Name: nuclear magnetic resonance spectroscopy for the identification of multiple phosphorylations of intrinsically disordered proteins
Uploaded: 2016-12-27T14:00:00.000+00:00
Duration: 12 min 47 s
Description: Watch this Scientific Journal Video about Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins at JoVE.com

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?...

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><tbody><tr><td>pET15B recombinant T7 expression plasmid</td><td>Novagen</td><td>69257</td><td>Keep at -20 &amp;deg;C</td></tr><tr><td>BL21(DE3) transformation competent &lt;em&gt;E. coli&lt;/em&gt; bacteria</td><td>New England Biolabs</td><td>C2527I</td><td>Keep at -80 &amp;deg;C</td></tr><tr><td>Autoclaved LB Broth, Lennox&amp;nbsp;</td><td>DIFCO</td><td>240210</td><td>Bacterial Growth Medium</td></tr><tr><td>MEM vitamin complements 100x</td><td>Sigma</td><td>58970C</td><td>Bacterial Growth Medium Supplement</td></tr><tr><td>&lt;sup&gt;15&lt;/sup&gt;N, &lt;sup&gt;13&lt;/sup&gt;C-ISOGRO complete medium powder</td><td>Sigma</td><td>608297</td><td>Bacterial Growth Medium Supplement</td></tr><tr><td>&lt;sup&gt;15&lt;/sup&gt;NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Sigma</td><td>299251</td><td>Isotope</td></tr><tr><td>&lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;6&lt;/sub&gt;-Glucose</td><td>Sigma</td><td>389374</td><td>Isotope</td></tr><tr><td>Protease inhibitor tablets&amp;nbsp;</td><td>Roche</td><td>5056489001</td><td>Keep at 4 &amp;deg;C</td></tr><tr><td>1 tablet in 1 ml is 40x solution that can be kept at -20 &amp;deg;C</td><td></td><td></td><td></td></tr><tr><td>DNaseI</td><td>EUROMEDEX</td><td>1307</td><td>Keep at -20 &amp;deg;C</td></tr><tr><td>Homogenizer (EmulsiFlex-C3)</td><td>AVESTIN</td><td></td><td>Lysis is realized at 4 &amp;deg;C</td></tr><tr><td>Pierce&amp;trade; Unstained Protein MW Marker</td><td>Pierce</td><td>266109</td><td></td></tr><tr><td>Active human MEK1 kinase, GST Tagged</td><td>Sigma</td><td>M8822</td><td>Keep at -80 &amp;deg;C</td></tr><tr><td>AKT&amp;Auml; Pure chromatography system</td><td>GE Healthcare</td><td></td><td>FPLC</td></tr><tr><td>HiTrap SP Sepharose FF (5 ml column)</td><td>GE Healthcare</td><td>17-5156-01</td><td>Cation exchange chromatography columns</td></tr><tr><td>HiPrep 26/10 Desalting</td><td>GE Healthcare</td><td>17-5087-01</td><td>Protein Desalting column</td></tr><tr><td>PD MidiTrap G-25</td><td>GE Healthcare</td><td>28-9180-08</td><td>Protein Desalting column</td></tr><tr><td>Tris D11, 97% D</td><td>Cortecnet</td><td>CD4035P5</td><td>Deuterated NMR buffer</td></tr><tr><td>5 mm Symmetrical Microtube SHIGEMI D&lt;sub&gt;2&lt;/sub&gt;O (set of 5 inner &amp;amp; outerpipe)&amp;nbsp;</td><td>Euriso-top</td><td>BMS-005B</td><td>NMR Shigemi Tubes</td></tr><tr><td>eVol kit-electronic syringe starter kit</td><td>Cortecnet</td><td>2910000</td><td>Pipetting</td></tr><tr><td>Bruker 900 MHz AvanceIII with a triple resonance cryogenic probehead</td><td>Bruker</td><td></td><td>NMR spectrometer for data acquisition</td></tr><tr><td>Bruker 600 MHz Avance with a triple resonance cryogenic probehead</td><td>Bruker</td><td></td><td>NMR spectrometer for data acquisition</td></tr><tr><td>TopSpin 3.1</td><td>Bruker</td><td></td><td>Acquisition and Processing software for NMR experiments</td></tr><tr><td>Sparky 3.114</td><td>UCSF (T. D. Goddard and D. G. Kneller)</td><td></td><td>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<su...

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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Unit 1

Protein Structure

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18.7K 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

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

This protocol describes the reconstitution of nucleosomes containing differentially isotope-labeled sister histones. At the same time, asymmetrically post-translationally modified nucleosomes can be generated after using a premodified histone copy. These preparations can be readily used to study modification crosstalk mechanisms, simultaneously on both sister histones, by using high-resolution NMR spectroscopy.

核小体的重建与差异同位素标记的姐妹组蛋白

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

科研

生物化学

Oligopeptide Competition Assay for Phosphorylation Site Determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

寡肽竞争测定磷酸化位点测定

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

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 ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human proteome. The highly flexible nature of these sequences allows them to form weak, long-range, and transient interactions with diverse biomolecular partners. Specific yet low-affinity interactions promote promiscuous binding and enable a single intrinsically disordered segment to interact with a multitude of target sites. Because of the transient nature of these interactions, they can be difficult to characterize by structural biology methods that rely on proteins to form a single, predominant conformation. Paramagnetic relaxation enhancement NMR is a useful tool for identifying and defining the structural underpinning of weak and transient interactions. A detailed protocol for using paramagnetic relaxation enhancement to characterize the lowly-populated encounter complexes that form between intrinsically disordered proteins and their protein, nucleic acid, or other biomolecular partners is described.

