, T. A., Sousa, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+YaeT:+conformational+flexibility+and+substrate+recognition.">Crystal structure of YaeT: conformational flexibility and substrate recognition.</a> Structure. 16, 1873-1881 (2008).</li><li>Kim, S., 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=Structure+and+function+of+an+essential+component+of+the+outer+membrane+protein+assembly+machine.">Structure and function of an essential component of the outer membrane protein assembly machine.</a> Science. 317, 961-964 (2007).</li><li>Geibel, S., Procko, E., Hultgren, S. J., Baker, D., Waksman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+energetic+basis+of+folded-protein+transport+by+the+FimD+usher.">Structural and energetic basis of folded-protein transport by the FimD usher.</a> Nature. 496, 243-246 (2013).</li><li>Phan, G., 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=Crystal+structure+of+the+FimD+usher+bound+to+its+cognate+FimC-FimH+substrate.">Crystal structure of the FimD usher bound to its cognate FimC-FimH substrate.</a> Nature. 474, 49-53 (2011).</li><li>van den Berg, B., Black, P. N., Clemons, W. M., Rapoport, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+long-chain+fatty+acid+transporter+FadL.">Crystal structure of the long-chain fatty acid transporter FadL.</a> Science. 304, 1506-1509 (2004).</li><li>Fairman, J. W., Noinaj, N., Buchanan, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structural+biology+of+beta-barrel+membrane+proteins:+a+summary+of+recent+reports.">The structural biology of beta-barrel membrane proteins: a summary of recent reports.</a> Curr Op Struct Bio. 21, 523-531 (2011).</li><li>Noinaj, N., Guillier, M., Barnard, T. J., Buchanan, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TonB-dependent+transporters:+regulation,+structure,+and+function.">TonB-dependent transporters: regulation, structure, and function.</a> Ann Rev of Micro. 64, 43-60 (2010).</li><li>Siburt, C. J., 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=Hijacking+transferrin+bound+iron:+protein-receptor+interactions+involved+in+iron+transport+in+N.+gonorrhoeae.">Hijacking transferrin bound iron: protein-receptor interactions involved in iron transport in N. gonorrhoeae.</a> Metallomics. 1, 249-255 (2009).</li><li>Cornelissen, C. N., Hollander, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TonB-Dependent+Transporters+Expressed+by+Neisseria+gonorrhoeae.">TonB-Dependent Transporters Expressed by Neisseria gonorrhoeae.</a> Front in Micro. 2 (117), (2011).</li><li>Dautin, N., Bernstein, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+secretion+in+gram-negative+bacteria+via+the+autotransporter+pathway.">Protein secretion in gram-negative bacteria via the autotransporter pathway.</a> Ann Rev of Micro. 61, 89-112 (2007).</li><li>Leo, J. C., Grin, I., Linke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+V+secretion:+mechanism(s)+of+autotransport+through+the+bacterial+outer+membrane.">Type V secretion: mechanism(s) of autotransport through the bacterial outer membrane.</a> Phil Trans of the Royal Soc of London. Series B, Biological Sciences. 367, 1088-1101 (2012).</li><li>Buchanan, S. K., Yamashita, S., Fleming, K. G., Engelman, E. H., Tamm, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Comprehensive Biophysics. 5, 139-163 (2011).</li><li>Buchanan, S. K., 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=Structure+of+colicin+I+receptor+bound+to+the+R-domain+of+colicin+Ia:+implications+for+protein+import.">Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import.</a> EMBO J. 26, 2594-2604 (2007).</li><li>Yamashita, S., 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=Structural+insights+into+Ail-mediated+adhesion+in+Yersinia+pestis.">Structural insights into Ail-mediated adhesion in Yersinia pestis.</a> Structure. 19, 1672-1682 (2011).</li><li>Aslanidis, C., de Jong, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligation-independent+cloning+of+PCR+products+(LIC-PCR).">Ligation-independent cloning of PCR products (LIC-PCR).</a> Nucleic Acids Res. 18, 6069-6074 (1990).</li><li>Haun, R. S., Serventi, I. M., Moss, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+reliable+ligation-independent+cloning+of+PCR+products+using+modified+plasmid+vectors.">Rapid reliable ligation-independent cloning of PCR products using modified plasmid vectors.</a> BioTechniques. 13, 515-518 (1992).</li><li>Rosenbusch, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+major+envelope+protein+from+Escherichia+coli.+Regular+arrangement+on+the+peptidoglycan+and+unusual+dodecyl+sulfate+binding.">Characterization of the major envelope protein from Escherichia coli. Regular arrangement on the peptidoglycan and unusual dodecyl sulfate binding.</a> J Biol Chem. 249, 8019-8029 (1974).</li><li>Barnard, T. J., Wally, J. L., Buchanan, S. K., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+of+integral+membrane+proteins.">Crystallization of integral membrane proteins.</a> Curr Prot in Prot Science. Chapter 17, Unit 17 19 (2007).</li><li>Ujwal, R., Abramson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+crystallization+of+membrane+proteins+using+the+lipidic+bicelle+method.">High-throughput crystallization of membrane proteins using the lipidic bicelle method.</a> JoVE. , e3383 (2012).