The NMR facilities were funded by the R&#233;gion Nord, CNRS, Pasteur Institute of Lille, European Community (FEDER), French Research Ministry and the University of Sciences and Technologies of Lille. We acknowledge support from the TGE RMN THC (FR-3050, France), FRABio (FR 3688, France) and Lille NMR and RPE Health and Biology core facility. Our research is supported by grants from the LabEx (Laboratory of Excellence) DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to Alzheimer's disease), EU ITN TASPPI and ANR BinAlz.

1.生产的15 N，13 C-头（图1） <ol><li>变换的pET15b-头重组T7表达质粒13,14到BL21（DE3）感受态大肠杆菌细菌细胞15。 注:该cDNA编码最长的（441个氨基酸残基）头同种型中的的pET15b质粒NcoI和XhoI限制性位点之间克隆。 <ol><li>轻轻混匀50微升感受态BL21（DE3）细胞，形成每质粒DNA微克1-5×10 7个菌落，用100ng质粒DNA在1.5ml塑料管中。 注:密码子用法优化的真核cDNA表达的细菌菌株不必需产生人头。 </li><li>放置在冰上的细胞混合物30分钟，然后热休克10秒，在42℃。放置管回冰上5分钟，加入1ml室温的LB（Luria液体-BERTANI）培养基。孵育在37℃下的细菌悬浮液用于下温和30分钟搅动。 </li></ol></li><li>传播用接种环将100μl细胞悬浮液均匀的到含有100微克/ ml氨苄青霉素的抗生素的LB培养基的琼脂平板上。 </li><li>在37℃下孵育15小时的选择板。 </li><li>保持在4℃的选择板，直到继续到培养步骤，对于一个最大2周大约的。 注:细菌培养物（50％甘油）中的甘油，保存于-80℃下，可以制备在稍后阶段开始培养。 </li><li>加入1ml的1M 硫酸镁 ，1毫升的100mM的CaCl 2，将10毫升100×MEM维生素补充，1毫升100毫克/毫升氨苄青霉素至1L高压灭菌的M9盐（6克Na 2 HPO 4，3克KH 2 PO 4 ，0.5克NaCl）。 注:白色的沉淀物会形成在加入氯化钙解决这一迅速消散的M9盐。 </li><li>溶解300毫克15 N，13 C-完全培养基1克15氯化铵和2g的13 C 6 -葡萄糖在10ml M9培养基中。过滤-消毒使用0.2微米的过滤器，直接进入M9培养基同位素溶液。 </li><li>暂停使用的pET15b-头的一个接种环将一个菌落在20毫升LB培养基中补充有100微克/毫升氨苄青霉素的转化的细菌从选择板。 </li><li>孵育在37℃的接种培养基中约6小时。 </li><li>测量在600nm（OD 600）上1毫升细菌培养物的十倍稀释在塑料分光计比色皿的光密度。 注:对应于OD 3.0-4.0 600的细菌培养物浊度指示存在正在达到饱和的生长阶段。 </li><li>将20毫升的饱和的LB培养物添加到1升补充有氨苄青霉素（100μg/ ml的终浓度）的M9培养基中，在2升的Erlenmeyer塑料挡板培养瓶中。 </li><li>放置培养瓶在可编程培养箱设定为10°℃和50转。编程培养箱提前切换到200rpm下和37℃，在第二天早晨。 </li><li>在一个塑料试管分光计1毫升细菌培养的措施OD 600。加入400微升的1M的IPTG（异丙基β-D-1-硫代半乳糖苷）原液（保持在-20℃）时的OD 600达到约1.0的值以诱导重组Tau蛋白的表达。 </li><li>继续在37℃下温育另外3小时。收集经离心细菌细胞以5,000 xg离心20分钟。 </li><li>冻结在-20℃将细菌沉淀。如果需要保持冷冻，直到纯化步骤，对于延长的时间。 </li></ol> 2.净化15 N，13 C-头（图2） <ol><li>在121℃下为15psi进行20分钟的高压釜阳离子交换（CEX）纯化缓冲器。存储缓冲区在4℃。 </li><li>解冻细菌细胞沉淀，并在45ml充分悬浮提取CEX的缓冲（50毫米那匹缓冲液pH 6.5，1毫米EDTA）新鲜补充蛋白酶抑制剂1×（1片）和DNA酶I（2000台）。 </li><li>使用高压均化器以20,000 psi的破坏细菌细胞。 