A protocol for the application of paramagnetic relaxation enhancement NMR spectroscopy to detect weak and transient inter- and intra-molecular interactions in intrinsically disordered proteins is presented.

顺磁弛豫增强，用于检测和表征固有无序蛋白质的自缔合

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

用于蛋白质磷酸化定量的优化单分子下拉测定

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

用于聚糖-蛋白质相互作用分析的先进 NMR 技术

Disentangling Glycan-Protein Interactions: Nuclear Magnetic Resonance (NMR) to the Rescue

The interactions of glycans with proteins modulate many events related to health and disease. In fact, the establishment of these recognition events and their biological consequences are intimately related to the three-dimensional structures of both partners, as well as to their dynamic features and their presentation on the corresponding cell compartments. NMR techniques are unique to disentangle these characteristics and, indeed, diverse NMR-based methodologies have been developed and applied to monitor the binding events of glycans with their associate receptors. This protocol outlines the procedures to acquire, process and analyze two of the most powerful NMR methodologies employed in the NMR-glycobiology field, 1H-Saturation transfer difference (STD) and 1H,15N-Heteronuclear single quantum coherence (HSQC) titration experiments, which complementarily offer information from the glycan and protein perspective, respectively. Indeed, when combined they offer a powerful toolkit for elucidating both the structural and dynamic aspects of molecular recognition processes. This comprehensive approach enhances our understanding of glycan-protein interactions and contributes to advancing research in the chemical glycobiology field.

作者聚焦:揭示聚糖分子识别的结构和动力学方面

Here, we present a protocol detailing the acquisition, processing, and analysis of a series of NMR experiments aimed at characterizing protein-glycan interactions in solution. Most common ligand-based and protein-based methodologies are outlined, which undoubtedly contribute to the fields of structural glycobiology and molecular recognition studies.

解开聚糖-蛋白质相互作用:核磁共振 （NMR） 来拯救

The interactions of glycans with proteins modulate many events related to health and disease. In fact, the establishment of these recognition events and ...

化学

<meta charset="utf8"></head><body>蛋白质动力学研究中 NMR 15N 弛豫和 hetNOE 实验的方案

NMR 15N Relaxation Experiments for the Investigation of Picosecond to Nanoseconds Structural Dynamics of Proteins

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide groups of the protein backbone or the methyl groups in side chains are used as reporters of structural dynamics in proteins. A structural dynamics study of the protein backbone of globular proteins on 15N labeled and fully protonated samples usually works well for proteins with a molecular weight of up to 50 kDa. When side chain deuteration in combination with transverse relaxation optimized spectroscopy (TROSY) is applied, this limit can be extended up to 200 kDa for globular proteins and up to 1 MDa when the focus is on the side chains. When intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs) are investigated, these weight limitations do not apply but can go well beyond. The reason is that IDPs or IDRs, characterized by high internal flexibility, are frequently dynamically decoupled. Various NMR methods offer atomic-resolution insights into structural protein dynamics across a wide range of time scales, from picoseconds up to hours. Standard 15N relaxation measurements overview a protein&#39;s internal flexibility and characterize the protein backbone dynamics experienced on the fast pico- to nanosecond timescale. This article presents a hands-on protocol for setting up and recording NMR 15N R1, R2, and heteronuclear Overhauser effect (hetNOE) experiments. We show exemplary data and explain how to interpret them simply qualitatively before any more sophisticated analysis.

<meta charset="utf8"></head><body>作者聚焦：通过 NMR 弛豫实验探索内在无序的蛋白质动力学

Nuclear magnetic resonance (NMR) spectroscopy can characterize structural protein dynamics in a residue-specific manner. We provide a hands-on protocol for recording NMR 15N R1 and R2 relaxation and {1H}-15N heteronuclear Overhauser effect (hetNOE) experiments, sensitive to the picoseconds to nanoseconds timescale.

NMR 15N 弛豫实验，用于研究蛋白质皮秒至纳秒的结构动力学

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide ...

核磁共振光谱法本质无序蛋白的磷酸化多元的鉴定

摘要

探索更多视频

此视频中的章节

核小体的重建与差异同位素标记的姐妹组蛋白

寡肽竞争测定磷酸化位点测定

固体核磁共振波谱法研究高分子组件的原子尺度结构

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

顺磁弛豫增强，用于检测和表征固有无序蛋白质的自缔合

用于蛋白质磷酸化定量的优化单分子下拉测定

解开聚糖-蛋白质相互作用:核磁共振（NMR）来拯救

解开聚糖-蛋白质相互作用:核磁共振（NMR）来拯救

解开聚糖-蛋白质相互作用:核磁共振（NMR）来拯救

NMR ¹⁵N 弛豫实验，用于研究蛋白质皮秒至纳秒的结构动力学

核磁共振光谱法本质无序蛋白的磷酸化多元的鉴定

摘要

探索更多视频

此视频中的章节

核小体的重建与差异同位素标记的姐妹组蛋白

寡肽竞争测定磷酸化位点测定

固体核磁共振波谱法研究高分子组件的原子尺度结构

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