作者感谢JJ博士范德克林克在选择和组装设备的帮助，以及F.凯笛博士和G. Bertho博士有益的讨论。交流是由瑞士国家科学基金会（授予PPOOP2_157547）的支持。我们承认从巴黎索邦太阳城（NMR @ COM中，DIM分析，威乐巴黎，基金会德拉RECHERCHE MEDICALE（FRM ING20130526708）和Parteneriat休伯特·居里安布朗库西32662QK融资。我们的团队是Equipex计划巴黎烯共振的一部分和CACSICE。

注意:使用商业软件进行所有数据分析。 1.准备偏光解决方案<ol><li>准备加入2ml 1.12 男性 13 C标记的丙酮酸钠（Na +的[CH 3 -CO- 13 COO] - ，衬底）溶液掺杂有TEMPOL自由基的33毫（4-羟基-2,2,6,6-四甲基-1-氧，偏光剂）4在2:1个D 2 O / D 6 -乙醇为13 C的意见。注意:处理必须采取预防措施，由于到D 6 -乙醇的易燃性，。 </li><li>验证自由基浓度是实现偏振方面的优化。 <ol><li>稍微改变在点1.1溶液的自由基浓度（例如，30毫米或35毫米）。 </li><li>确定迭代固态增强（见步骤3），并找到自由基浓度，导致最大的增强。 </li></ol></li></ol> 2. Polarizat离子<ol><li>通过降低技能冷却过程中的低温恒温器DNP温度。按此低温恒温器软件接口（图2D）上的"冷却"按钮。 </li><li>在通过填充过程的DNP低温恒温器收集液态氦。按此低温恒温器软件接口（图2D）上的"填充"按钮。 </li><li>放置300微升优化偏振溶液从设置有DNP偏振器样品容器步骤1.1。通过在液氮浴轻轻浸渍冻结与内部的样本的样本容器。 </li><li>消除液氮可存在于通过提取从液氮浴中的样本容器和旋转倒置的冷冻过程（步骤2.3）后的样品容器。 注:此步骤是为了获得样品的透彻溶解关键（见步骤5）。 </li><li>将样品放入低温恒温器。 注:此圣EP，下面的子步骤构成，必须迅速地进行（即，在小于10-20秒），以防止熔化的样品。 <ol><li>插入在样品架的样本容器。插入在主低温恒温器插入件的样品保持器。插入在样品架的微波波导。 </li></ol></li><li>通过启动1K的降温过程降低低温恒温器的温度。按此低温恒温器软件接口（图2D）上的"1K冷却"按钮。 </li><li>上打开微波源和照射样品。按此低温恒温器软件接口（图2D）上的"MW-ON"按钮。 </li></ol> 3.固态核磁共振测量<ol><li>由90°旋转的连接器逆时针方向并拉动它断开分光计线圈核磁共振控制台检测通道的同轴电缆。堵塞检测通道的同轴电缆的低温恒温器的同轴连接器NMR线圈。插入低温恒温器NMR线圈的母连接器的NMR控制台电缆的插入式连接器，注意定向销;力推和90°连接器顺时针旋转。 </li><li>测量在固态在低温恒温器现场使用具有在T = 5分钟的间隔反复未校准低翻转角脉冲的简单脉冲获取序列核磁共振热信号。建立测量后，写在软件"ZG"的命令行，然后按"Enter"键键盘键。请参阅核磁共振仪操作手册上的光谱仪的操作和测量设置的进一步细节。 </li><li>测量DNP-NMR增强信号。引发如在第2部分所述的DNP过程后步骤3.2的重复操作。 </li><li>由90°旋转的连接器逆时针方向并拉动它断开低温恒温器核磁共振线圈核磁共振控制台检测通道同轴电缆。堵塞DETE的同轴电缆ction通道分光计线圈的同轴连接器。插入谱仪线圈的母连接器的NMR控制台电缆的插入式连接器，注意定向销;力推和90°连接器顺时针旋转。 </li></ol> 4.优化主磁铁场（&#39;匀场"）的同质性<ol><li>在分光计放置装有混合物的500微升在以前的实验（即，后步骤6.2）所产生的管。 </li><li>通过改变电流的垫补梯度线圈搜索最​​大执行NMR波谱仪垫补&#39;锁定&#39;氘的信号。选择通过推动核磁共振控制台上的相应按钮相关的匀场线圈。 <ol><li>旋转核磁共振控制台上的旋钮，以确定增加锁定信号的旋转方向。继续旋转，直到信号达到最大值。选择另一个垫补线圈和重复的最大化联合国直到所有垫补线圈​​当地最大发现。请参阅核磁共振仪的操作手册了解更多细节。最后，除去用于锁定样品。 </li></ol></li></ol> 5.解散<ol><li>插入5毫升D 2 O中的溶出插入锅炉。加压并达到了T≈180-200°C，直到由电阻丝的方式加热D 2 O操作。按此低温恒温器软件接口（图2D）上的"准备加热器"按钮。 </li><li>样品极化过程完成之后（例如，后3个T DNP），略微加压高于室温压力的低温恒温器。按此低温恒温器软件接口（图2D）上的"SS操作"按钮。取下解散插入微波波导。 </li><li>向下滑动的溶解插入物（ 图3B）到样品保持器（图3A-C）。溶解插入件具有到达样本保持器的底部，并必须牢固地推以与样本容器气密连接，以避免在低温恒温器D 2 O中泄漏。 </li><li>按硬件触发（图3C-h）至开始溶解序列，气动阀的操作的预先确定的定时序列。 </li></ol> 6.注射<ol><li>解散地方一个5毫米的NMR管，含有500微升样品（ 例如D 2 O为前<img alt="Equation13" src="/files/ftp_upload/53548/53548equation13.jpg" width="29" />在从步骤8在步骤9的酶活性的测量步骤7或LDH溶液），在11.74ŤNMR波谱仪的等角点测量（见步骤4）。 </li><li>刚解散（步骤5）后，放置在NMR波谱仪在步骤6.1的样品混合500μl的超极化溶液。 <ol><li>手动进样:收集超极化溶液在2.5米长的传输管中放置在靠近NMR波谱仪磁铁玻璃烧杯中的结束。通过定制的毛细管系统手动注入500微升在NMR样品管超极化液。 </li><li>自动注射:在溶解过程之前，传输管连接到分离用于传送氦气的超极化溶液的自动化定制的注射装置。该装置自动提供500微升超极化溶液进入样品管。 </li></ol></li></ol> 7.液态核磁共振测量<ol><li>溶解后，通过一系列的1.5秒​​间隔以跟随基板（A）的磁化的进展和产物（B）10°翻转角脉冲获取序列测量来自在光谱仪样品核磁共振超极化信号。建立测量后，写在软件"ZG"的命令行，然后按＆＃39，输入"键盘键。请参阅核磁共振仪操作手册上的光谱仪的操作和测量设置的进一步细节。 </li><li>一旦磁化了一系列由90 T = 3个T 1≈3分钟间隔°翻转角度脉冲获得序列得到充分的放松（即后T = 10吨1），测量核磁共振热信号。建立测量后，写在软件"ZG"的命令行，然后按"Enter"键键盘键。请参阅核磁共振仪操作手册上的光谱仪的操作和测量设置的进一步细节。 </li></ol> 8.制备酶含有样品（具体为丙酮酸到乳酸变换） <ol><li>制备具有下述配方的反应缓冲液:50mM在0.1M磷酸盐缓冲液还原的烟酰胺腺嘌呤二核苷酸（NADH），1mM的乙二胺四乙酸（EDTA），0​​.1mM的二硫苏糖醇（DTT），pH值=7.0。 </li><li>制备兔肌肉的LDH溶液在反应缓冲液新鲜制备的20微克/毫升牛血清白蛋白，BSA 1单位/毫升。 </li><li>混合478微升反应缓冲液（8.1步），20微升D 2 O中，以便谱仪场稳定（&#39;锁定&#39;）和2微升LDH溶液（7.2步）。 </li><li>代替步骤8.3准备的500微升溶液体积为5 mm的NMR管和管位置到500 MHz的核磁共振波谱仪。 </li></ol> 9.完成酶促反应速率测量程序<ol><li>制备偏振样品（步骤1）和偏振它（步骤2）。 </li><li>执行匀场在光谱仪（步骤4）和制备酶样品（步骤8）。 </li><li>执行的溶解（步骤5）和喷射（步骤6）。 </li><li>测量与由1.5秒（步骤7）间隔开10°的翻转角的脉冲采集一系列从超极化样品的信号。此步骤允许测量超极化信号衰减和代谢物的信号积累。 </li></ol> 10.配件<ol><li>在步骤9.4获得的每一谱，识别对应分别到基板（A）和产物（B）和整合它们的峰（确定峰的面积）。峰积分给磁化时间过程信号（M（A，B）（T），见公式2）。 </li><li>使用方程的溶液（2），确定:F =（R A + K eff与值 ）从集成在步骤10.1衬底信号的一个简单的指数拟合。 </li><li>使用值F =（R A + K EFF ）在步骤10.2确定的，从与函数拟合通过积分方程获得的产物信号M B（t）的确定ķEFF 和 R B（2）。参照代表性的成果段（EnzymatIC的活动和代谢测量）对反应建模，装配和 RF校正的细节。 </li></ol>