3-4通行证是必要的。离心机以20,000×g离心40分钟以除去不溶性物质。 </li><li>使用水浴加热15分钟，细菌细胞提取物在75℃。 注意:一白色沉淀几分钟后观察到的。 </li><li>离心在15,000rpm×g离心20分钟，并保持上清液含有热稳定Tau蛋白。 </li><li>在-20℃储存，直到下面的纯化步骤，如果需要的话。 </li><li>在填充作为使用快速蛋白质液相色谱（FPLC）系统（ 图3A）5ml的床柱强烈CEX树脂进行阳离子交换色谱。 <ol><li>设定流速2.5毫升/分钟。 </li><li>在CEX平衡柱子的缓冲区</li><li>装入60-70毫升heated-提取使用样品泵头含有，或者A泵，这取决于系统。收集用于分析，以验证Tau蛋白被有效地结合到树脂（见2.8）流过。 </li><li>与CEX洗涤树脂缓冲，直到在280吸光度是回到基准值。 </li><li>洗脱头使用由CEX B缓冲液的逐渐增加而获得的三步骤NaCl梯度的柱（CEX用1M NaCl的缓冲液）。程序的FPLC如下:梯度至25％的CEX B缓冲液中10个柱体积（CV），以达到250 mM氯化钠，第二步70％的CEX B缓冲液中5 CV达到500 mM氯化钠的第一步骤，和第三步骤2 CV 100％CEX B缓冲液，以达到1 M氯化钠。在洗脱步骤收集1.5毫升分数。 </li></ol></li><li>分析10微升在洗脱步骤通过SDS-PAGE（12％SDS-丙烯酰胺凝胶）和考马斯染色（ 图3A）16 中收集的级分。检查在柱上装载步骤，以及通过ANALY查懋10微升流通的。 </li><li>选择含头的级分，并汇集这些级分的下一个步骤。 </li><li>在执行牛头含合并的级分（ 图3 B）的缓冲交换。 <ol><li>平衡在50mM碳酸氢铵（易失性缓冲区），使用FPLC系统53毫升G25树脂的填充床（26×10厘米）的脱盐柱。 </li><li>设定流量至5毫升/分。通过5ml的注射环注入在列的头样品。收集对应于吸收峰在280nm处的级分。 </li><li>重复注射3-4次，这取决于初始CEX池的体积。 </li></ol></li><li>通过使用色谱的峰面积在280纳米（1毫克头的对应于140 MAU *毫升）计算纯化的Tau蛋白的量。 注:Tau蛋白的在280nm的消光系数是7,550 M -1 -1。头不包含任何色氨酸残基。 </li><li>池中的所有头部分。 </li><li> Aliquot是样品放入含有1至5mg头相当于管。使溶液的体积相比是很小的管（例如，5毫升溶液在50ml管）的容积选择这些管。 </li><li>冲用针在管帽的孔。在-80℃冷冻头的样品。 </li><li>牛头冷冻干燥样品。冻干Tau蛋白可以保持在-20℃的很长一段时间。 </li></ol> 3. 15 N-头的体外磷酸化<ol><li>溶解于500μl磷酸缓冲液（50毫摩尔的Hepes·KOH，pH值8.0，12.5毫氯化镁 ，1毫摩尔EDTA，50毫摩尔NaCl）5毫克冻干头的。 </li><li>添加2.5mM的ATP（25微升的100mM储备溶液保持在-20℃），1毫摩尔DTT（1微升的1M储备溶液保持在-20℃），1毫EGTA（2微升0.5M的股票的溶液），1×蛋白酶抑制剂鸡尾酒（25微升溶解1片而获得的40倍的库存在1ml磷酸缓冲液）和181，M活化的His-ERK2（250微升在保护缓冲器的10mM Hepes，pH值7.3，1毫摩尔DTT，5mM的MgCl 2的，100毫摩尔NaCl和10％甘油，保存于-80℃）中的总样品体积1毫升 注:激活了他，ERK2可以在内部5,8通过与MEK激酶磷酸化做好准备。 </li><li>孵育在37℃下3小时。 </li><li>加热在75℃下的样品15分钟以灭活ERK激酶。 </li><li>离心以20,000 xg离心15分钟。收集和保存上清。 </li><li>脱盐使用3.45毫升G25树脂的填充床（1.3×2.6厘米），其适用于1毫升样品的列中的蛋白质样品放入50mM碳酸氢铵。 </li><li>运行在12％的SDS-PAGE 16 2.5微升蛋白质样品的同时检查它的完整性和有效的磷酸化（ 图4）。 </li><li>冻干磷酸化Tau蛋白样品。储存在-20℃的粉末。 </li></ol> 4. NMR光谱采集（网络连接古尔5） <ol><li>溶解4毫克的冻干15 N，13 C的ERK磷酸化，头于400μlNMR缓冲液（50mM NAPI或50mM的氘化的三- D11 .CL，pH值6.5，30 mM氯化钠，2.5毫摩尔EDTA和1mM DTT）。 </li><li>加入5％D 2 O的核磁共振波谱仪的领域锁定和1毫米第三次会议（3-（三甲基硅基）丙酸-2,2,3,3-D 4磺酸钠））内部的核磁共振信号的参考。