</li><li>Faham, S., Ujwal, R., Abramson, J., Bowie, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+Aspects+of+Membrane+Proteins+Crystallization+in+Bicelles.">Practical Aspects of Membrane Proteins Crystallization in Bicelles.</a> Curr Topics in Membranes. 63, 111-127 (2009).</li><li>Li, D., Boland, C., Walsh, K., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+robot+for+high-throughput+crystallization+of+membrane+proteins+in+lipidic+mesophases.">Use of a robot for high-throughput crystallization of membrane proteins in lipidic mesophases.</a> JoVE. , e4000 (2012).</li><li>Liu, W., Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+of+membrane+proteins+in+lipidic+mesophases.">Crystallization of membrane proteins in lipidic mesophases.</a> JoVE. , (2011).</li><li>Buchanan, S. K. <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+refolding+of+an+80-kDa+iron+transporter+from+the+outer+membrane+of+Escherichia+coli.">Overexpression and refolding of an 80-kDa iron transporter from the outer membrane of Escherichia coli.</a> Biochem Soc Trans. 27, 903-908 (1999).</li><li>Buchanan, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-barrel+proteins+from+bacterial+outer+membranes:+structure,+function+and+refolding.">Beta-barrel proteins from bacterial outer membranes: structure, function and refolding.</a> Curr Op in Struct Bio. 9, 455-461 (1999).</li><li>Stanley, A. M., Fleming, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+process+of+folding+proteins+into+membranes:+challenges+and+progress.">The process of folding proteins into membranes: challenges and progress.</a> Arch of Biochem and Biophysics. 469, 46-66 (2008).</li><li>Pautsch, A., Schulz, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+outer+membrane+protein+A+transmembrane+domain.">Structure of the outer membrane protein A transmembrane domain.</a> Nature Struct Bio. 5, 1013-1017 (1998).</li><li>Cowan, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+OmpF+porin+in+a+tetragonal+crystal+form.">The structure of OmpF porin in a tetragonal crystal form.</a> Structure. 3, 1041-1050 (1995).</li><li>Ujwal, 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=The+crystal+structure+of+mouse+VDAC1+at+2.3+A+resolution+reveals+mechanistic+insights+into+metabolite+gating.">The crystal structure of mouse VDAC1 at 2.3 A resolution reveals mechanistic insights into metabolite gating.</a> PNAS. 105, 17742-17747 (2008).</li><li>Robert, 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=Assembly+factor+Omp85+recognizes+its+outer+membrane+protein+substrates+by+a+species-specific+C-terminal+motif.">Assembly factor Omp85 recognizes its outer membrane protein substrates by a species-specific C-terminal motif.</a> PLoS Bio. 4, e377 (2006).</li><li>Paramasivam, N., Habeck, M., Linke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+C-terminal+insertional+signal+in+Gram-negative+bacterial+outer+membrane+proteins+species-specific+or+not?.">Is the C-terminal insertional signal in Gram-negative bacterial outer membrane proteins species-specific or not?.</a> BMC Genomics. 13, 510 (2012).</li><li>Agah, S., Faham, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+of+membrane+proteins+in+bicelles.">Crystallization of membrane proteins in bicelles.</a> Methods in Mol Bio. 914, 3-16 (2012).</li><li>Ujwal, R., Bowie, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallizing+membrane+proteins+using+lipidic+bicelles.">Crystallizing membrane proteins using lipidic bicelles.</a> Methods. 55, 337-341 (2011).</li><li>Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipidic+cubic+phase+technologies+for+membrane+protein+structural+studies.">Lipidic cubic phase technologies for membrane protein structural studies.</a> Cur Op in Struct Bio. 21, 559-566 (2011).</li><li>Luecke, H., Schobert, B., Richter, H. T., Cartailler, J. P., Lanyi, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+bacteriorhodopsin+at+1.55+A+resolution.">Structure of bacteriorhodopsin at 1.55 A resolution.</a> J Mol Biol. 291, 899-911 (1999).</li><li>Kato, H. E., 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=Crystal+structure+of+the+channelrhodopsin+light-gated+cation+channel.">Crystal structure of the channelrhodopsin light-gated cation channel.</a> Nature. 482, 369-374 (2012).</li><li>White, J. F., 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=Structure+of+the+agonist-bound+neurotensin+receptor.">Structure of the agonist-bound neurotensin receptor.</a> Nature. 490, 508-513 (2012).</li><li>Cherezov, 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=High-resolution+crystal+structure+of+an+engineered+human+beta2-adrenergic+G+protein-coupled+receptor.">High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor.</a> Science. 318, 1258-1265 (2007).</li><li>Rasmussen, S. G., 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=Crystal+structure+of+the+beta2+adrenergic+receptor-Gs+protein+complex.">Crystal structure of the beta2 adrenergic receptor-Gs protein complex.</a> Nature. 477, 549-555 (2011).</li><li>Haupert, L. M., Simpson, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+of+protein+crystallization+trials+by+second+order+nonlinear+optical+imaging+of+chiral+crystals+(SONICC).">Screening of protein crystallization trials by second order nonlinear optical imaging of chiral crystals (SONICC).</a> Methods. 55, 379-386 (2011).</li></ol>

