这项工作的经费是由国家健康与医学研究委员会，澳大利亚（NHMRC项目赠款1011352 MEC和澳门赛马会，独立研究机构基础设施支援计划IRIISS拨款，以WEHI）和维多利亚州政府（VESKI创新奖学金MEC;维多利亚州政府运营基础设施的支持WEHI）。 MEC是一个英国女王伊丽莎白二世的澳大利亚研究委员会研究员。 EFXB承认，从诺玛·希尔达·舒斯特在墨尔本大学的奖学金的支持。

1。克隆的结构和设计感兴趣的序列克隆到PMM-肽用HindIII和BamHI限制性位点（参见图1）的载体（可以按要求提供）。按以下顺序：一个HindⅢ位点的双链DNA，插入应纳入CNBr裂解; E 的一个单一的蛋氨酸密码子大肠杆菌密码子优化的编码序列的肽，一个终止密码子，一个BamHⅠ位点。应消除所有其他的肽中的蛋氨酸和二肽序列天冬 - 亲应避免，因为它也可以在酸性条件下裂解。 一个独特的半胱氨酸残基应被引入每个肽序列，根据所需的定位，在最终的复合物的二硫化物交联，和所有其他半胱氨酸应突变为丝氨酸。该质粒携带用于选择卡那霉素抗性盒。 2。表达trpLE-pepti的去融合<ol><li>化妆和无菌过滤器M9/Kan最小的生长培养基中（0.1毫的CaCl 2，2mM的用MgSO 4，3克/升KH 2 PO 4，7.5克/升的Na 2 HPO 4·2H 2 O，0.5克/升的NaCl， 1克/升NH 4 Cl的 ，4的g / L葡萄糖，50mg / L的硫酸卡那霉素）。补充中枢完成A提供B族维生素和微量金属元素：1片溶解在40毫升H 2 O混合30分钟，离心除去不溶物和无菌过滤器的橙色上清锌。在4℃下在黑暗中，长达2个星期，并使用4 ml / L的在M9存储原液。 注：15 N-富集的NH 4 Cl的 ，13 C-富集的葡萄糖或其他稳定的同位素标记的前体可以被包括在M9培养基中，在此阶段，如果需要的话。 </li><li>接种到100毫升的新鲜转化BL21（DE3）菌株E.发酵大肠杆菌中M9/Kan介质。在37℃下生长过夜以200rpm振荡。 </li><li>第二天早晨，检查起动文化的OD 600 -这应该是在2或更大的范围内。稀释到1升中心区补充的M9培养基中的总体积为100毫升发酵。分裂成两个2升的折流板的烧瓶（在每个烧瓶中的500毫升培养），生长于37℃下摇动，在140的转速，直到OD 600达到0.6。 </li><li>振动筛温度设定到18°C，并继续振荡1小时，使文化完全冷却后方可上岗。 </li><li>以诱导前样品进行SDS-PAGE分析（相当于50微升从培养在OD 600 = 1），然后添加IPTG至终浓度0.1mM的诱导表达的trpLE-肽融合。在18°C/140转过夜（16-20小时），在IPTG诱导下继续保持增长。 </li></ol> 3。包涵体的制备及镍基绑定<ol><li>在基本培养基上过夜表达培养最终OD 600是可变的，昭民联是在2.5和5.0之间。 SDS-PAGE分析以用样品（相当于50微升从培养物的OD 600 = 1），并以5,000×g离心20分钟收集细胞。 </li><li>来自1 L培养在50ml裂解缓冲液中（50mM的Tris-HCl 20mM的pH为7.5，β-ME，0.2M NaCl）中重悬细胞沉淀。可以冷冻贮存，购买处理的细胞悬浮液。 </li><li>通过超声处理裂解细胞，在冰上在最大输出为1分钟，休息5分钟，在冰上。我们使用Misonix的超声波仪，3000（最大输出功率600瓦），一个半英寸高强度更换的提示。重复为三个周期的超声波处理/冷却。 </li><li>通过离心收集不溶性包含体（IB）的材料，在20,000 xg离心15分钟，并弃去上清液。浅色的细胞碎片松散的，薄层顶部致密的IB丸可以是可见的，这可以使用温和搅拌的水或缓冲液与被冲走。步骤3.3和3.4可重复，以提高纯度的IB部分，如果德斯黎红色。