添加10微升完全蛋白酶抑制剂混合物的40倍原液的。 </li><li>使用电子注射器长针或巴斯德吸管采用5 mm NMR管转移的样品。用柱塞关闭NMR管。删除由活塞运动被困柱塞和液体之间的任何气泡。 </li><li>将核磁共振管在旋转。调整其垂直位置与用于所使用的NMR探头适当规喷丝，使得大多数样品溶液将是核磁共振线圈的内侧。 </li><li>启动空气流量:点击我抬n个磁铁控制系统窗口。小心地与在气流管内的离心器在磁体孔的顶部。停止空气流量（点击电梯 ），并让管下降到磁体中的探头内的地方。 </li><li>设定温度至25℃（298K）。 </li><li>执行半自动调谐和探针头的匹配来优化电力传输。 ATMM键入命令行上。 </li><li>锁用记录在氘信道的样本的D 2 O中的信号的光谱仪的频率。在磁铁控制系统窗口中单击锁定 。 </li><li>启动垫补过程在样品的位置，以优化的磁场的均匀性。键入topshim贵在命令行打开垫片窗口。点击开始垫片窗口。检查剩余B0标准变化值，以确认垫片是最优的（小于2 Hz的频率是好的）。 </li><li>校准质子的P1参数（长在微秒射频脉冲），这是必要的，以获得一个90°旋转质子自旋。瞄准使用水的质子的一维谱（ 图6）的360°脉冲。 </li><li>调整频率通过设置O1参数（单位为Hz），以在一维光谱质子水频率（ 图6）的偏移。 </li><li>开始采集一维质子谱（与水的信号抑制水门序列脉冲序列，例如zggpw5）来验证从样品（ 图7）的信号。适应扫描次数的相对蛋白质浓度。 ZG型在命令行上开始采集。 </li><li>设置附加参数收购二维[1 H，15 N] HSQC谱（脉冲序列hsqcetfpf3gpsi， 图8-9）。 <ol><li>对于一个15 N，13 C标记的样本中，15 N间接的进化过程中分离13℃。 </li><li&gt;设置点中的1 H（F2），和15 N（F1），尺寸谱宽（PPM）的数量和。 注:适应的采集数据点到光谱仪场的数目，以保持每点的类似赫兹的数，并限制去耦次:在900兆赫和600兆赫2048点使用3,072点，在通过1 H维。 </li><li>优化在脉冲序列的其他参数，对应于延迟，脉冲长度，偏移频率，功率电平。 ASED在命令行中键入显示相关实验的所有参数。 </li></ol></li><li>收购了3D的设定参数[1 H，15 N，13 C]在600兆赫频谱HNCACB（脉冲序列hncacbgpwg3d， 图10A）。 </li><li>设定参数为一个三维[1 H，15 N，15 N] HNCANNH实验（脉冲序列hncannhgpwg3d）在600兆赫。设置点的数量为2048的1 H和64和128分的两个15 N维。定义光谱宽度为14，25，百万分之一（ppm）25份集中在4.7，119，119 ppm的在1小时，15 N和15 N尺寸。收购与16扫描持续时间是1天22小时。 </li></ol> 5.磷酸化位点鉴定<ol><li>使用采集和处理NMR软件过程谱。 <ol><li>执行数据（ 图7）的傅里叶变换。类型英尺为一维谱，XFB用于2D谱或ft3d用于3D谱，在命令行上。 </li><li>相位和参考的所有光谱（ 图7C）使用交互式窗口。 </li></ol></li><li>识别在2D HSQC潜在对应于磷酸化丝氨酸和苏氨酸残基（ 图9，红色框）兴趣共振。 </li><li>提取平面（ 即二维1 H- 13 C谱）从使用的在2D兴趣谐振的15 N的化学位移的三维1 H 15 N- 13 C谱。使用尺寸2（W2）的光标来选择相应于平面（W1-W3）而被可视化的15个 N分频（ 图10B）。 </li><li>接"CA"的谐振频率和的"i"的"CB"13 C-核和的"i-1"的残基（更弱的信号组相比，这些第i残基的）为每个[1 H，15 N]共振通过点击共振，处于指针模式菜单中的HNCACB三维频谱的兴趣查找/添加高峰，在一个峰值列表文件中添加化学位移值。 <ol><li>识别我渣型，pSER的或PTHR，由峰值列表化学位移比较CA的已知值和pSER的和PTHR 17 CB化学位移。 </li><li>一个特征性+2 ppm的转变o确定在i + 1的位置上的Pro残基的存在F中的CA化学位移值18。 </li><li>比较对应于第i-1残基，以预测头氨基酸残19在第i-1位，以确定残余物的性质，化学位移的一个表的CA和CB共振的化学位移值。 </li></ol></li><li>匹克的&#39;我&#39;15 N核共振频率和"I-1"残基在HNCANNH频谱每个感兴趣[1 H，15 N]共鸣。 </li><li>比较15 N的化学位移值的Tau蛋白20的化学位移分配- 23。 </li><li>比较确定的二肽与头序列来定义序列特异性分配。 </li></ol>