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，有一些目标，特别是对线粒体和叶绿体，这可能需要用于表达和纯化的其他方法。因此，这里的方法应适用于该从革兰氏阴性细菌包括外膜蛋白大多数项目，但表达水平纯化样品S和量将取决于目标OMP变化。 有对β桶的OMPs的用于结构研究的表达两种常用的方法，（1） 在体内的表达直接进入外膜和（2）表达的包涵体在体外重折叠。对于体内表达方式，β桶外膜蛋白有针对性地E.外膜大肠杆菌在那里它们被本地折叠并直接从膜分离。表达的膜是优选的方法，因为目标是更可能被正确折叠。虽然这种方法通常产生整体蛋白质的水平较低，它避免了有时在体外复性相关的并发症。许多β桶的OMPs以这种方式对于结构确定4,5,11被成功表达。成功是使用依赖于低水平组成型表达由T7启动推进系统经常实现R，这依赖于感应，但其他系统 （ 即 IPTG，阿拉伯糖）表达也经常与成功使用。 表达直接进入外膜要求目标蛋白为具有指示核糖体新生链复合到二段易位子，对新生的β桶OMP到周质的分泌的N-末端信号序列。信号序列被切割的内膜的周质侧，使得信号序列先于任何N-末端标签添加到目标是非常重要的。初生的β桶OMP然后由伴侣通过周质的BAM复杂插入陪同下走进外膜2,3。 对于不能被过度表达到膜的目标，另一种方法是表达成用于在体外复性28-30包涵体。这种方法通常导致高水平的和健壮的表达ØF中的靶蛋白。然而，它可以是具有挑战性的，以确定重折叠条件下，作为重折叠可以是无效率的。此外，也可以是难以当目标β桶的OMP被适当折叠以测定。不过，也有已经成功重折叠用于结构研究，包括外膜蛋白A 31，艾里19，OmpF的32和VDAC 33蛋白质的许多例子。对于不在所有（天然或成包涵体）表达或表达，但不正确组装到膜（本机）的克隆，人们可以尝试突变的β桶到更接近该大肠杆菌的大肠杆菌 34,35。在桶结构域的β型信号的氨基酸序列为识别与组件由BAM复杂和变化的β型信号序列重要可以显著影响目标β桶OMP 11,34的两个适当的生物合成和表达水平35。适当的集成的目标β-巴尔埃尔OMP可以通过筛选膜分馏和去污剂提取，随后通过热修改性测定法来监测。 在洗涤剂胶束存在膜蛋白的结晶是最古老的技术，让传统的可溶性蛋白质结晶设备和战略调整方便。通过掩蔽膜用洗涤剂分子埋入区域，浓缩蛋白可以以类似的方式，以可溶性蛋白（ 例如，混有结晶母液，并包含在引起该蛋白质下降的缓慢脱水蒸汽扩散装置）进行处理。而在概念上很简单，洗涤剂的特点和属性上正常结晶挑战顶部添加复杂性显著层。具体地，洗涤剂的分子性质必须凭经验针对给定的目标蛋白质，包括微胶粒的大小，喷墨头组极性（阴离子，阳离子，非离子，或两性离子），并且烃链长度，而每个这些影响在溶液中的目标膜蛋白的稳定性。这种方法的缺点包括非天然的化学环境中，可以形成晶体接触，和洗涤剂浓度问题表面区域的电势遮蔽。 Bicelles是 ​​组装成模仿类似于在细胞36,37发现膜的双层结构的单个颗粒的脂质和两亲物（ 如，清洁剂或短链脂质）的混合物中。两亲物掩模此双层在那里在其边缘以类似的方式暴露于在洗涤剂结晶的胶束的疏水性芯。这提供了一个更自然般的环境，以帮助稳定靶蛋白。 膜蛋白靶也可以与脂质立方相（LCP）的方法38结晶。 LCP是由脂质和水的混合形成的中间相，在其中连续双层是由溶剂通道的两个非交叉网络的渗透。这种三维结构允许脂质嵌入膜蛋白的扩散，在大体上天然样的环境，并允许晶体接触到蛋白质的疏水和亲水表面之间形成并同时增强其排序减小结晶的总溶剂含量。自20世纪90年代引进，LCP技术已确定的许多难以捉摸的膜蛋白靶结构的关键，如视紫红质39,40和G蛋白偶联受体41-43。在高通量筛选的LCP的使用需要特殊的规定（ 即，特定的LCP机器人，气密注射器，LCP分配工具，三明治结晶板等 ），为粗油酸酯通常用于LCP不能由传统的纳升的液体处理来处理机器人。 凝胶过滤色谱法是极为重要的一步对于一般的膜蛋白的结晶，因为它产生对如何稳定选择的去污剂是对膜蛋白靶标，其可以通过色谱进行可视化的信息。通过在空隙体积，保留时间和峰的形状的样品的量进行比较，样品的整体稳定性和单分散性可以被访问。理想的样品将具有很少没有在空隙体积丢失样本和将具有与高斯分布的单一对称洗脱峰。洗涤剂-C 8 E 4（0.8％），OG（1.0％），和LDAO（0.05％）的常规用于成功β桶的OMPs的结晶和是好的开始。理想的情况下，小规模实验来进行比较洗涤剂几个清净剂或它们的混合物，以确定哪些是最合适的结晶。那些被发现是最稳定，然后用于目标β巴的大规模制备rrel OMP和结晶试验。 一旦结晶目标β桶的OMP，铅优化的形成（ 即，添加剂筛选，冷冻筛选，洗涤剂添加剂筛分等 ）和类似的可溶性蛋白靶的其他技术可以遵循与一些区别。然而，也有一些与膜蛋白时是非常有用的最新进展。具体地，膜蛋白结晶往往很难其母液中检测用于各种原因。膜蛋白常形成比较小的晶体和误报误导能水晶的优化。 LCP技术尤其存在额外的挑战，因为在该晶体生长的高粘性，常常不透明的环境。策略来解决这些问题包括使用与光学显微镜结合使用紫外线显微镜（UV），允许自然荧光蛋白结晶加以区别˚F罗非荧光盐和洗涤剂晶体（ 图7）。挑战仍然存在，但是，在沉淀剂的领域内形成蛋白质晶体的阳性鉴定。晶体也可通过的最手性晶体的倍频效应开发当由飞秒激光扫描脉冲成像来检测，如通过SONICC技术44来实现。此高分辨率，高对比度的技术可以用来从模糊条件区分亚微米的晶体。 收获膜蛋白使用标准结晶学技术完成的，特别是用于洗涤剂和bicelle结晶。循环用手用低倍率的显微镜和一个水晶安装环（ 即尼龙纤维，丝，或聚合物）进行。过量的溶剂和低温保护的排汗之前暴跌收获β桶OMP晶体当低温液体冻结还标准程序。然而，收获LCP生长的晶体的ING呈现特别的问题，特别是如果直接从夹层板收获，晶体可能难以访问，并且可以不通过显微镜容易地观察到。另外，在LCP生长的晶体有时必须在散装收获由于LCP混合物不能容易地在循环中分离出来。 数据收集β桶外膜蛋白可以与只有少数其他注意事项可溶性蛋白质晶体进行。同时通过洗涤剂和bicelle方法生长晶体的尺寸通常比得上那些用可溶性蛋白生长，由LCP的方法生长的晶体几乎总是显著小。另外，由于样品从LCP基质收获通常包含多个晶体，难以观察，必须利用基于迪夫拉同步加速器源具有微型波束和环光栅扫描功能，可以系统地扫描整个循环来定位晶体的位置ction（ 图7）。 LCP晶体的小尺寸也使得它们特别容易受到辐射损伤。因此，从多个晶体数据经常合并，以便收集完整的数据集。 一旦井衍射结晶得到和一个完整的数据集收集，结构确定为β桶的OMPs可以用相同的程序作为用于可溶性蛋白来实现，牢记膜蛋白结晶通常表现出更高的溶剂含量。如同所有结晶的目标，无论是可溶性蛋白或膜蛋白，每间都设有自己的挑战，为此，没有一个单一的管道可以直接向所有目标。因此，它是主研究员（县）相应地调整这些一般的协议来保证他/她的项目成功的工作。