应采取的沉淀和上清材料的样品SDS-PAGE分析，以确认trpLE-TM的融合本土化，包涵体。 </li><li>将IB颗粒溶解在胍溶液（6M的盐酸胍，50mM的pH为8.0的Tris-HCl，200 mM氯化钠，1％的Triton X-100，5mM的β-ME），使用25-50毫升每升培养处理。这一步将需要好几个小时，与搅拌，偶有轻度超声。 </li><li>在75,000-100,000 xg离心1小时和20℃，溶胞产物通过离心清除IB滗析出上清液，通过0.2微米的膜过滤器。 </li><li>联合清零IB裂解液，在50毫升锥形管或更大范围内的可以封闭的容器安全，与镍亲和树脂已与水和平衡到胍溶液洗涤。使用3-4毫升结算的每公升树脂处理文化的融合，表达了类似的程度，，作为trpLE-DAP12TM在这里显示（ 图3，泳道2）。批量绑定孵育过夜房T<html> emperature与温和的混合。 </li></ol> 4。列的氧化交联<ol><li> IB裂解液/镍树脂悬浮加载到一个空的重力流柱与多孔床的支持，不断增加，直到整个体积流量通过。搜集和整理一边穿过峰，这可能仍包含未绑定的融合蛋白。 </li><li>用5个床体积的含有5mMβ-ME的尿素水溶液（8M尿素，50mM的pH为8.0的Tris-HCl，200mM NaCl的），通过重力流的镍树脂洗净。 </li><li>镍树脂，再与洗净的5个床体积的尿素溶液， 没有 β-ME的。 </li><li>镍洗净树脂用5柱床体积的尿素水溶液，已补充用20μMCuSO 4的氧化型谷胱甘肽和2mM三分之一时间。此冲洗后，关闭柱出口，并在室温下孵育30分钟，以允许最大的二硫键的形成。 </li></ol> 5。 TFA洗脱和定量分析内容“&gt; 注意：三氟乙酸（TFA）导致严重的烧伤与皮肤接触和烟雾有强烈刺激性。集中TFA只应使用经批准的化学通风柜的用眼保护和非乳胶手套。检查所有的化学相容性所用材料，聚丙烯兼容此协议的所有步骤。 <ol><li>洗出来的氧化剂的尿素溶液与10个床体积的水，并通过施加真空线柱出口干燥柱床。 </li><li>关闭柱出口增加1.5床体积的整齐（99-100％）TFA。搅拌镍树脂与一个小抹刀将确保甚至暴露于酸，孵育5分钟。打开柱出口，收集的酸性洗脱液，轻轻加压如有必要，用注射器或压缩空气管线推所有的液体。 </li><li>重复酸洗脱步骤与1.5床体积的纯TFA。快速工作，在此步骤中，以避免完全溶解的beadeð琼脂糖基质。蛋白质产量可确定在这一点上，按照比尔 - 朗伯方程和理论的trpLE肽序列的摩尔消光系数。稀释的TFA洗脱液（1:20至1:50）的三氟乙醇（TFE）的样品，并测量在280nm处的吸光度，使用TFA / TFE，在相同的稀释作为空白。 </li></ol> 6。溴化氰消化 注意：溴化氰（CNBr）是剧毒，只有在经批准的化学通风柜应处理。个人防护装备，包括防护眼镜，实验室外套，非乳胶手套是绝对必要的。溴化氰反应水分，并应提请冷却至室温后再开。请联系您的机构安全办公室中/出售的CNBR污染的解决方案和材料上的说明。 <ol><li>通过滴加水稀释到80％的最终的TFA浓度的酸的洗脱液，同时轻轻混合。如果沉淀物的形式，日体积小（0.5〜1毫升）的纯TFA，直至溶液澄清，然后重新正确与水的80％。取5微升预消化的样品进行SDS-PAGE分析，在液氮中冻结，并立即冷冻干燥样品。 </li><li>到约0.2克/毫升，添加的CNBr晶体照顾权衡的有毒化学品的安全封闭的，预先称重的试管中，或使用化学通风柜内部的平衡。轻轻混匀，直至完全溶解。冲洗和惰性气体（氮或氩气流）下密封反应容器中，并在室温下孵育3-4小时，避光。 </li><li> 5微升，消化后的样品进行SDS-PAGE分析，并立即冻结和冻干。