The authors have nothing to disclose.

我们已经使用NMR光谱法表征酶改性头样品。重组表达和这里描述的全长人Tau蛋白纯化可以类似地用于产生突变体头或头域。同位素是需要核磁共振光谱富集蛋白质，因此有必要重组表达。磷酸化位点的识别，需要共振分配和15 N，13 C双重标记的蛋白质。定同位素的成本，良好的产率，需要在该重组表达的步骤。葡萄糖是在M9培养基中细菌的生长，因此13 C 6 -葡萄糖的量可以增加至4每升生长介质以提高产率的限制因素。完整的中，MEM维生素除了不是强制性的，但有利于刺激经济增长，提高成品率。由于完整的标记介质的成本高，这款产品只作为培养基supplemen吨。细菌生长在M9培养基缓慢。通常，使用M9媒体细菌培养后约4小时的潜伏期辅以诱导3％完全培养基到达时间。的OD 1.6-1.8 600通常是在培养结束时获得。重组Tau蛋白的预期收益率是每公升细菌培养约15毫克。采用可编程孵化器允许方便地安排在工作日的时间生产蛋白质，细菌沉淀和蛋白生产分析管理的集合。 样品浓度是很重要的，以获得良好的质量光谱。一个典型的头样本足够的频谱2D将包含1毫克200微升（ 即 100μM）Tau蛋白。对于3D光谱，在300微升至少200μM的要求，条件是低温探针头被使用。获得了高场分光计，如本研究中所使用的900兆赫的仪器将提供更好的信号 - 噪声并减少对样品浓度（ 图8）的限制。鉴于头是一个大的紊乱的蛋白质，其NMR谱的特征是相当大的信号的重叠，和一个高场NMR谱仪也将在分辨率方面（ 图8）的最佳选择。然而，对应于磷酸化位点的共振是在光谱的不同区域，很容易用600兆赫光谱甚至检测（比较图9 B和图9C）。另外，由于它的无序性，Tau蛋白是蛋白水解（ 图8B）敏感。 缓冲区的灭菌建议限制牛头降解。蛋白酶抑制剂的NMR样品的加成有助于期间，可以从小时持续至数天，这取决于脉冲序列数据采集周期，以保护头对抗降解。低pH值（ 即 pH值低于7.0）需要AVoid的蛋白质的质子和水的质子，从而导致信号增宽之间太快交换。样品pH值也必须被很好地控制，以确保光谱的可重复性。实际上，由于pSER的和PTHR的pKa值是接近用于核磁共振光谱的最佳pH，磷酸化残基的化学位移对pH变化非常敏感。在NMR样品的pH值的调整可以直接使用pH计用pH微电极，适于小体积进行。头是一个可溶性蛋白，并在标准条件下不聚集。聚阴离子，如硫酸肝素，并且在37℃下温育另外可头样品中引发聚合。在这种情况下，给定的聚集体的固体性质，在相应的NMR光谱的大部分共振拓宽超出检测。即使是tau蛋白磷酸化的样本不会聚集在这里描述的NMR数据采集的条件。 相比于抗体分析，NMR提供了一个O翻译后修饰的verall视图。质谱还可以用于鉴定蛋白质样品的磷酸化模式。同位素标记是没有必要的这种技术，并且要求的样本量要小得多。然而，具有多磷酸，如头蛋白的表征，仍然具有挑战性。相邻的磷酸化将在鉴定自下而上的策略产生等压肽。然后完成识别需要通过样品的蛋白水解获得的肽的MS / MS测序。核磁共振的一个优点在于该技术的固有定量性质。的NMR信号的强度可以被链接到本化学基团的量在特定的位点。因此，我们可以定义化学修饰的在每个站点的比例。然而，最近在MS应用的进展表明，头多磷酸化的MS分析是可行的24，甚至在适当正常化25后定量的方式。 这里，我们提出头磷酸化激活的ERK2，但该方法可用于与其它激酶以及6,7,26磷酸- 28。动力学实验可以执行，其可以有助于朝着蛋白基板28限定激酶特异性- 31。磷酸化的研究不限于重组激酶和细胞或组织提取物的激酶活性可以分析6,32,28。一个有趣的发展是利用细胞 NMR 原位修改33,34学习。相反地，核磁共振也适应良好的解决磷酸酶的特异性，如通过由PP2A磷酸35研究磷酸化tau脱磷酸化所示。 NMR谱可以通过在2D谱中的抑制剂化合物机智的存在下蛋白质底物的磷酸化信息进行比较来施加激酶抑制剂的表征h，从对照实验6日的频谱。 使用NMR光谱学的兴趣，相比更敏感的MS技术，驻留在广泛的应用利用这里描述的，而不是仅仅其分析能力的协议。它已被证明以确定磷酸化位点，以便能够特异性磷酸化与使用NMR该主要研究的结构或功能上的修改链接至关重要。 tau蛋白磷酸化样品的核磁共振光谱允许探索的磷酸化对暂态本地二级结构和改性头36,37的动态合奏的全球重组的结构性影响。功能方面包括头的蛋白质伙伴7,38,39和聚集由tau蛋白磷酸8二者相互作用的调节。其特征在于用NMR磷酸化Tau蛋白样品可进一步用于破译磷酸依赖性相互作用，为例如用14-3-3蛋白40，以及改造的蛋白质-蛋白质相互作用抑制剂41,42。核磁共振允许相互作用位点（多个）的定义在残余物中的水平，并在磷酸这种相互作用的依赖。此外，tau蛋白磷酸化的核磁共振光谱是一个关键的方法来表征脯氨酸顺:反异构酶中Pin1，参与头村第43条的一个重要磷酸依赖性酶- 45。另外，通过NMR的磷酸化分析，不仅可以向流离失所者还要球状蛋白46被应用。最后，其它类型的头翻译后修饰，例如乙酰化22,40,41可以通过NMR进行研究。这里所描述的协议已被证明关键，以更好地理解头的在生理和病理条件的功能和结构的调节。