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 border="0" cellpadding="0" cellspacing="0" width="804">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:175px;">Crystallization Robot</td>
			<td style="width:159px;">TTP Labtech, Art Robbins</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;">Any should work here, except for LCP crystallization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">PCR thermocycler</td>
			<td style="width:159px;">Eppendorf, BioRad</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:175px;">Media Shaker</td>
			<td style="width:159px;">New Brunswick, Infors HT</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">UV-vis spectrometer</td>
			<td style="width:159px;">Eppendorf</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">SDS-PAGE apparatus</td>
			<td style="width:159px;">BioRad</td>
			<td style="width:227px;">1645050, 1658005</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">SDS-PAGE and native gels</td>
			<td style="width:159px;">BioRad, Life Technologies</td>
			<td style="width:227px;">4561084, EC6035BOX (BN1002BOX)</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">AkTA Prime</td>
			<td style="width:159px;">GE Healthcare</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">AkTA Purifier</td>
			<td style="width:159px;">GE Healthcare</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Microcentrifuge</td>
			<td style="width:159px;">Eppendorf</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Centrifuge (low-medium speed)</td>
			<td style="width:159px;">Beckman-Coulter</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Ultracentrifuge (high speed)</td>
			<td style="width:159px;">Beckman-Coulter</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">SS34 rotor</td>
			<td style="width:159px;">Sorvall</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Type 45 Ti rotor</td>
			<td style="width:159px;">Beckman-Coulter</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Type 70 Ti rotor</td>
			<td style="width:159px;">Beckman-Coulter</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Dounce homogenizer</td>
			<td style="width:159px;">Fisher Scientific</td>
			<td style="width:227px;">06 435C</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Emulsiflex</td>
			<td style="width:159px;">Avestin</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Dialysis tubing</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:227px;">D9652</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">LCP tools</td>
			<td style="width:159px;">Hamilton, TTP Labtech</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">VDX 24 well plates</td>
			<td style="width:159px;">Hampton Research</td>
			<td style="width:227px;">HR3-172</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:175px;">Sandwich plates</td>
			<td style="width:159px;">Hampton Research, Molecular Dimensions</td>
			<td style="width:227px;">HR3-151, MD11-50 (MD11-53)</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Grace Crystallization sheets</td>
			<td style="width:159px;">Grace Bio-Labs</td>
			<td style="width:227px;">875238</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">HiPrep S300 HR column</td>
			<td style="width:159px;">GE Healthcare</td>
			<td style="width:227px;">17-1167-01</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Q-Sepharose column</td>
			<td style="width:159px;">GE Healthcare</td>
			<td style="width:227px;">17-0510-01</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:175px;">Crystallization screens</td>
			<td style="width:159px;">Hampton Research, Qiagen, Molecular Dimensions</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Gas-tight syringe (100 mL)</td>