摘要反应转移用截留分子量为3.5 kDa的或更少的再生纤维素透析盒或管（醋酸纤维素溶解在浓酸），根据预期的肽复合物的大小。透析过夜，4升的水在化学通风柜，以减少的TFA浓度和分解任何未反应的CNBr。留出空间，在透析磁带或管道的量显着增加（高达两到三倍），在透析过夜。 </li><li>取出透析的透析盒或管（大部分蛋白质会析出时，反式脂肪酸的浓度降低）悬浮沉淀的反应溶液和悬浮液转移到50毫升锥形管。冻结并冷冻干燥以除去水和溴化氰/ TFA的痕迹。此步骤是可能需要数天。 注意：透析的消化反应和透析用水应继续被视为有毒适当的预防措施和处理/处置。 </li><li>预诱导和收获阶段，包涵体上清液和沉淀，TFA洗脱液和溴化氰消化的材料的培养的样品可以通过SDS-PAGE分析。准备样品通过加热至95℃，在Invitrogen公司的NuPAGE LDS SAMPLË缓冲区。 TFA洗脱液和消化样品应准备在还原和非还原条件​​下的氧化交联，以促进产品的识别和评价。 </li><li>分离出的样品在12％的NuPAGE预制双 - 三丙烯酰胺凝胶使用MES-SDS电泳缓冲液为最小的消化产品的最佳分辨率。 </li></ol> 7。二硫键交联的肽复合物的反相HPLC纯化<ol><li>溶解冻干消化产品100毫克到2-3毫升整齐甲酸和负载到制备规模（21.2×150毫米）中的Agilent ZORBAX SB-300 C3 PrepHT端在溶剂A中（通常为10-20％乙腈或5列-10％异丙醇在水中含0.1％TFA）。 </li><li>洗脱梯度的溶剂B（用0.1％TFA的乙腈或异丙醇）中的至少5倍柱体积的摘要产品。建议选择最优梯度条件不同的肽序列进行讨论。 </li><li>采取50-100微升样品从每个HPLC级分的SDS-PAGE分析和冻结并立即冷冻干燥。高效液相色谱法溶剂的去除是快速的，应在1-2小时完成。 </li><li>准备冷冻干燥的HPLC样品进行SDS-PAGE，通过加热至95℃，Invitrogen公司的NuPAGE LDS样品缓冲液中， 与没有还原剂 。冷却，平均每个样品拆分成2个离心管中，加入100 mM的DTT他们重新加热简要之一。 </li><li>分隔的还原和非还原的HPLC样品，在12％的NuPAGE预制双 - 三丙烯酰胺凝胶与MES-SDS运行缓冲液，如上。小，疏水性多肽往往不考马斯染色以及可能无法运行在预期的分子量。然而，还原和非还原的样品相比，应便于识别的交联的肽纯度的产品和评估。 </li><li>分析的候选肽的部分，由基质辅助激光解吸电离飞行时间，飞行（MALDI-F）质谱（见讨论），以确定该物种的利益，并确认其预期的质量。 </li><li>结合，冻结和冻干HPLC含有纯的，交联的TM肽复合物的级分，并采取干重量为最终产物，以确定产量。异丙醇含量高的分数可能不会保持冻结。如果肽级分干燥的膜，而不是一个蓬松的冻干产品，重新溶解膜完全纯的六氟异丙醇（HFIP）的一个小体积中，冻结并再次冻干。的的HFIP过的产品应该是一个小的，白色的圆锥体，轻松放倒，称重。 </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Advances+in+the+Application+of+Solution+NMR+Spectroscopy+to+Multi-Span+Integral+Membrane+Proteins.">Recent Advances in the Application of Solution NMR Spectroscopy to Multi-Span Integral Membrane Proteins.</a> Prog. Nucl. Magn. Reson. Spectrosc. 55, 335-360 (2009).</li></ol>