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作者宣称，他们已没有竞争经济利益

溶解DNP核磁共振实验的关键点是:（i）偏振的实现为基材的水平，它决定所必需的实验最低的产品浓度以及能够进行和（ii）的寿命信号的采集次数磁化的，相比于偏振和检测位点之间，并以基板转化率传输的持续时间。本文描述的溶出DNP设置的喷射系统允许在低至3-4秒样品转移。虽然转印不如在由S. Bowen和C. Hilty提出的方法为快速，丙酮酸26极化损失由于在低场的中等纵向弛豫限制。 [1- 13 C]丙酮酸，以其悠久的羧酸核T 1，允许通过测量LDH通量浓度低至10 -3单位/毫升。 该<html>使用溶解DNP表明之一可以是敏感于更低[1- 13 C]丙酮酸浓度，和较低的酶转化率得到高的信噪比。所达到的极化水平，得到使用低倾倒角α= 10°的液态NMR波谱仪要采集超过200实验用显著信噪比。这使得优化空间两者中的重复次数和翻转角度而言。在固态极化的积聚一些分子与一些偏振剂（TEMPO，OXO63）4等极化以及不这样做，对于那些还没有完全了解的原因。实验性试验是确定的偏振步骤是否成功的唯一途径。为了提高偏光度，可以探索采用不同的自由基4和不同的技术依托"交叉极化"的应用程序。25 内容】"&gt;基和底物浓度以及对DNP试样中的溶剂组合物的进一步优化可以尝试以改善偏振。该技术被限制为分子，其中一个核或能够维持偏振后的溶解可以是一组核识别。极化能持续或者作为在高磁场或在LLS的形式离域上的两个或更多个耦合的细胞核的单核征态之间的不平衡。对于第一种选择，探针核必须是从具有高的其他核遥远旋磁比，如质子。如果这样的位置没有天然发现，富集在活性核的附近质子的分子或更换在分离的站点NMR-活性核通过氘核，以降低磁偶极子的强度，是必要的为了获得LLS，磁耦合的核团内的理论分析可以为了找到最佳的手段增刊出27,28进行ORT两极分化。这个策略已被证明是成功的小分子如氨基酸29和可以应用于参与感兴趣的代谢循环的其它分子。在实验过程中更好地维护磁化，溶解DNP与LLS的激励相结合的承诺延长测量时间跨度为其它酶促反应。20 这里所描述的DNP-NMR实验适于在癌细胞丙酮酸代谢的测量。6通过溶解DNP增强NMR酶活性的实时测量可以通过DNP增强MRI帮助癌症的诊断目前的努力，在已经使用诊所。12 DNP-NMR增强的分子特异性使得它首选的分子靶点和转换的产品区分的方法。未来的改进将专注于其他分子示踪剂的评估代谢转化30 </SUP&gt;易被相关用于MRI诊断，以及对获得延长观察时间窗口。