			<td style="width:159px;">Hamilton&#160;</td>
			<td style="width:227px;">????</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质粒转化到BL21（DE3）感受态细胞，并用来生长5ml的起子培养单个菌落至OD 600〜0.5。含750毫升TB培养基十二烧瓶然后用1ml的发酵剂培养物接种，并让其在20°C 3天生长（以220rpm振荡）。然后收获细胞，产生约150-200g总细胞糊的，这是闪冷却在液氮中，并储存在-80ºC直至使用。 为YiuR的纯化，将20g单元的方式进行（1.8毫KH 2 PO 4，2.7毫米氯化钾，137mM的氯化钠，pH值7.4的10mM 的 Na 2 HPO 4，）通过在RT用DNase I搅拌下再悬浮在120毫升1×PBS中的并添加AEBSF。裂解是由两个穿过均化器进行的。然后，裂解液离心在12000 XG 10分钟（降速完整的细胞和包涵体）。将上清液在235,000 xg离心再次离心60分钟，用含有膜级分（YiuR）沉淀。该膜然后再悬浮在100毫升1×PBS中的用一个Dounce匀浆。 YiuR是溶解/使用Elugent以5％的最终浓度的膜中提取，在4ºC搅拌过夜（高达16小时）。溶解的膜，然后在371000 XG再次离心60分钟，用苏pernatant含有溶解组分包括YiuR。隔离YiuR，IMAC是用缓冲液A（1×PBS，0.1％DDM）和缓冲液B（1×PBS中，1M咪唑，0.1％DDM）中，用30-50 mM咪唑的浓度并用250-500毫洗脱进行。以除去N-末端的10倍，组氨酸标签，与TEV孵育在1×PBS缓冲液透析期间在4ºC进行HIS蛋白酶过夜。然后将样品通过一个IMAC柱再次，流程通过浓缩，透析，然后进一步分离使用在离子交换塔的0-1.0摩尔NaCl梯度在50mM的Tris-HCl，pH值7.5。然后将含有YiuR级分合并，浓缩并辗过使用的25mM的Tris-HCl，pH值7.5，200mM的NaCl和0.05％LDAO凝胶过滤柱。然后将含有YiuR的馏分被汇集，并浓缩至10mg / ml的结晶。 然后，进行广泛的基质筛选和条件决定这产生了一些初步衍射晶体。进一步的优化导致其中使用收集原生的X射线衍射数据更大的晶体。 图8中所示的晶体是代表性晶体之一可以达到使用这里提出的方法之一。从洗涤剂筛选和bicelles晶体可以像通常为可溶性蛋白质得到的晶体为大;然而，那些从LCP几乎总是要小得多。数据不够好用于分子置换而导致结构测定，以2.65的决议。 <img alt="图1" src="/files/ftp_upload/53245/53245fig1.jpg" /> 图 1. 两种类型的完全整合的膜蛋白是α螺旋和β桶 。这里示出的是各自与β2肾上腺素受体（PDB代码2RH1，α螺旋）和的TonB依赖性转运BtuB（PDB代码1NQE的例子，β桶）。最上面一行显示外视图，而下面一行显示膜视图。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53245/53245fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53245/53245fig2.jpg" /> 图2.β 桶外膜蛋白提供许多不同的功能，可以有多样的结构 。而α螺旋膜蛋白可以包含一个或多个跨膜结构域，β桶的OMPs的范围从8-26链和每条链紧密与相邻链相互作用。外残基，其与膜相互作用，通常是疏水性的，而内部的残基，其与溶剂相互作用，典型地是极性和亲水性的。顶行示出的胞外视图，中间一行示出了膜视图和底部行示出了periplasmiC查看。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53245/53245fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53245/53245fig3.jpg" /> 图3. 两种常见β桶的OMPs的实例 。左侧时，的TonB依赖性转运FEPA（PDB代码1FEP）的结构，描绘了细胞外（上），膜（中间）和周质（底部）的意见。该β桶域以绿色表示，而该插件域为洋红色。对，自转运ESTA（PDB代码3KVN）的结构，描绘外（上），膜（中）和周质（下）的景色。该β桶域金表示，而乘客域为蓝色。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53245/53245fig3large.jpg" target="_blank">请点击这里查看大VERSI</a>这个数字的。 <img alt="图4" src="/files/ftp_upload/53245/53245fig4.jpg" /> 图4. 克隆和表达流水线生产β桶的OMPs的示意图。示意图用于克隆和表达通过天然膜表达（左）的靶OMP的管道或通过表达成包涵体为重折叠（右）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53245/53245fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="如图5" src="/files/ftp_upload/53245/53245fig5.jpg" /> 图5. 纯化管线β桶的OMPs的隔离的示意图。示意图用于已直接表达插入的OM外膜蛋白的提取的管道。这里，目标OMP必须提取从膜溶解通过用适当的洗涤剂编辑，然后通过IMAC纯化，离子交换层析和凝胶过滤。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53245/53245fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/53245/53245fig6.jpg" /> 图6. 用于生长β桶的OMPs的晶体结晶管道的示意图。三种方法可用于β桶的OMPs的结晶 包括洗涤剂筛选，bicelles和脂质立方相（LCP）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53245/53245fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/53245/53245fig7.jpg" /&gt; 图 7. 晶体的新工具已显著贡献构建β桶外膜蛋白的决心 。例子包括结晶机器人/ LCP机器人（A），UV显微镜（B），手性晶体的二阶非线性成像（SONICC）可视化（C），机器人冰球/机器人（D），光栅扫描/矢量/螺旋数据收集方法（E ）和微聚焦光束（F）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53245/53245fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/53245/53245fig8.jpg" /> 图8. 从协议所产生的晶体的代表性图像。从洗涤剂筛选和bicelles晶体可结晶大通常为可溶性蛋白质获得的;然而，那些从LCP几乎总是要小得多。比例尺= 100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53245/53245fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图9" src="/files/ftp_upload/53245/53245fig9.jpg" /> 图9. 从结构到晶体结构测定从 鼠疫杆菌 。用于YiuR结构测定的总管道假定的疫苗目标YiuR，说明采取克隆的步骤，表达，纯化，结晶，解决了结构。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53245/53245fig9large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about From Constructs to Crystals Ã¢â‚¬â€œ Towards Structure Determination of ÃŽÂ²-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