作者宣称，他们没有竞争的金融利益。

根据我们的经验， 表达的融合trpLE-TM。trpLE-TM融合低表达丰富的培养基中在37℃下，和这里所描述的培养条件为许多不同的序列，含有一至四个TM域，产率范围已证明是成功的为50〜150 mg / L的纯的，完整的融合。融合编码三-或四-TM G蛋白偶联受体片段（人CCR5 TM1-TM3和TM4-TM7分别）或人类信号肽肽酶（4 TM域;见文献15）的芯催化片段代表在此范围内的最低的表达的水平，而这里所使用的序...

该协议的细节一个过程中，我们已开发生产出二硫键稳定的复合物的跨膜肽（TM）采用溶液NMR结构研究。这个过程需要利用所提供的强大的表达ΔtrpLE1413融合系统（见下文），并允许使用先进的稳定同位素标记技术，现代化的多维NMR实验TM肽复合物的定义组成。我们采用这些技术来确定的NMR结构淋巴细胞活化免疫受体如何从多个膜蛋白亚基组装TM域之间的相互作用（1）最近检讨发现重要的新信息。这些技术适用于许多其他的膜蛋白的系统，以及广泛的下游除了溶液的NMR分析方法。虽然这里给出的例子利用本地的半胱氨酸残基创建一个复杂的，模仿自然的二硫键键合的蛋白，设计是同样适合于创建工程二硫键稳定的复合物，通常是由较弱，均聚物和杂二聚体TM配合物的非共价相互作用，如保持在一起表皮生长因子受体（EGFR）家族蛋白2-4或异源二聚体αβ整合复合物5,6。 非常疏水的肽序列，如那些来自脂质双层跨越TM蛋白的部分是极为困难的课题，生物化学和生物物理研究。除了是非常具有挑战性的操作使用标准的蛋白质和多肽化学技术，他们往往对细胞的毒性，因此难以产生重组。我们7,8和9-11有显着的成功表达这样的困难在细菌的肽序列帧中的羧基端来自大肠杆菌的修改版本的ΔtrpLE1413序列融合到大肠杆菌色氨酸操纵12。 〜13 kDa的trpLE由该序列编码的多肽可以在T7启动子下的高的水平，并产生完全定位于包涵体的问题有关的毒性和/或不稳定的规避。该序列通过加入一种氨基末端9-组氨酸标记13和消除从trpLE序列内部蛋氨酸和半胱氨酸残基，14允许trpLE-肽融合到由金属离子亲和层析法进行纯化和消化使用溴化氰（CNBr改性），以释放所需的肽序列。我们已经成功地使用这个系统来表达更多的十几家不同序列的trpLE融合膜蛋白片段包含多达四个TM域名（7,8和未公布结果;也见讨论部分）。 “在本协议中的关键创新是确定的条件下，其中的的非结构化和难溶trpLE-TM融合可以有效地二硫键交联的背景下，一个精简的工作流程。高收益的表达，处理和纯化的多肽产品的几个方面也得到了彻底的优化，这里提出的建议同样适用于非二硫键交联（单体）TM肽产品的生产。