其中一个在21 世纪医疗保健的主要挑战是神经退行性疾病如阿尔茨海默病（AD）。头是一个微管相关蛋白，刺激微管（MT）的形成。头被均等地参与了一些神经变性疾病，所谓τ病变，其中最有名的是广告。在这些疾病中成对螺旋丝（的PHF）头自聚集，并发现修改由翻译后后修饰，如磷酸1个多残留。 Tau蛋白的磷酸化在MT稳定和功能丧失的病理表征AD神经元及其生理功能的调节都牵连。 此外，Tau蛋白，在神经元病中集成的PHF时，则不约而同地过度磷酸2。不同于含有2-3个磷酸基团正常头，在对的PHF tau蛋白含有5至9 phosphatË组3。 tau蛋白对应既能在一些网站和被称为磷酸化位点病理其他站点的磷酸化增加了化学计量。然而，重叠的AD和磷酸化的正常成人图案之间存在，尽管在4级量化的差别。具体怎么磷酸化事件的影响作用和Tau功能障碍仍是未知。我们的目标是通过翻译后修饰在分子水平上破译头监管。 深化头的分子方面的理解，我们必须解决的技术难题。首先，头在溶液中分离出来时，一个内在的无序蛋白（IDP）。这样的蛋白质缺乏在生理条件下明确定义的三维结构并需要特定的生物物理方法来研究它们的功能（S）和结构特性。头是为不断增长的国内流离失所者类的一个范例，经常发现关联疾病，如神经退行性疾病，从而增加利息，了解他们的基本功能分子参数。其次，tau蛋白磷酸表征是一个分析的挑战，随着时间最长的441氨基酸头亚型的序列80的潜在磷酸化位点。一些抗体已经开发针对头的磷酸化表位，并用于在神经元或脑组织检测病理头的。磷酸化事件可以采取由脯氨酸的激酶靶向至少有20处地点，其中大多数是在富含脯氨酸的区域内近在咫尺。定性（哪些网站？）和定量（化学计量学什么？）特性是很难甚至最新的MS技术5。 核磁共振光谱可用于研究是高度构成构象合奏的动态系统紊乱的蛋白质。高分辨率核磁共振光谱是APPLI编调查Tau蛋白的两个结构和功能。另外，该头的磷酸化信息的复杂性导致了使用NMR对磷酸化位点6的识别分子的工具和新的分析方法的发展- 8。核磁共振作为分析方法允许在全局方式识别的头磷酸化位点，在一个单一实验中的所有的单点修饰的可视化和量化磷酸并入的程度。因为虽然tau蛋白磷酸的研究在文献中比比皆是，他们大多已与抗体进行，留下了磷酸化的完整个人资料大程度的不确定性，因此个别磷酸化事件的真正影响这一点是至关重要的。重组激酶，包括PKA，糖原合酶激酶3β（GSK3β），细胞周期蛋白依赖性激酶2 /细胞周期蛋白A（CDK2 / CycA），细胞周期蛋白依赖性激酶5（CDK5）/ P25行为ivator蛋白质，细胞外信号调节的激酶2（ERK2）和微管亲合性 - 调节激酶（MARK），其显示向头磷酸化活性，可以以活性形式来制备。另外，头突变体，其允许产生与充分表征的磷酸化模式特定Tau蛋白同种型用于破译头的磷酸化的代码。然后NMR谱来表征酶改性头样品6 - 8。虽然在体外头的磷酸化是比伪磷酸更具挑战性，例如通过选定的丝氨酸/苏氨酸的突变成谷氨酸（谷氨酸）残基，该方法有其优点。实际上，无论是磷酸化的结构影响也没有相互作用参数总是可以通过谷氨酸模拟。一个例子是围绕磷酸丝氨酸202（pSer202）观察到的转基序/磷酸苏205（pThr205），这是不符合谷氨酸突变9再现。 中通过重组ERK2激酶磷酸化体外了详细的方案提出。 ERK2是通过磷酸化而活化有丝分裂原活化蛋白激酶/ ERK激酶（MEK）10 - 12。另外修改，同位素标记的Tau蛋白的制备中，用于翻译后修饰的识别核磁共振策略进行说明。

<table border="0" cellpadding="0" cellspacing="0" width="820">
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			<td height="21" style="height:21px;width:328px;">pET15B recombinant T7 expression plasmid</td>
			<td style="width:177px;">Novagen</td>
			<td style="width:119px;">69257</td>
			<td style="width:196px;">Keep at -20&#176;C</td>
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			<td style="width:177px;">New England Biolabs</td>
			<td style="width:119px;">C2527I</td>
			<td style="width:196px;">Keep at -80&#176;C</td>
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			<td height="21" style="height:21px;width:328px;">Autoclaved LB Broth, Lennox&#160;</td>
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			<td style="width:119px;">240210</td>
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			<td height="41" style="height:41px;width:328px;">MEM vitamin complements 100X</td>
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			<td height="41" style="height:41px;width:328px;">15N, 13C-ISOGRO complete medium powder</td>
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			<td style="width:119px;">608297</td>
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			<td style="width:119px;">389374</td>
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			<td height="61" rowspan="2" style="height:61px;width:328px;">Protease inhibitor tablets&#160;</td>
			<td rowspan="2" style="width:177px;">Roche</td>
			<td rowspan="2" style="width:119px;">5056489001</td>
			<td style="width:196px;">Keep at 4&#176;C</td>
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			<td height="41" style="height:41px;width:196px;">1 tablet in 1ml is 40X solution that can be kept at -20&#176;C</td>
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			<td height="21" style="height:21px;width:328px;">DNaseI</td>
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			<td style="width:119px;">1307</td>
			<td style="width:196px;">Keep at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Homogenizer (EmulsiFlex-C3)</td>
			<td style="width:177px;">AVESTIN</td>
			<td style="width:119px;"></td>
			<td style="width:196px;">Lysis is realized at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Pierce&#8482; Unstained Protein MW Marker</td>
			<td style="width:177px;">Pierce</td>
			<td style="width:119px;">266109</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Active human MEK1 kinase, GST Tagged</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">M8822</td>
			<td style="width:196px;">Keep at -80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">AKT&#196; Pure chromatography system</td>
			<td style="width:177px;">GE Healthcare</td>
			<td style="width:119px;"></td>
			<td style="width:196px;">FPLC</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:328px;">HiTrap SP Sepharose FF (5 mL column)</td>
			<td style="width:177px;">GE Healthcare</td>
			<td style="width:119px;">17-5156-01</td>
			<td style="width:196px;">Cation exchange chromatography columns</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:328px;">HiPrep 26/10 Desalting</td>
			<td style="width:177px;">GE Healthcare</td>
			<td style="width:119px;">17-5087-01</td>
			<td>Protein Desalting column</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:328px;">PD MidiTrap G-25</td>
			<td style="width:177px;">GE Healthcare</td>
			<td style="width:119px;">28-9180-08</td>
			<td style="width:196px;">Protein Desalting column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Tris D11, 97% D</td>
			<td style="width:177px;">Cortecnet</td>
			<td>CD4035P5</td>
			<td style="width:196px;">Deuterated NMR buffer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:328px;">5 mm Symmetrical Microtube SHIGEMI D2O ( set of 5 inner &#38; outerpipe)&#160;</td>
			<td style="width:177px;">Euriso-top</td>
			<td style="width:119px;">BMS-005B</td>
			<td style="width:196px;">NMR Shigemi Tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">eVol kit-electronic syringe starter kit</td>
			<td style="width:177px;">Cortecnet</td>
			<td style="width:119px;">2910000</td>
			<td style="width:196px;">Pipetting</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:328px;">Bruker 900MHz AvanceIII with a triple resonance cryogenic probehead</td>
			<td style="width:177px;">Bruker</td>
			<td style="width:119px;"></td>
			<td style="width:196px;">NMR spectrometer for data acquisition</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:328px;">Bruker 600MHz DMX600 with a triple resonance cryogenic probehead</td>
			<td style="width:177px;">Bruker</td>
			<td style="width:119px;"></td>
			<td style="width:196px;">NMR spectrometer for data acquisition</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:328px;">TopSpin 3.1</td>
			<td style="width:177px;">Bruker</td>
			<td style="width:119px;"></td>
			<td style="width:196px;">Acquisition and Processing software for NMR experiments</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:328px;">Sparky 3.114</td>
			<td style="width:177px;">UCSF (T. D. Goddard and D. G. Kneller)</td>
			<td style="width:119px;"></td>
			<td style="width:196px;">NMR data Analysis software</td>
		</tr>
	</tbody>
</table>