Nanyang Technological University

Research

Education

JoVE Journal

JoVE Core

Biology

Chemistry

Cellular Processes

Cell Structure and Function

SCIENTISTS IN ACTION

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

Determination of the Gas-phase Acidities of Oligopeptides

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue located at or near the N-terminus of a helix is often more acidic than that at or near the C-terminus 1-6. Although extensive experimental studies on the acid-base properties of peptides have been carried out in the condensed phase, in particular in aqueous solutions 6-8, the results are often complicated by solvent effects 7. In fact, most of the active sites in proteins are located near the interior region where solvent effects have been minimized 9,10. In order to understand intrinsic acid-base properties of peptides and proteins, it is important to perform the studies in a solvent-free environment.
We present a method to measure the acidities of oligopeptides in the gas-phase. We use a cysteine-containing oligopeptide, Ala3CysNH2 (A3CH), as the model compound. The measurements are based on the well-established extended Cooks kinetic method (Figure 1) 11-16. The experiments are carried out using a triple-quadrupole mass spectrometer interfaced with an electrospray ionization (ESI) ion source (Figure 2). For each peptide sample, several reference acids are selected. The reference acids are structurally similar organic compounds with known gas-phase acidities. A solution of the mixture of the peptide and a reference acid is introduced into the mass spectrometer, and a gas-phase proton-bound anionic cluster of peptide-reference acid is formed. The proton-bound cluster is mass isolated and subsequently fragmented via collision-induced dissociation (CID) experiments. The resulting fragment ion abundances are analyzed using a relationship between the acidities and the cluster ion dissociation kinetics. The gas-phase acidity of the peptide is then obtained by linear regression of the thermo-kinetic plots 17,18.
The method can be applied to a variety of molecular systems, including organic compounds, amino acids and their derivatives, oligonucleotides, and oligopeptides. By comparing the gas-phase acidities measured experimentally with those values calculated for different conformers, conformational effects on the acidities can be evaluated.

The determination of the gas-phase acidities of cysteine-containing oligopeptides is described. The experiments are performed using a triple quadrupole mass spectrometer. The relative acidities of the peptides are measured using collision-induced dissociation experiments, and the quantitative acidities are determined using the extended Cooks kinetic method.

寡肽测定气相酸性

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue ...

科研

化学

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and lignin. Layer-by-layer (LbL) assembly process which relies on the assembly of oppositely charged molecules from aqueous solutions was used to build a freestanding composite film of isolated wood polymers of lignin and oxidized nanofibril cellulose (NFC). To facilitate the assembly of these negatively charged polymers, a positively charged polyelectrolyte, poly(diallyldimethylammomium chloride) (PDDA), was used as a linking layer to create this simplified model cell wall. The layered adsorption process was studied quantitatively using quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. The results showed that layer mass/thickness per adsorbed layer increased as a function of total number of layers. The surface coverage of the adsorbed layers was studied with atomic force microscopy (AFM). Complete coverage of the surface with lignin in all the deposition cycles was found for the system, however, surface coverage by NFC increased with the number of layers. The adsorption process was carried out for 250 cycles (500 bilayers) on a cellulose acetate (CA) substrate. Transparent free-standing LBL assembled nanocomposite films were obtained when the CA substrate was later dissolved in acetone. Scanning electron microscopy (SEM) of the fractured cross-sections showed a lamellar structure, and the thickness per adsorption cycle (PDDA-Lignin-PDDA-NC) was estimated to be 17 nm for two different lignin types used in the study. The data indicates a film with highly controlled architecture where nanocellulose and lignin are spatially deposited on the nanoscale (a polymer-polymer nanocomposites), similar to what is observed in the native cell wall.