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>溴化氰</td><td> ALDRICH </td><td> P.No，CAS-506-C91492-68-3 </td><td> 有害物质。危险品。非常吸入毒性，与皮肤接触和吞食。与酸接触会产生剧毒气体。对水生生物有剧毒，可能对水生环境造成长期的不良影响。 </td></tr><tr><td>三氟乙酸</td><td> SIGMA-ALDRICH </td><td> P.CODE 1000984387，CAS编号76-05-1 </td><td> 有害物质。危险品，造成严重灼伤。吸入有害。对水生生物有害，可能对水生环境造成长期的不良影响。 </td></tr><tr><td> 2 - 巯基乙醇</td><td> SIGMA-ALDRICH </td><td> P.No M7154，CAS编号60-24-2 </td><td> 有害物质。危险品 。吸入，皮肤接触及若食入。对皮肤有刺激性。严重伤害眼睛的危险。皮肤接触可能引起过敏。对水生生物有剧毒，可能对水生环境造成长期的不良影响。 </td></tr><tr><td> 1,1,1,3,3,3 - 六氟-2 - 丙醇</td><td> SIGMA-ALDRICH </td><td>产品编号52512，CAS编号。 920-66-1 </td><td> 有害物质。危险品 。吸入有害，如果吞食。引致灼伤。 </td></tr><tr><td>蚁酸</td><td>默克</td><td> K41186564 </td><td>易燃液体和蒸气。造成严重皮肤灼伤和眼损伤。 </td></tr><tr><td>尿素</td><td> UNIVAR，AJAX FINECHEM </td><td>产品型号，817，CAS编号57-13-6 </td><td>当加热时，十二月omposes二氧化碳和氨;如果烧掉，发出少量的氮氧化物。会导致红肿和刺激皮肤和眼睛。 </td></tr><tr><td>盐酸胍</td><td> AMRESCO </td><td> P.No-M110，CAS号码：50-01-1 </td><td>如果吞下将是有害的，造成严重眼刺激，使皮肤有刺激性，急性口服毒性，皮肤刺激性，对眼睛有刺激性。 </td></tr><tr><td> TRITON X-100 </td><td> SIGMA </td><td>产品编号T8532 CAS编号：9002-93-1 </td><td>的Triton X-100是一种非离子洗涤剂，100％活性成分，通常被用在以溶解蛋白质的生化应用。的Triton X-100具有无抗菌性能，并考虑了相对温和的非变性洗涤剂</td></tr><tr><td>他选择镍亲和凝胶</td><td> SIGMA-ALDRICH </td><td>产品目录号P6611 </td><td> IS-选择镍亲和凝胶是一种固定化金属离子亲和层析（IMAC）产品。他的选择尼克EL亲和凝胶是一种专有的四齿螯合，组氨酸含有蛋白质的特异性结合，目的是用镍珠状琼脂糖收取。 </td></tr><tr><td> （ - ） - 谷胱甘肽，氧化</td><td> SIGMA-ALDRICH </td><td>目录NUM 150568 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Misonix S-3000超声波仪</td><td> QSONICA </td><td> S-3000（已停产） </td><td>最大功率输出600瓦。 2英寸可更换的尖探针采用2英寸的高强度，高容量的提示和一系列的高强度，低容量的提示。最近的机型，目前该公司的Q500和Q700。 </td></tr><tr><td> RP-HPLC：生物DuoFlow层析系统，软件5.3版</td><td> Bio-Rad公司实验室</td><td>目录编号760-0047，配置编号：AU500571，序列号：484BR3705 </td><td>肽结合反相HPLC色谱柱在高AQueous流动相，洗脱RP HPLC柱高有机流动相。在反相高效液相色谱法分离的肽根据其疏水性。肽可通过运行的有机溶剂的线性梯度分离。 </td></tr><tr><td>准备HT C3 ZORBAX 300SB分析HPLC柱，21.2×150毫米，5微米的颗粒大小</td><td>安捷伦</td><td>产品编号：895150-909 </td><td>反相HPLC柱</td></tr><tr><td>的NuPAGE 12％的Bis-Tris凝胶</td><td> Life Technologies公司</td><td> NP0341BOX </td><td>前投凝胶蛋白电泳</td></tr><tr><td>幻灯片-A仪G2透析的带3.5K MWCO </td><td> Thermo Scientific的</td><td>产品编号：87724 </td><td>样品透析</td></tr></tbody></table>

production of disulfide-stabilized transmembrane peptide complexes for structural studies

脂质嵌入的膜蛋白，α-螺旋结构域之间的物理相互作用起着至关重要的作用，在膜蛋白复合体的折叠和装配和跨膜（TM）信令和细胞表面蛋白水平的调节，例如动态过程。了解这些关联的特定序列的结构特点，需要复杂的生物物理和生物化学分析TM肽复合物。然而，TM域的极端疏水性使得它们非常难以操纵使用标准的肽化学技术，生产合适的学习材料常常被证明是令人望而却步具有挑战性的。确定的条件下，肽可以采用稳定的螺旋构象，并形成复合物， 自发地增加了更深一层的难度。在这里，我们提出了一种程序，均聚物或杂二聚体TM肽复合物，从材料的生产是在大肠杆菌中表达的关口，从而允许稳定同位素标签用于核磁共振（NMR）或其他应用程序相对廉价的非天然存在的氨基酸的掺入。在此方法中的关键的创新是作为共价结合 （二硫键交联的）组件改组为洗涤剂，脂质或其他膜-模拟物时，可以形成稳定的，化学计量的和均质的结构，TM配合物的生产和纯化。我们还提出了精心优化的程序也同样适用，无论是生产单TM域或交联复合物的表达和纯化，并提供这些方法来适应新的TM序列的意见。