动态核极化（DNP），1,2-旨在加强核自旋极化， 即与"向上"和"向下"的自旋人群（P =不平衡的技术[N↑ - N↓] / [N↑+ N ↓]），最早是在1950年推出。核自旋如13 C能在良好的条件被极化到 P = 10 -1，典型地在1的K的量级的温度下和在3.357磁场T. 3,4-一种用于生物应用的突破是在2000年初与溶解的DNP的发展其由在过热水 ​​中溶解的冷冻极化样品，同时保持在低温下获得的高核极化水平。5所述的液状NMR信号增强由一个因子10 3 -10 4相比共同热极化RT NMR条件。因此溶解DNP提供了一种方法，以在实时原位非侵入测量生化反应速率，允许通过NMR监测动力学与1秒或更小的时间分辨率。6 - 10它也成为可能检测非常低浓度的分析物11。 间的非侵入性的分子成像方式，超极化的核磁共振是，允许同时测量基底和其代谢产物进行实时的唯一技术。溶解DNP用在各种科学领域从体外核磁共振到临床MRI 12和最有前景的应用热情接收都涉及到在现场监测代谢。13,14溶出DNP的主要限制在于，一个时间上后的纵向弛豫时间五倍常数 T 1的顺序，增强极性化都将丢失。因此，有必要使用分子轴承表现出相对长 T 1的核自旋。为了延长极化增强的时间跨度，慢慢松弛核自旋态称为寿州（LLS），可使用15 - 17 LLS是不敏感的对内偶极-偶极相互作用，因此它们的特征弛豫时间常数 ，T LLS，可以更长于 T 1。18数十分钟的磁化寿命，因此，以1小时可以得到，19,20和LLS已经提出了用于两磁共振波谱（MRS）和MRI。21 需要用于通过超极化NMR研究酶反应速率来仔细优化的要点是:（i）最大化固态极化和（ii）的超极化溶液从在传送过程中最小化的偏振损耗偏光片到核磁共振波谱仪。本文介绍了一个特制的溶解DNP设备和喷射系统的适应性研究酶促反应。的特点和设置的性能将与知名的和广泛使用的超极化基板[1- 13 C]的丙酮酸证明。这使得监控反应过程几分钟;第二，它的核心作用这一选择的原因主要是，第一，它的自然长13 C纵向弛豫时间（在高磁场和温度超过293度牛逼 1&gt; 50秒）癌症的代谢。13,14使用溶解DNP NMR和定制开发喷射系统，丙酮酸通过乳酸脱氢酶（LDH）催化的氧化可以在未标记的乳酸9,22的初始池的或不加入未标记乳酸盐存在进行监控，如下图所示。它已经显示在vi测定的[1- 13 C]乳酸盐信号VO（包括细胞）的超极化[1- 13 C]丙酮酸的喷射以下的主要原因是丙酮酸和乳酸之间，而不是乳酸产生一个快速标签交换6 这里我们提出了实时生产[1- 13 C]乳酸从注入到包含LDH一个NMR管中但最初没有乳酸超极化[1- 13 C]丙酮酸盐。 系统描述 有在溶解DNP设置两个主要部分（ 图1）:对DNP偏振器和NMR波谱仪。对DNP偏振器的主要元件是低温恒温器，以在一个泵送氦浴样品冷却到大约1K的。低温恒温器被插入在一个3.35Ť超导磁铁，并且具有保证具有在磁铁（图1）的等角点的偏振样品的几何形状。低温恒温器内，所述样品（a）的由核磁共振线圈（B）包围，以测量偏振buildup，包含在过模微波腔体（C）。整个样本被保持在一个泵送氦浴（D）低的温度和通过所述波导的微波照射。整个系统由定制软件（图2D）进行管理。 硬件和执行DNP所需的深冷设备和随后的溶解仍然是一个技术挑战。新DNP低温恒温器23,24的开发和测试，以确定其低温性能，然后快速降温，氦保持时间和最小的总消耗的氦操作过程中进行了优化。 低温恒温器由两个部分组成。低温恒温器的第一部分是绝缘杜瓦（图2A），可以在顶端部大致分开（一个）的尾部，或者样品的空间（b）中，与外真空室（OVC）保持在高真空和壳体的下辐射屏幕（c）所示。低温恒温器的第二部分是在主SERT（图2B），放入绝缘杜瓦，其中所有的流量的规定进行管理。液态氦通过传输线（一）从外部存储杜瓦到来，是在分离器（B）中冷凝的第一级，中间室中使用既能保持低温恒温器冷的顶端部分，并除去蒸馏的氦在转移过程中。分离器压力是通过围绕低温恒温器的顶部缠绕的毛细管（C）的泵送降低;冷氦在此毛细管的流用于冷却在绝缘杜瓦（OVC）的挡板（d）和所述辐射屏。将样品置于和偏振中的样本的空间。样品空间被连接到通过另一个毛细管（e）该分离器，围绕主低温恒温器插入件的尾部缠绕。此毛细管可以通过从外部手动操作的针阀被打开或关闭。 为了实现DNP PR过程中使用的温度低ocess，液氦需要在低温恒温器样品空间被收集和其压力降低到毫巴范围。所需的低温恒温器的操作的操作是通过一个相当复杂的泵送系统具有三套泵，监测，并与电子和电机械仪器（图2C）的不同点操作的执行。低温恒温器OVC需要由第一泵系统泵送至在高真空。该系统由通过旋转泵（一）备份涡轮分子泵。液态氦从通过低温恒温器输送线路入口到低温恒温器分离器的存储杜瓦（B）传送。隔板具有连接到所述第二泵送组的出口。这组由35 立方米 /小时隔膜泵（三）。这条线，可除去从杜瓦和分离冷却过程中传输过程中煮氦气。在分离器中收集的液体氦然后可通过盖传送到样本空间上述illary管。从分离器传输液氦到样本空间，并随后以较低的采样空间压力毫巴范围内，250 米 3 /小时罗茨构成的第三泵系统泵由65 米 3 /小时旋转泵（四）备份通过手动蝶阀（E）连接到低温恒温器。 所有的真空系统的操作控制，并通过一个电气定制设备（F）调节。此装置控制低温恒温器分离器（克）和样品空间（H）网点，第二/第三泵送系统（C，D），一个压缩氦瓶（i）和外部之间真空管线连接。 （f）和外部之间的通信通过一个单向阀（J）。电 - 气动装置（F），以及所有的系统参数和溶出硬件通过用普通PC定制的电子设备接口的USB控制和操作。最后，所有的系统，通过电子装置中，是通过在相关操作是通过一个接口使用软件按键发起定制独立的软件（图2D）进行管理。 管理样品并测量被用于一系列插入物的固体状态（ 图3A）的NMR信号积聚。为了准备极化冷冻切片机，将（一）主要样本插入，进入低温恒温器。主样品插入件设置有放置在过模镀金微波腔内的NMR线圈（b）中。预冷冻含有溶液的基片进行极化的在液氮温度（偏光溶液）在适宜的样本容器，并将其放置在玻璃纤维样本保持器（C）的端底部​​。样品架滑入主要样本插入到达磁体等中心。插入在样品支架上的镀金波导（d）所示。