The objective of this research was to form synthetic plant cell wall tissue using layer-by-layer assembly of nanocellulose fibrils and isolated lignin assembled from dilute aqueous suspensions.&#160; Surface measurement techniques of quartz crystal microbalance and atomic force microscopy were used to monitor the formation of the polymer-polymer nanocomposite material.

朝仿生学木:纳米纤维素，木质素的装配式自由站立电影和合成聚阳离子

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and ...

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for characterizing lyophilized formulations of protein therapeutics. Labeling followed by suitable proteolytic digestion allows the protein structure and interactions to be mapped with peptide-level resolution. Since the protein structural elements are stabilized by a network of chemical bonds from the main-chains and side-chains of amino acids, specific labeling of atoms in the amino acid residues provides insight into the structure and conformation of the protein. In contrast to routine methods used to study proteins in lyophilized solids (e.g., FTIR), ssHDX-MS and ssPL-MS provide quantitative and site-specific information. The extent of deuterium incorporation and kinetic parameters can be related to rapidly and slowly exchanging amide pools (Nfast, Nslow) and directly reflects the degree of protein folding and structure in lyophilized formulations. Stable photolytic labeling does not undergo back-exchange, an advantage over ssHDX-MS. Here, we provide detailed protocols for both ssHDX-MS and ssPL-MS, using myoglobin (Mb) as a model protein in lyophilized formulations containing either trehalose or sorbitol.

Here, we present detailed protocols for solid-state amide hydrogen/deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS) for proteins in solid powders. The methods provide high-resolution information on protein conformation and interactions in the amorphous solid-state, which may be useful in formulation design.

质谱途径研究蛋白质结构和相互作用的冻干粉

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for ...

Synthesis and Exfoliation of Discotic Zirconium Phosphates to Obtain Colloidal Liquid Crystals

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and engineers. Growth of such anisotropic nanocrystals through a simple chemical method is a challenging task. In this study, we fabricate discotic nanodisks of zirconium phosphate [Zr(HPO4)2&#183;H2O] as a model material using hydrothermal, reflux and microwave-assisted methods. Growth of crystals is controlled by duration time, temperature, and concentration of reacting species. The novelty of the adopted methods is that discotic crystals of size ranging from hundred nanometers to few micrometers can be obtained while keeping the polydispersity well within control. The layered discotic crystals are converted to monolayers by exfoliation with tetra-(n)-butyl ammonium hydroxide [(C4H9)4NOH, TBAOH]. Exfoliated disks show isotropic and nematic liquid crystal phases. Size and polydispersity of disk suspensions is highly important in deciding their phase behavior.

A two dimensional model material of discotic zirconium phosphate was developed. The inorganic crystal with lamellar structure was synthesized by hydrothermal, reflux, and microwave-assisted methods. On exfoliation with organic molecules, layered crystals can be converted to monolayers, and nematic liquid crystal phase was formed at sufficient concentration of monolayers.

合成与盘状磷酸盐锆剥落获取胶体液晶

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and ...

X-ray Powder Diffraction in Conservation Science: Towards Routine Crystal Structure Determination of Corrosion Products on Heritage Art Objects

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder diffraction (XRPD) is presented in detail via two case studies.
The first material under investigation was sodium copper formate hydroxide oxide hydrate, Cu4Na4O(HCOO)8(OH)2&#8729;4H2O (sample 1) which forms on soda glass/copper alloy composite historic objects (e.g., enamels) in museum collections, exposed to formaldehyde and formic acid emitted from wooden storage cabinets, adhesives, etc. This degradation phenomenon has recently been characterized as &#34;glass induced metal corrosion&#34;.
For the second case study, thecotrichite, Ca3(CH3COO)3Cl(NO3)2&#8729;6H2O (sample 2), was chosen, which is an efflorescent salt forming needlelike crystallites on tiles and limestone objects which are stored in wooden cabinets and display cases. In this case, the wood acts as source for acetic acid which reacts with soluble chloride and nitrate salts from the artifact or its environment.
The knowledge of the geometrical structure helps conservation science to better understand production and decay reactions and to allow for full quantitative analysis in the frequent case of mixtures.

Modern high resolution X-ray powder diffraction (XRPD) in the laboratory is used as an efficient tool to determine crystal structures of long-known corrosion products on historic objects.

X射线粉末衍射在保护科学:迈向文物艺术品腐蚀产物的常规晶体结构测定

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder ...

Highly Stable, Functional Hairy Nanoparticles and Biopolymers from Wood Fibers: Towards Sustainable Nanotechnology