trpLE融合实现的表达水平是可变的和严重依赖于连接的肽的氨基酸序列， 图3示出了预诱导的SDS-PAGE分析（泳道1）和时间的收获（泳道2）从文化，取得了约120毫克的纯的，完整trpLE DAP12TM融合从1升的文化和4毫升镍基样品。所有的trpLE DAP12TM融合的定位包涵体的沉淀（泳道4），而不是为上清液（泳道3）。 约70％的镍-纯化trpLE DAP12TM融合是二硫键交联的二聚体形式（ <...

Watch this Scientific Journal Video about Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies at JoVE.com

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

膜嵌入蛋白结构域之间的相互作用的生物物理和生物化学的研究面临许多技术上的挑战，首先是获得适当的学习材料。本文描述了一种用于生产和纯化二硫键稳定的的跨膜肽复合物，是适合用于结构分析通过溶液的核磁共振（NMR）和其他的分析应用的协议。

生产二硫键稳定的跨膜肽复合物的结构研究

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

production-disulfide-stabilized-transmembrane-peptide-complexes-for

Research

JoVE Journal

Chemistry

14.0K 次观看.  The Walter and Eliza Hall Institute of Medical Research. 膜嵌入蛋白结构域之间的相互作用的生物物理和生物化学的研究面临许多技术上的挑战，首先是获得适当的学习材料。本文描述了一种用于生产和纯化二硫键稳定的的跨膜肽复合物，是适合用于结构分析通过溶液的核磁共振（NMR）和其他的分析应用的协议。

视频: Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

稳态，稳定前的状态，单营业额动力学测量DNA糖基化酶活性

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

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 ...

Thin-layer Chromatographic (TLC) Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the chromatograms to microbial suspensions (e.g. fungi or bacteria in broth or agar), allowing time for the microbes to grow in a humid environment, and visualizing zones with no microbial growth.&#160;The effectiveness of this screening method, known as bioautography, depends on both the quality of the chromatographic separation and the care taken with microbial culture conditions.&#160;This paper describes standard protocols for TLC and contact bioautography&#160;with a novel application to amino acid-fermenting bacteria. The extract is separated on flexible (aluminum-backed) silica TLC plates, and bands are visualized under ultraviolet (UV) light.&#160;Zones are cut out and incubated face down onto agar inoculated with the test microorganism.&#160;Inhibitory bands are visualized by staining the agar plates with tetrazolium red.&#160;The method is applied to the separation of red clover (Trifolium pratense cv. Kenland) phenolic compounds and their screening for activity against Clostridium sticklandii, a hyper ammonia-producing bacterium (HAB) that is native to the bovine rumen.&#160;The TLC methods apply to many types of plant extracts&#160;and other bacterial species (aerobic or anaerobic), as well as fungi, can be used as test organisms if culture conditions are modified to fit the growth requirements of the species.

Thin-layer Chromatographic TLC Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

Methods are described for thin-layer chromatographic (TLC) separation of plant extracts and contact bioautography to identify antibacterial metabolites. The methods are applied to the screening of red clover phenolic compounds inhibiting hyper&#160;ammonia-producing bacteria (HAB) native to the bovine rumen.

薄层色谱（TLC）分离和植物提取物的生物测定识别抗菌化合物

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the ...