波导允许从外部的微波源产生的微波以最小的损失吨至游澳样本。 为低温恒温器管理的定做软件自动处理，当点击相应的接口按钮，不同的操作一样冷却（低温恒温器温度接近降低到液体氦温度），填充（低温恒温器充满液体氦到预先确定的电平），冷却至 T≈1 K（液态氦浴泵送至达到的最低温度可能），加压的附加 ​​步骤（低温恒温器被稍微高于在P房压力加压= 10-30毫巴，以允许低温恒温器开口不会危害由空气中的低温恒温器的污染），并溶解（自动程序以溶解DNP样品，将所得的超极化溶液转移到测量部位，即核磁共振光谱仪）。 进行偏振照射微波的样品在94千兆赫（在偏振场 B 0 </sUB&gt; = 3.35 T）。一个样本后3个T DNP，其中T是DNP极化产生时间:T DNP是由于震级在给定场和温度固态靶核的纵向弛豫时间的顺序相同才算完全极化。在我们所有的实验中，样品被极化超过5吨的DNP。 在的极化时间结束时，将样品必须被溶解在为了一个的RT溶液用于酶活性的测量。在溶解过程中，加入5ml的过热D 2从溶解插入物（ 图3B）的锅炉O均为由压缩氦气（P = 6-8巴）推到到达DNP增强样品和溶解。将所得的超极化溶液通过压缩氦气压出溶解的插入，通过溶解插入插座（图3C-B </s仲&gt;），2毫米内径特氟隆传输管。所需的溶解过程的时间是300毫秒。23需要从DNP偏振器的样品转移至NMR波谱仪站点的时间是大约3秒。 使用溶解插入物（ 图3B）进行溶解过程。溶解插入是由一个电子 - 气动组件（a）的，含在气动组件的锅炉和样本容器更衣室（c）中，它允许防漏耦合与样品之间的连接管的碳纤维棒（二）容器，以及背出到出口。电-气动组件（图3C）被用于生产，并通过碳纤维棒驱动过热D 2 O中的样品容器，然后以提取从低温恒温器的超极化溶液。电 - 气动组件由用于控制共之间的连接气动阀（一）的mpressed氦（P = 6-8巴）线（b）中，其中D 2 O中，通过阀（D）中喷射出的锅炉（c）和所述出口（e）通过所述的碳纤维棒（F）。该系统由一个压力G，温度计和在锅炉（c）一种加热电阻丝，一触发（h）及用于连接系统与电子管理装置的连接盒（ⅰ）完成。 对DNP低温恒温器和NMR波谱仪是由传输线， 即，溶解被触发当两个内径在其内部的超极化溶液通过加压氦气压（P = 6-8巴）的PTFE管相连。 溶解序列由下列操作组成:在第一300毫秒，过热D 2 O中被推到样品容器，以便熔化和溶解该超极化的冷冻溶液中。此后，超极化溶液从低温恒温器被PRES的平均提取surized（P = 6-8栏）氦气，通过2个内径聚四氟乙烯管推（图3C-E），可在注射与任一步骤中描述的步骤进行测量点6.2.1或6.2步0.2。 溶解DNP核磁共振设置的第二个组件是NMR波谱仪。在本文所描述的设置中，NMR波谱仪在一个场 B 0 = 11.7特斯拉操作。一个5毫米的NMR探头来测量解散后超极化的信号。该核磁共振波谱仪通过核磁共振控制台操作，使用了固态和液态核磁共振测量，以及公司提供的软件XWinNMR。一个典型的测定由低翻转角硬脉冲（或者校准，为liquidstate或未校准的，固态的测量），接着在信号采集。 固态热极化信号与DNP-的Si的测量gnal集结使用定制的13 C在线圈耦合到核磁共振波谱仪的DNP偏振器（ 图3AB）的现场进行。在这种特定情况下的NMR波谱仪不执行信号锁定。当固态测量进行，以避免显著扰动偏振，采集之间的时间延迟应足够长，比至 0.5吨的DNP大致更长。 固态增强被定义为<img alt="公式4" src="/files/ftp_upload/53548/53548equation4.jpg" width="100" />哪里<img alt="Equation5" src="/files/ftp_upload/53548/53548equation5.jpg" width="31" />在超极化信号（步骤3.3获得）和<img alt="公式6" src="/files/ftp_upload/53548/53548equation6.jpg" width="23" />是固态信号（在泵送液体氦温度在步骤3.2在热平衡获得）（图4A）。此参数Defines可用于NMR实验的最大极化之前，超极化溶液的转移过程中不可避免的损失。测量是使用一个未校准的低翻转角脉冲的简单脉冲采集序列进行。脉冲校准通常跳过固态测量。 类似的方法可用于确定在液体状态下的超极化的信号增强。在这种情况下，放置在分光计管注射前的样本（步骤6.2）组成的500μl的D 2 O的溶解和注射后，有监视两个重要参数。第一个是在NMR波谱仪站点的超极化增强， <img alt="Equation7" src="/files/ftp_upload/53548/53548equation7.jpg" width="95" /> （图4B），其中<img alt="Equation8" src="/files/ftp_upload/53548/53548equation8.jpg" width="30" />是信号只是超注射后<html>偏振光溶液（步骤7.1获得）和<img alt="公式9" src="/files/ftp_upload/53548/53548equation9.jpg" width="20" />是热极化信号（步骤7.2获得）。第二个是纵向弛豫时间T 1（图4B，插图），与基板和各代谢产物（通过在步骤7.1获得的指数拟合信号获得）相关联。这两个参数定义必要获得足够的信噪比（SNR）和对代谢转换的测量可用的时间窗的最小底物浓度。固态极化之间的比例<img alt="Equation10" src="/files/ftp_upload/53548/53548equation10.jpg" width="30" />和liquidstate极化<img alt="Equation11" src="/files/ftp_upload/53548/53548equation11.jpg" width="29" />给出了超极化溶液转移过程中的极化损失，由于松弛的估计。值ation12"SRC ="/文件/ ftp_upload / 53548 / 53548equation12.jpg"宽度="80"/&gt;应在不存在松弛的损失被观察到。