nuclear magnetic resonance spectroscopy for the identification of multiple phosphorylations of intrinsically disordered proteins

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

图3A示出了在洗脱梯度过程中观察到280纳米的大吸收峰。这个峰对应于纯化的Tau蛋白的丙烯酰胺凝胶色谱以上所见。 图3B示出在280nm和电导率的峰的很好地分离的吸收峰，从而确保该蛋白质的脱盐是有效的。 图4示出了通过SDS-PAGE分析观察到多种蛋白磷酸化的16特性蛋白凝胶迁移（比较泳道2和3）。 图6示出了具有增加的脉冲长度（以微秒）的一系列质子（1H）1D光谱。设置将旋转90°1小时的自旋磁化，对应于转动360°的脉冲，脉冲的长度在实际中被使用，因为它更容易通过最小化信号来校准。当360°脉冲长度充分界定水质子的信号为空。对应于360的值76;旋转然后通过4. P1在这个实验分为是10.5微秒。放大区域:剩余信号的频率被用于定义O1P参数（频率为1小时偏移）。 图7 A.示出了自由感应衰减（FID），可视化，以确保检测到的NMR信号。 图7B。显示了一个不正确的相位1D 1小时的频谱，通过出现不对称峰共振所看到。 图7C示出了具有良好信噪比的一维1小时光谱，表明基本采集参数被正确设置，并且可以检测到来自蛋白质样品的信号。 图8显示2D 1 H，重组15 15 N-头N个HSQC光谱在900兆赫;具有良好的感光度和分辨率和图8B 图8A。与样品中检测的蛋白水解，如图由附加的pe的外观在光谱（蓝框）AKS。 图9显示2D 1 H，重组15 15 N碧桃图9A氮HSQC光谱在600 MHz时，其敏感性不错，但相比于图8A分辨率小。在600 MHz的图9B中 ，示出了在光谱对应于磷酸化的残基（红色框）额外的峰出现。在900 MHz 图9C中 ，示出对应于磷酸化的残基（红色框）在光谱的峰。分辨率比图9B中更好。 图10 示出从用于评估一个成功的实验三维NMR光谱突起。 图10B显示了良好的信号强度允许检测来自i和i-1残基13 C信号1 H- 15 N- 13 C 3D谱中提取的1 H- 13 C面。 图1:重组蛋白的生产和同位素标记的主要步骤方案。细菌改造重组蛋白生产步骤，在协议中的第1段所述进行了概述。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55001/55001fig1large.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/55001/55001fig2.jpg" /> 图2: 重组Tau蛋白纯化的主要步骤方案。从细菌细胞裂解步骤重组蛋白纯化在协议中的第2款所述进行了概述。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55001/55001fig2large.jpg" target="_blank">请点击此处查看大图</a>这个数字。 <img alt="图3" src="/files/ftp_upload/55001/55001fig3.jpg" /> 图3:协议的液相色谱步骤。加热细菌提取物（A），阳离子交换层析分离。在280纳米，260纳米和电导率的吸光度分别对应于固体和黑色虚线和红色虚线。所收集的级分的12％SDS-PAGE分析的色谱图的上方示出。的Tau蛋白的（B）的脱盐成适合于冷冻干燥的缓冲器。纯化的Tau蛋白的由峰面积（2,260 MAU *毫升）所估计的量为16毫克头的。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55001/55001fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55001/55001fig4.jpg"/&gt; 图4:牛头的12％SDS-PAGE分析。泳道1，分子量标记;第2泳道，10微克牛头;第3泳道，10微克ERK磷酸化的Tau蛋白。头，作为其他流离失所者，运行在SDS-PAGE上的异常的方式，在表观分子量约60kDa的而不是预期的46 kDa的。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55001/55001fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55001/55001fig5.jpg" /> 图5:用于NMR样品制备，NMR谱数据采集和数据处理的主要步骤方案。从NMR样品制备到数据采集和处理步骤，在协议中的第4段所述进行了概述。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55001/55001fig5large.jpg" target="_blank">请点击此处查看大versio</a>这个数字的n个。 <img alt="图6" src="/files/ftp_upload/55001/55001fig6.jpg" /> 图 6: 核磁共振数据采集的P1参数的设置。这个参数的不同样品之间和主要依赖于盐浓度。 80％H 2 O的D 2 O中的标准1小时垂头曲线所示。用30秒的再循环延迟单扫描光谱收集和水平绘制。脉冲（P1）从1微秒到55微秒为1微秒的步骤而变化。在理论上，该信号应该是最大为90°脉冲和等于零为180°脉冲。然而，在实践中，辐射阻尼引起的不对称性和相位失真的问题，这使得它难以直接确定90°或180°的脉冲。第二零点对应于360°脉冲持续时间。扩大区域表示为360°的残差信号<html>脉冲用来定义O1P频率参数。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55001/55001fig6large.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/55001/55001fig7.jpg" /> 图7:NMR数据的处理。 （ 一 ）在时域自由感应信号衰减。 1D氢谱（B）从FID的傅立叶变换从A组到频域，但不正确的相位（PHC0 -206°）产生。 （C）的相控（PHC0 -113°）和参考（在0ppm TMSP信号）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55001/55001fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> "/&gt; 图8:2D 1 H，重组15 15 N-头N个HSQC光谱在900兆赫。 3,072和416采集的数据点用的14光谱宽度，并在1 H（F2），和15 N（F1），尺寸为25ppm分别记录。 