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. Compared to other analytical separation techniques, such as chromatography, CE is cheap, robust and effectively requires no sample preparation (for a number of complex natural matrices or polymeric samples). CE is fast and can be used to follow the evolution of mixtures in real time (e.g., chemical reaction kinetics), as the signals observed for the separated compounds are directly proportional to their quantity in solution.
Here, the efficiency of CE is demonstrated for monitoring the covalent grafting of peptides onto chitosan films for subsequent biomedical applications. Chitosan&#39;s antimicrobial and biocompatible properties make it an attractive material for biomedical applications such as cell growth substrates. Covalently grafting the peptide RGDS (arginine - glycine - aspartic acid - serine) onto the surface of chitosan films aims at improving cell attachment. Historically, chromatography and amino acid analysis have been used to provide a direct measurement of the amount of grafted peptide. However, the fast separation and absence of sample preparation provided by CE enables equally accurate yet real-time monitoring of the peptide grafting process. CE is able to separate and quantify the different components of the reaction mixture: the (non-grafted) peptide and the chemical coupling agents. In this way the use of CE results in improved films for downstream applications.
The chitosan films were characterized through solid-state NMR (nuclear magnetic resonance) spectroscopy. This technique is more time-consuming and cannot be applied in real time, but yields a direct measurement of the peptide and thus validates the CE technique.

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

毛细管电泳监视肽接枝壳聚糖膜实时

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

Controlled Photoredox Ring-Opening Polymerization of O-Carboxyanhydrides Mediated by Ni/Zn Complexes

Here, we describe an effective protocol that combines photoredox Ni/Ir catalysis with the use of a Zn-alkoxide for efficient ring-opening polymerization, allowing for the synthesis of isotactic poly(&#945;-hydroxy acids) with expected molecular weights (&#62;140 kDa) and narrow molecular weight distributions (Mw/Mn &#60; 1.1). This ring-opening polymerization is mediated by Ni and Zn complexes in the presence of an alcohol initiator and a photoredox Ir catalyst, irradiated by a blue LED (400 - 500 nm). The polymerization is performed at a low temperature (-15 &#176;C) to avoid undesired side reactions. The complete monomer consumption can be achieved within 4 - 8 hours, providing a polymer close to the expected molecular weight with narrow molecular weight distribution. The resulted number-average molecular weight shows a linear correlation with the degree of polymerization up to 1000. The homodecoupling 1H NMR study confirms that the obtained polymer is isotactic without epimerization. This polymerization reported herein offers a strategy for achieving rapid, controlled O-carboxyanhydrides polymerization to prepare stereoregular poly(&#945;-hydroxy acids) and its copolymers bearing various functional side-chain groups.

Controlled Photoredox Ring-Opening Polymerization of O-Carboxyanhydrides Mediated by Ni/Zn Complexes

A protocol for the controlled photoredox ring-opening polymerization of O-carboxyanhydrides mediated by Ni/Zn complexes is presented.

镍/锌配合物介导的O-Carboxyanhydrides 的控制 Photoredox 环开聚合

Here, we describe an effective protocol that combines photoredox Ni/Ir catalysis with the use of a Zn-alkoxide for efficient ring-opening polymerization, ...

Preparation of Stable Bicyclic Aziridinium Ions and Their Ring-Opening for the Synthesis of Azaheterocycles

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the aziridine ring. The utility of bicyclic aziridinium ions, specifically 1-azoniabicyclo[3.1.0]hexane and 1-azoniabicyclo[4.1.0]heptane tosylate highlighted in the aziridine ring openings by the nucleophile with the release of the ring strain to yield the corresponding ring-expanded azaheterocycles such as pyrrolidine, piperidine and azepane with diverse substituents on the ring in regio- and stereospecific manner. Herein, we report a simple and convenient method for the preparation of the stable 1-azabicyclo[4.1.0]heptane tosylate followed by selective ring opening via a nucleophilic attack either at the bridge or at the bridgehead carbon to yield piperidine and azepane rings, respectively. This synthetic strategy allowed us to prepare biologically active natural products containing piperidine and azepane motif including sedamine, allosedamine, fagomine and balanol in highly efficient manner.

Bicyclic aziridinium ions such as 1-azoniabicyclo[4.1.0]heptane tosylate were generated from 2-[4-tolenesulfonyloxybutyl]aziridine, which was utilized for the preparation of substituted piperidines and azepanes via regio- and stereospecific ring-expansion with various nucleophiles. This highly efficient protocol allowed us to prepare diverse azaheterocycles including natural products such as fagomine, febrifugine analogue and balanol.

稳定笼 Aziridinium 离子的制备及其开环 Azaheterocycles 的合成

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the ...

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 ...