<table><tbody><tr><td>DNP polarizer</td><td>Vanderklink s.a.r.l (Switzerland)</td><td>///</td><td>Cryostat and electronic equipment for sample polarization</td></tr><tr><td>Vacuum system components</td><td>Edwards vacuum (France)</td><td>Various</td><td> &lt;p&gt;- turbomolecular pumping setup&lt;/p&gt; &lt;p&gt;- membrane pumping setup&lt;/p&gt; &lt;p&gt;- high capacity roots pumping system&lt;/p&gt; &lt;p&gt;- vacuum fittings and components&lt;/p&gt; </td></tr><tr><td>DNP 3.35T Magnet</td><td>Bruker (France)</td><td></td><td></td></tr><tr><td>500 MHz NMR Spectrometer</td><td>Bruker (France)</td><td></td><td></td></tr><tr><td>Origin 8.0</td><td>OriginLab (US)</td><td></td><td>Data analysis software</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SODIUM PYRUVATE-1-13C, 99 ATOM % 13C</td><td>Sigma Aldrich (France)</td><td>490709</td><td></td></tr><tr><td>ETHANOL-D6, ANHYDROUS, 99.5 ATOM % D</td><td>Sigma Aldrich (France)</td><td>186414</td><td></td></tr><tr><td>&amp;nbsp;4-Hydroxy-TEMPO 97%</td><td>Sigma Aldrich (France)</td><td>176141</td><td></td></tr><tr><td>Deuterium oxide</td><td>Sigma Aldrich (France)</td><td>151882</td><td></td></tr><tr><td>reduced nicotinamide adenine dinucleotide (NADH)</td><td>Sigma Aldrich (France)</td><td></td><td></td></tr><tr><td>ethylene-diaminetetraacetic acid (EDTA)</td><td>Sigma Aldrich (France)</td><td></td><td></td></tr><tr><td>dithiothreitol (DTT)</td><td>Sigma Aldrich (France)</td><td></td><td></td></tr><tr><td>phosphate buffer, pH&amp;nbsp;=&amp;nbsp;7.0</td><td>Sigma Aldrich (France)</td><td></td><td></td></tr><tr><td>LDH enzyme in&amp;nbsp;</td><td>Sigma Aldrich (France)</td><td>L-2500</td><td></td></tr><tr><td>bovine serum albumin, BSA</td><td>Sigma Aldrich (France)</td><td></td><td></td></tr></tbody></table>