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While metal-based nanoparticles are associated with synthetic and environmental hassles, cellulose introduces a green, sustainable alternative for nanoparticle synthesis. Here, we present the chemical synthesis and separation procedures to produce new classes of hairy nanoparticles (bearing both amorphous and crystalline regions) and biopolymers based on wood fibers. Through periodate oxidation of soft wood pulp, the glucose ring of cellulose is opened at the C2-C3 bond to form 2,3-dialdehyde groups. Further heating of the partially oxidized fibers (e.g., T = 80 &#176;C) results in three products, namely fibrous oxidized cellulose, sterically stabilized nanocrystalline cellulose (SNCC), and dissolved dialdehyde modified cellulose (DAMC), which are well separated by intermittent centrifugation and co-solvent addition. The partially oxidized fibers (without heating) were used as a highly reactive intermediate to react with chlorite for converting almost all aldehyde to carboxyl groups. Co-solvent precipitation and centrifugation resulted in electrosterically stabilized nanocrystalline cellulose (ENCC) and dicarboxylated cellulose (DCC). The aldehyde content of SNCC and consequently surface charge of ENCC (carboxyl content) were precisely controlled by controlling the periodate oxidation reaction time, resulting in highly stable nanoparticles bearing more than 7 mmol functional groups per gram of nanoparticles (e.g., as compared to conventional NCC bearing &#60;&#60; 1 mmol functional group/g). Atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) attested to the rod-like morphology. Conductometric titration, Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), dynamic light scattering (DLS), electrokinetic-sonic-amplitude (ESA) and acoustic attenuation spectroscopy shed light on the superior properties of these nanomaterials.

Synthesis schemes to prepare highly stable wood fiber-based hairy nanoparticles and functional cellulose-based biopolymers have been detailed.

高稳定性，功能性毛纳米粒子和木纤维生物聚合物:迈向可持续纳米技术

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While ...

From Molecules to Materials: Engineering New Ionic Liquid Crystals Through Halogen Bonding

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid crystals (ILCs). Taking advantages of the high specificity of XB for haloperfluorocarbons and the ability of anions to act as XB-acceptors, we obtained supramolecular complexes based on 1-alkyl-3-methylimidazolium iodides and iodoperfluorocarbons, overcoming the well-known immiscibility between hydrocarbons (HCs) and perfluorocarbons (PFCs). The high directionality of the XB combined with the fluorophobic effect, allowed us to obtain enantiotropic liquid crystals where a rigid, non-aromatic, XB supramolecular anion acts as mesogenic core.
X-ray structure analysis of the complex between 1-ethyl-3-methylimidazolium iodide and iodoperfluorooctane showed the presence of a layered structure, which is a manifestation of the well-known tendency to segregation of perfluoroalkyl chains. This is consistent with the observation of smectic mesophases. Moreover, all the reported complexes melt below 100 &#176;C, and most are mesomorphic even at room temperature, despite that the starting materials were non-mesomorphic in nature.
The supramolecular strategy reported here provides new design principles for mesogen design allowing a totally new class of functional materials.

A protocol for the synthesis of a new type of mesogens, based on the halogen-bonded supramolecular anion [CnF2n+1-I&#183;&#183;&#183;I&#183;&#183;&#183;I-CnF2n+1]&#8722;, is reported.

从分子到材料: 通过卤素键合工程新的离子液体晶体

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid ...

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational spectroscopy and time-resolved electron diffraction. Liquid crystal phase is an important state of matter that exists between the solid and liquid phases and it is common in natural systems as well as in organic electronics. Liquid crystals are orientationally ordered but loosely packed, and therefore, the internal conformations and alignments of the molecular components of LCs can be modified by external stimuli. Although advanced time-resolved diffraction techniques have revealed picosecond-scale molecular dynamics of single crystals and polycrystals, direct observations of packing structures and ultrafast dynamics of soft materials have been hampered by blurry diffraction patterns. Here, we report time-resolved IR vibrational spectroscopy and electron diffractometry to acquire ultrafast snapshots of a columnar LC material bearing a photoactive core moiety. Differential-detection analyses of the combination of time-resolved IR vibrational spectroscopy and electron diffraction are powerful tools for characterizing structures and photoinduced dynamics of soft materials.

Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

观测 Photoresponsive 液晶结构动力学的新方法

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational ...

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local variations in calcium ion concentration. The main molecular components involved in these processes have been identified; however, the specific interplay between calcium ion gradients and the lipids within the cell membrane is far less known, mainly due to the complex nature of biological cells and the difficultly of observation schemes. To bridge this gap, a synthetic approach is successfully implemented to reveal the localized effect of calcium ions on cell membrane mimics. Establishing a mimic to resemble the conditions within a cell is a severalfold problem. First, an adequate biomimetic model with appropriate dimensions and membrane composition is required to capture the physical properties of cells. Second, a micromanipulation setup is needed to deliver a small amount of calcium ions to a particular membrane location. Finally, an observation scheme is required to detect and record the response of the lipid membrane to the external stimulation. This article offers a detailed biomimetic approach for studying the calcium ion-membrane interaction, where a lipid vesicle system, consisting of a giant unilamellar vesicle (GUV) connected to a multilamellar vesicle (MLV), is exposed to a localized calcium gradient formed using a microinjection system. The dynamics of the ionic influence on the membrane were observed using fluorescence microscopy and recorded at video frame rates. As a result of the membrane stimulation, highly curved membrane tubular protrusions (MTPs) formed inside the GUV, oriented away from the membrane. The described approach induces the remodeling of the lipid membrane and MTP production in an entirely contactless and controlled manner. This approach introduces a means to address the details of calcium ion-membrane interactions, providing new avenues to study the mechanisms of cell membrane reshaping.

We present a technique for contactless micromanipulation of vesicles, using localized calcium ion gradients. Microinjection of a calcium ion solution, in the vicinity of a giant lipid vesicle, is utilized to remodel the lipid membrane, resulting in the production of membrane tubular protrusions.

局部钙离子梯度对大泡囊膜重塑的响应

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local ...