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing metabolic reactions, DNA repair and replication. Therefore, a detailed understanding of these processes provides critical information on how cells function. Integrative structural MS methods offer structural and dynamical information on protein complex assembly, complex connectivity, subunit stoichiometry, protein oligomerization and ligand binding. Recent advances in integrative structural MS have allowed for the characterization of challenging biological systems including large DNA binding proteins and membrane proteins. This protocol describes how to integrate diverse MS data such as native MS and ion mobility-mass spectrometry (IM-MS) with molecular dynamics simulations to gain insights into a helicase-nuclease DNA repair protein complex. The resulting approach provides a framework for detailed studies of ligand binding to other protein complexes involved in important biological processes.

Mass spectrometry (MS) has emerged as an important tool for the investigation of structure and dynamics of macromolecular assemblies. Here, we integrate MS-based approaches to interrogate protein complex formation and ligand binding.

综合结构质谱分析蛋白质结构和蛋白质配体配合物

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing ...

Preparation of SNS Cobalt(II) Pincer Model Complexes of Liver Alcohol Dehydrogenase

Chemical model complexes are prepared to represent the active site of an enzyme. In this protocol, a family of tridentate pincer ligand precursors (each possessing two sulfur and one nitrogen donor atom functionalities (SNS) and based on bis-imidazole or bis-triazole compounds) are metallated with CoCl2&#183;6H2O to afford tridentate SNS pincer cobalt(II) complexes. Preparation of the cobalt(II) model complexes for liver alcohol dehydrogenase is facile. Based on a quick color change upon adding the CoCl2&#183;6H2O to acetonitrile solution that contains the ligand precursor, the complex forms rapidly. Formation of the metal complex is complete after allowing the solution to reflux overnight. These cobalt(II) complexes serve as models for the zinc active site in liver alcohol dehydrogenase (LADH). The complexes are characterized using single crystal X-ray diffraction, electrospray mass spectrometry, ultra-violet visible spectroscopy, and elemental analysis. To accurately determine the structure of the complex, its single crystal structure must be determined. Single crystals of the complexes that are suitable for X-ray diffraction are then grown via slow vapor diffusion of diethyl ether into an acetonitrile solution that contains the cobalt(II) complex. For high quality crystals, recrystallization typically takes place over a 1 week period, or longer. The method can be applied to the preparation of other model coordination complexes and can be used in undergraduate teaching laboratories. Finally, it is believed that others may find this recrystallization method to obtain single crystals beneficial to their research.

Preparation of SNS CobaltII Pincer Model Complexes of Liver Alcohol Dehydrogenase

The preparation of SNS pincer cobalt(II) model complexes of liver alcohol dehydrogenase is presented here. The complexes can be prepared by reacting the ligand precursor with CoCl2&#183;6H2O and can then be recrystallized by allowing diethyl ether to slowly diffuse into an acetonitrile solution that contains the cobalt complex.

制备SNS钴（II）针质模型复合物肝酒精脱氢酶

Chemical model complexes are prepared to represent the active site of an enzyme. In this protocol, a family of tridentate pincer ligand precursors (each ...

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery vehicles and enzyme scaffolds. Due to their biocompatibility and tunability on the genetic level, Elastin-like proteins (ELP) are ideal building blocks for biotechnological and biomedical applications. Nevertheless, the assembly of protein based supramolecular structures with distinct physiochemical properties and good encapsulation potential remains challenging.
Here we provide two efficient protocols for guided self-assembly of amphiphilic ELPs into supramolecular protein architectures such as spherical coacervates, fibers and stable vesicles. The presented assembly protocols generate Protein Membrane-Based Compartments (PMBCs) based on ELPs with adaptable physicochemical properties. PMBCs demonstrate phase separation behavior and reveal method dependent membrane fusion and are able to encapsulate chemically diverse fluorescent cargo molecules. The resulting PMBCs have a high application potential as a drug formulation and delivery platform, artificial cell, and compartmentalized reaction space.

At the interface of organic and aqueous solvents, tailored amphiphilic elastin-like proteins assemble into complex supramolecular structures such as vesicles, fibers and coacervates triggered by environmental parameters. The described assembly protocols yield Protein Membrane-Based Compartments (PMBCs) with tunable properties, enabling the encapsulation of various cargo.