dissolution dynamic nuclear polarization instrumentation for real-time enzymatic reaction rate measurements by nmr

基于NMR的调查的主要限制是低灵敏度。这提示对于长采集时间，从而防止代谢转换的实时NMR测量。通过溶解DNP极化绕过灵敏度的一部分发出得益于大外的平衡核磁化从电子到核自旋极化转移而产生。得到高的NMR信号可用于实时监测化学反应。超极化NMR的缺点在于可用于信号采集，这通常是核自旋的纵向弛豫时间常数 ，T 1，或的量级，在有利的情况下，在有限的时间窗口中，与相 ​​关的松弛时间常数的量级加上核的单线态，T LLS。内源性分子和代谢率的细胞摄取可以提供对肿瘤发展和药物反应的基本信息。怒江瓣先前超极化NMR研究已经证明丙酮酸的关联性作为用于在体内监测酶活性的代谢底物。这项工作提供了用于酶反应的研究所需的实验装置和方法的详细描述，尤其是在乳酸脱氢酶（LDH）的存在丙酮酸至乳酸的转化率，通过超极化NMR鉴定。

使用溶解DNP核磁共振信号增益 对DNP效果在于不成对电子自旋的高极化的传送，典型地为稳定的自由基的分子，以NMR-活性核时，样品的微波辐射下。最常用的自由基是TAM（OXO63）和TEMPOL。使用TEMPOL 4极化过程可以通过"交叉极化&#39;进行优化。25 为了获得在固体状态下的最大核极化优化稳定的自由基的浓度是该技术的成功是至关重要的。最佳TEMPOL浓度被认为是在本研究的实验条件下33毫米。所选择的衬底的该偏振足以实时跟踪其酶促转化。 该polari的磁场ZER安装在巴黎笛卡尔为 B 0 = 3.35 T和本系统的组件上方（图1 - 3）进行了描述。低温恒温器的设计保证了最终样品的位置与磁铁等中心重合。偏振片的磁场为倾斜并仅使用超导匀场线圈，以代替低温恒温器匀。一个1×0.5×0.5厘米水柱样品的最终质子线宽为23千赫。我们评估<img alt="Equation14" src="/files/ftp_upload/53548/53548equation14.jpg" width="29" />和<img alt="Equation13" src="/files/ftp_upload/53548/53548equation13.jpg" width="29" />为[1 13 C]丙酮酸盐。这样做，有必要首先确定13 C DNP-增强信号<img alt="Equation5" src="/files/ftp_upload/53548/53548equation5.jpg" width="31" />在在溶解（图4A）前偏振片站点固态。解散后，我们测得的[1-13 C]丙酮酸超极化信号<img alt="Equation8" src="/files/ftp_upload/53548/53548equation8.jpg" width="30" />及其衰变在分光计站点。液体状态的信号用500微升D 2 O中的如核磁共振谱仪样品在测试测量解散。这使我们能够确定固态DNP增强（图4B，插图），液体核磁共振极化水平（图4B）和在转移过程中的极化损失。信号之间的比率在步骤3.3测量和步骤3.2定义固态增强， <img alt="Equation14" src="/files/ftp_upload/53548/53548equation14.jpg" width="29" /> 。在步骤7.1所测量的第一信号和来自步骤7.2的信号之间的比值限定了液状超极化增强， <img alt="Equation13" src="/files/ftp_upload/53548/53548equation13.jpg" width="29" /> 。 从第3步的数据，S在图4A 和图4A（小图）ummarized，表明该技术允许极化13 C在[1- 13 C]丙酮酸达<img alt="Equation14" src="/files/ftp_upload/53548/53548equation14.jpg" width="29" /> = 22±5，对应的 13 C偏振<img alt="Equation10" src="/files/ftp_upload/53548/53548equation10.jpg" width="30" /> = 1.5±0.3％。 这种两极分化水平是常见的MRS 条件 （例如，11.74 T和300 K），碳热极化超过一千次。从第7步数据， 在图4B和图4B（插图）总结，允许确定[1- 13 C]丙酮酸液态13 C超极化， <img alt="Equation11" src="/files/ftp_upload/53548/53548equation11.jpg" width="29" /> = 1±0.2％。在固态丙酮酸得到的增强是足以用于实验的目的，虽然更高的增强已经使用不同的基团表明5的时间来自步骤7.1跑道数据拟合给出了松弛时间常数的测量。由500微升的DNP增强溶液和500μl的D 2 O中的混合物丙酮酸纵向弛豫时间， 呈丁1 75±5秒后= 射频校正（见公式（3））。 酶活性及代谢测试 的13 C标记的底物（A）中溶解的DNP核磁共振检测可以用于实时酶促转化动力学遵循并观察产物的形成（B）中: <img alt="公式1" src="/files/ftp_upload/53548/53548equation1.jpg" />随即超极化基板（A）的注射后，产物（B）的信号为空。然后，酶转化（1）开始，以产生（B）。的13 C核的磁化不会受到不同的化学位移和分子的化学变化。然而，新的环境可能会导致不同的纵向弛豫率在B的13 C的产物信号的时间当然可以在三个步骤来定性地分开:在开始时，酶转化转化为B产生B中的信号的增加;经过一定时间后，依赖于实验条件下，磁化损失，由于放松，平衡这种增加和B的信号达到最大;最后，在较长的时间，B的信号由于纵向弛豫衰减。