16扫描每F1增量记录，导致4小时30分钟的实验的持续时间。 （ 一 ）质量好头样品（B）头样本显示退化通过附加共振出现在频谱（高场1 H，低场15 N）的特定区域发现，这里的盒装为蓝色。这最后一个样品没有蛋白酶抑制剂制备。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55001/55001fig8large.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/55001/55001fig9.jpg" /> 图9:2D 1 H，重组15 15 N-头N个HSQC谱。 （A）磷酸化的头，600 MHz频谱（B）磷酸化tau蛋白，600 MHz频谱和（C）磷酸化tau蛋白，900 MHz频谱。在光谱的特定区域的其他谐振，在红色这里盒装，在磷酸化tau光谱中观察到。这些共振，这对应于质子酰胺（1 H 15 N），pSER的和PTHR残基的相关性，很容易在该区域可视化约8.5至9.5 ppm的1 H和117至125 ppm的15 N中，散装的外侧1 H时，未磷酸化头频谱15 N的相关性。 （A和B）分别对应于光谱在600兆赫，分别在14和25ppm的光谱宽度2048和256个数据点被记录在1 H（F2），和15 N（F1）的尺寸，获得的。32扫描被使用，和采集的总时间为2小时44分钟，并在900兆赫，3,072和416在14光谱宽度和数据点25ppm的被记录在1 H（F2），和15 N（C）（ F1）的尺寸，分别为。 48扫描被使用，以及收购的总时间为6小时37分钟。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55001/55001fig9large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图10" src="/files/ftp_upload/55001/55001fig10.jpg" /> 图10:3D 1 H，15 N，13 C NMR谱，预测和提取二维平面。 13 C（F1）分别2048，72，和256个数据点被记录在1 H（F3），15 N（F2）和尺寸。光谱宽度是14，25和61 ppm的，集中于4.7，119和41 ppm的在1 H，15 N和13 C尺寸，分别为。使用扫描16次收购的期限为4天，6小时。对应的傅立叶（A）立方体代表在转变600MHz的获得ERK-tau蛋白磷酸化的HNCACB频谱的三维数据。二维1 H，15 N和1 H，13 C平面沿13 C中的三维数据和15 N维的投影分别获得。数据处理和代表使用NMR采集和处理软件来完成。 （B）2D 1 H，13 C飞机从3D 1小时提取，15 N，121.8处ppm的15 N化学位移13 C NMR数据集。变焦（右侧）集中在9.38 ppm的的1小时化学位移同时显示残我（pThr153）和i-1（Ala152）的13 CA和CB 13共振。 13 CB共振到S的宽度别名因pectral窗口。利用NMR分析软件进行图形表示，峰采摘。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55001/55001fig10large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins at JoVE.com

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

核磁共振光谱法本质无序蛋白的磷酸化多元的鉴定

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

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18.6K 次观看.  Lille University. We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

视频: Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

A蛋白制备方法在蛋白质与核余子交互使用LC-MS / MS分析的高通量鉴定

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

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生物化学

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

固体核磁共振波谱法研究高分子组件的原子尺度结构

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

单分子力谱中蛋白质共价固定化的研究

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. Here, a protocol for detecting and measuring such interactions is described using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The globular protein of interest is labeled with 15N and titrated with vimentin protein in solution. A two-dimensional NMR spectrum is acquired to detect interactions by observing changes in peak shape or chemical shifts, and to elucidate effects of solution conditions including salt levels, which influence vimentin quaternary structure. If the protein of interest binds the filamentous ligand, the binding interaction is quantified by MST using the purified proteins. The approach is a straightforward way for determining whether a protein of interest binds a filament, and for assessing how alterations, such as mutations or solution conditions, affect the interaction.

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy NMR and Microscale Thermophoresis MST

Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments.

用核磁共振 (nmr) 和微尺度热能 (mst) 测量球状和丝状蛋白的相互作用

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

利用单击化学辅助 rna-路特捕获 (无花果) 策略捕获和鉴定 rna 结合蛋白

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR and sensitivity-enhanced DNP experiments. The sample processing procedure of plant biomass is also detailed. This method allows the measurement of a series of 1D and 2D 13C-13C/15N correlations spectra, which enables high-resolution structural elucidation of complex biomaterials in their native state, with minimal perturbation. The isotope-labeling can be examined by quantifying the intensity in 1D spectra and the polarization transfer efficiency in 2D correlation spectra. The success of dynamic nuclear polarization (DNP) sample preparation can be evaluated by the sensitivity enhancement factor. Further experiments examining the structural aspects of the polysaccharides and proteins will lead to a model of the three-dimensional architecture. These methods can be modified and adapted to investigate a wide range of carbohydrate-rich materials, including the natural cell walls of plants, fungi, algae and bacteria, as well as synthesized or designed carbohydrate polymers and their complex with other molecules.

A protocol for preparing 13C,15N-labeled fungal and plant samples for multidimensional solid-state NMR spectroscopy and dynamic nuclear polarization (DNP) investigations is presented.

动态核极化固态核磁共振法制备结构澄清用真菌和植物材料

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

成熟水稻的横向切片（米 苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and have the potential to influence the sensitization process either through directly stimulating the immune system or altering the biophysical properties of the allergenic protein. In order to control for these variables, techniques are required for the removal of endogenously bound ligands and, if necessary, replacement with lipids of known composition. The cockroach allergen Bla g 1 encloses a large hydrophobic cavity which binds a heterogeneous mixture of endogenous lipids when purified using traditional techniques. Here, we describe a method through which these lipids are removed using reverse-phase HPLC followed by thermal annealing to yield Bla g 1 in either its Apo-form or reloaded with a user-defined mixture of fatty acid or phospholipid cargoes. Coupling this protocol with biochemical assays reveal that fatty acid cargoes significantly alter the thermostability and proteolytic resistance of Bla g 1, with downstream implications for the rate of T-cell epitope generation and allergenicity. These results highlight the importance of lipid removal/reloading protocols such as the one described herein when studying allergens from both recombinant and natural sources. The protocol is generalizable to other allergen families including lipocalins (Mus m 1), PR-10 (Bet v 1), MD-2 (Der p 2) and Uteroglobin (Fel d 1), providing a valuable tool to study the role of lipids in the allergic response.

This protocol describes the removal of endogenous lipids from allergens, and their replacement with user-specified ligands through reverse-phase HPLC coupled with thermal annealing. 31P-NMR and circular dichroism allow for the rapid confirmation of ligand removal/loading, and the recovery of native allergen structure.

从脂质绑定蛋白和过敏原中去除和替换内源性利根

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...

NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest advantage of NMR-based fragment screening is its ability not only to detect binders over 7-8 orders of magnitude of affinity but also to monitor purity and chemical quality of the fragments and thus to produce high quality hits and minimal false positives or false negatives. A prerequisite within the FBS is to perform initial and periodic quality control of the fragment library, determining solubility and chemical integrity of the fragments in relevant buffers, and establishing multiple libraries to cover diverse scaffolds to accommodate various macromolecule target classes (proteins/RNA/DNA). Further, an extensive NMR-based screening protocol optimization with respect to sample quantities, speed of acquisition and analysis at the level of biological construct/fragment-space, in condition-space (buffer, additives, ions, pH, and temperature) and in ligand-space (ligand analogues, ligand concentration) is required. At least in academia, these screening efforts have so far been undertaken manually in a very limited fashion, leading to limited availability of screening infrastructure not only in the drug development process but also in the context of chemical probe development. In order to meet the requirements economically, advanced workflows are presented. They take advantage of the latest state-of-the-art advanced hardware, with which the liquid sample collection can be filled in a temperature-controlled fashion into the NMR-tubes in an automated manner. 1H/19F NMR ligand-based spectra are then collected at a given temperature. High-throughput sample changer (HT sample changer) can handle more than 500 samples in temperature-controlled blocks. This together with advanced software tools speeds up data acquisition and analysis. Further, application of screening routines on protein and RNA samples are described to make aware of the established protocols for a broad user base in biomacromolecular research.

Fragment-based screening by NMR is a robust method to rapidly identify small molecule binders to biomacromolecules (DNA, RNA, or proteins). Protocols describing automation-based sample preparation, NMR experiments &#38; acquisition conditions, and analysis workflows are presented. The technique allows for optimal exploitation of both 1H and 19F NMR-active nuclei for detection.

基于核磁共振的片段筛选，样品最少，自动化程度最高

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest ...

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

纳米微分扫描荧光仪，用于在基于碎片的铅发现中筛选

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...