在酶的饱和的假说，忽略后面的转换，并考虑到磁松弛，酶转化过程中的两种分子种类（A和B）的磁化可通过凑来描述PLED微分方程系统:22 <img alt="方程2" src="/files/ftp_upload/53548/53548equation2.jpg" /> 其中 ，M（A，B）（t） 为分子种类A和B的磁化，分 ​​别中 ，k eff为通过酶反应的信号传输和 R的有效转化率常数（A，B）是表观纵向弛豫速率常数的分别在分子种类A和B，观测核R（A，B）占两个纯纵向磁化弛豫和射频脉冲的效果。: <img alt="Equation3" src="/files/ftp_upload/53548/53548equation3.jpg" /> 其中 T 1，（A，B）是纵向弛豫分别在分子种类A和B的本地分子环境细胞核，的时间常数。θ 和 τ分别是射频翻转角和两个信号采集之间的延迟。 在这项研究中，基片A和产品B分别[1- 13 C]丙酮酸和[1- 13 C]乳酸盐，分别与目的是测量[1- 13 C]的条件下的乳酸生产乳酸脱氢酶在没有（同位素未标记）乳酸存在于在实验开始时的溶液。测量包括在记录在 T = 21°C的简单pulseacquire序列的一系列13 C谱。校准后的10°翻转角度脉冲用于连续激励（第9步）。两个相继脉冲之间的延迟为τ= 1.5秒。核磁共振采集序列的超极化SOLUT之前开始几几十秒离子注入。在注射后的样品管中的丙酮酸底物浓度为25〜35毫米。在步骤10中的乳酸为丙酮酸转换速度为10 -3单位/毫升的LDH浓度进行测定。从乳酸盐和丙酮酸记录信号合身在公式（2）（图5中虚线）的模型和屈服ķ 有效= 0.9±0.1×10 -3 秒 -1（图5）。这个值与由比[紫胶] / [比利]在时间0秒（图5，小图）测定反应初速度一致。 <img alt="图1" src="/files/ftp_upload/53548/53548fig1.jpg" /> 图1 的实验装置的示意图 。偏振片（左）由放置到位于一个低温恒温器（D）内的微波腔（C）的宽孔3.35Ť超导磁体。周边T A定做NMR线圈（B）他样品（一）用于原位监控核自旋极化。氦浴温度保持在1.12±0.03ķ而样品是在微波频率νMW = 94 GHz的照射。该系统允许对固态样品的偏振期间进行NMR测定。极化样品溶解在过热的水，并通过传输线到以前放在相邻的核磁共振谱仪（右）内的样品管与压缩氦气推。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53548/53548fig1large.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/53548/53548fig2.jpg" /> 图2.低温恒温器和真空系统原理图。 （A）低温恒温器的绝缘杜瓦; （B）哭<html> ostat主要插入; （C）真空系统的连接; （D）与用于执行步骤2和协议一节的步骤5中描述的具体操作按钮的管理软件界面的屏幕截图。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53548/53548fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53548/53548fig3.jpg" /> 图3.样品处理和溶解。 （A）样品处理插入; （B）的溶解插入件; （C）溶解插入电子气动连接的详细信息。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53548/53548fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> "&gt; <img alt="图4" src="/files/ftp_upload/53548/53548fig4.jpg" /> 图4. 固态极化堆积，超极化的信号衰减。溶出DNP信号。 （A）一个DNP极化过程中1.12酸钠[1- 13 C]丙酮酸解决方案固态核磁共振极化积聚信号（十字架，配有一个指数函数S（T）=一λEXP（-t / T b）+时间常数 T b = 270秒）和放松（圆圈，S（t）的b = A·EXP（T / T b）+ b，T SS 1 = 840秒）; （A，插图）在固态DNPenhanced和热极化（放大10倍）磁化信号之间比较（εSS = 22±5中，对应于一个固态极化 P SS = 1.5±0.3％）; （B）的溶解和AVERA后记录在第一频谱之间的比较GED光谱（放大100倍）从热极化13 C获得使用1024瞬变在室温旋转（ε= 1000±200）。溶解过程需要3.3秒和在自动注射约2秒与40％估算信号损失。用于实验进行传送通过手动注射样品，由于较长喷射延迟附加损失估计至30％; （B，插图）在没有乳酸脱氢酶（T 1 = 75±5秒）解散后丙酮酸超极化信号衰减。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53548/53548fig4large.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/53548/53548fig5.jpg" /> 由于enzyma [1- 13 C]乳酸信号图5. LDH活性和肿瘤细胞的新陈代谢效果。内置了在10 -3 U /的LDH浓度抽动转换ml的后跟信号衰减，由于13 C磁化纵向的放松。红色线显示了贴合在式（2）中所述的模型包括松弛和酶转化的影响。 [1- 13 C]乳酸和[1-13℃]与时间 t = 0（红线）的反应速度外推丙酮酸浓度（插图）的比例。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53548/53548fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR at JoVE.com

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

The sensitivity enhancement provided by dissolution dynamic nuclear polarization (DNP) enables following metabolic processes in real time by NMR and MRI. The characteristics and performances of a dedicated dissolution DNP setup designed for study enzymatic reactions are discussed.

溶出动态核极化仪器用NMR实时酶反应速率测量

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of ...

dissolution-dynamic-nuclear-polarization-instrumentation-for-real

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10.6K 次观看.  Université Paris Descartes. The sensitivity enhancement provided by dissolution dynamic nuclear polarization (DNP) enables following metabolic processes in real time by NMR and MRI. The characteristics and performances of a dedicated dissolution DNP setup designed for study enzymatic reactions are discussed.

视频: Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have emerged in conjunction with the state-of-the-art positron emission tomography with 18F-fluorodeoxyglucose ([18F]-FDG PET). 13C magnetic resonance spectroscopic imaging (13CMRSI) is a minimally invasive imaging method that enables the monitoring of metabolism in vivo and in real time. As with any other method based on 13C nuclear magnetic resonance (NMR), it faces the challenge of low thermal polarization and a subsequent low signal-to-noise ratio due to the relatively low gyromagnetic ratio of 13C and its low natural abundance in biological samples. By overcoming these limitations, dynamic nuclear polarization (DNP) with subsequent sample dissolution has recently enabled commonly used NMR and magnetic resonance imaging (MRI) systems to measure, study, and image key metabolic pathways in various biological systems. A particularly interesting and promising molecule used in 13CMRSI is [1-13C]pyruvate, which, in the last ten years, has been widely used for in vitro, preclinical, and, more recently, clinical studies to investigate the cellular energy metabolism in cancer and other diseases. In this article, we outline the technique of dissolution DNP using a 3.35 T preclinical DNP hyperpolarizer and demonstrate its usage in in vitro studies. A similar protocol for hyperpolarization may be applied for the most part in in vivo studies as well. To do so, we used lactate dehydrogenase (LDH) and catalyzed the metabolic reaction of [1-13C]pyruvate to [1-13C]lactate in a prostate carcinoma cell line, PC3, in vitro using 13CMRSI.

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

Dynamic nuclear polarization with subsequent sample dissolution has enabled real-time studies of metabolism in biological systems. Hyperpolarized [1-13C]pyruvate was used to study lactate dehydrogenase activity in a prostate carcinoma cell line in vitro.

超极化<sup&gt; 13</sup&gt;ç代谢磁共振波谱与成像

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have ...

科研

癌症研究

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.

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, ...

生物化学

Use of a Multi-compartment Dynamic Single Enzyme Phantom for Studies of Hyperpolarized Magnetic Resonance Agents

Imaging of hyperpolarized substrates by magnetic resonance shows great clinical promise for assessment of critical biochemical processes in real time. Due to fundamental constraints imposed by the hyperpolarized state, exotic imaging and reconstruction techniques are commonly used. A practical system for characterization of dynamic, multi-spectral imaging methods is critically needed. Such a system must reproducibly recapitulate the relevant chemical dynamics of normal and pathological tissues. The most widely utilized substrate to date is hyperpolarized [1-13C]-pyruvate for assessment of cancer metabolism. We describe an enzyme-based phantom system that mediates the conversion of pyruvate to lactate. The reaction is initiated by injection of the hyperpolarized agent into multiple chambers within the phantom, each of which contains varying concentrations of reagents that control the reaction rate. Multiple compartments are necessary to ensure that imaging sequences faithfully capture the spatial and metabolic heterogeneity of tissue. This system will aid the development and validation of advanced imaging strategies by providing chemical dynamics that are not available from conventional phantoms, as well as control and reproducibility that is not possible in vivo.

A multi-compartment dynamic phantom is used to simulate some biology of interest for metabolic studies using hyperpolarized magnet resonance agents.

一个多腔动态单一酶幻影超极化磁共振剂研究的用途

Imaging of hyperpolarized substrates by magnetic resonance shows great clinical promise for assessment of critical biochemical processes in real time. Due ...

工程学

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 ...

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the &#181;s-ms Timescale

Protein conformational dynamics play fundamental roles in regulation of enzymatic catalysis, ligand binding, allostery, and signaling, which are important biological processes. Understanding how the balance between structure and dynamics governs biological function is a new frontier in modern structural biology and has ignited several technical and methodological developments. Among these, CPMG relaxation dispersion solution NMR methods provide unique, atomic-resolution information on the structure, kinetics, and thermodynamics of protein conformational equilibria occurring on the &#181;s-ms timescale. Here, the study presents detailed protocols for acquisition and analysis of a&#160;15N relaxation dispersion experiment. As an example, the pipeline for the analysis of the &#181;s-ms dynamics in the C-terminal domain of bacteria Enzyme I is shown.

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the  &#181;s-ms Timescale

Here, a detailed description of the protocol implemented in the laboratory for acquisition and analysis of 15N relaxation dispersion profiles by solution NMR spectroscopy is provided.

15N CPMG 放松分散，用于研究 μs-ms 时间尺度上的蛋白质符合性动力学

Protein conformational dynamics play fundamental roles in regulation of enzymatic catalysis, ligand binding, allostery, and signaling, which are important ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

快速扫描电子顺磁共振打开新的途径成像生理重要参数<em&gt;在体内</em

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

生物工程

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-(1H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.

This work details robust basic routines on how to prepare isotope-labeled membrane protein samples and analyze them at high-resolution with modern solid-state NMR spectroscopy methods.

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy ...

化学

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various factors affect the relaxation rates, such as buffer composition, solution pH, temperature, and magnetic field. In this last regard, the spin-lattice relaxation time can be measured at clinical field strengths, but at lower fields, where these compounds are dispensed from the polarizer and transported to the MRI, the relaxation is even faster and difficult to measure. To have a better understanding of the amount of magnetization lost during transport, we used fast field-cycling relaxometry, with magnetic resonance detection of 13C nuclei at ~0.75 T, to measure the nuclear magnetic resonance dispersion of the spin-lattice relaxation time of hyperpolarized [1-13C]pyruvate. Dissolution dynamic nuclear polarization was used to produce hyperpolarized samples of pyruvate at a concentration of 80 mmol/L and physiological pH (~7.8). These solutions were rapidly transferred to a fast field-cycling relaxometer so that relaxation of the sample magnetization could be measured as a function of time using a calibrated small flip angle (3&#176;-5&#176;). To map the T1 dispersion of the C-1 of pyruvate, we recorded data for different relaxation fields ranging between 0.237 mT and 0.705 T. With this information, we determined an empirical equation to estimate the spin-lattice relaxation of the hyperpolarized substrate within the mentioned range of magnetic fields. These results can be used to predict the amount of magnetization lost during transport and to improve experimental designs to minimize signal loss.

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

We present a protocol to measure the magnetic field dependence of the spin-lattice relaxation time of 13C-enriched compounds, hyperpolarized by means of dynamic nuclear polarization, using fast field-cycled relaxometry. Specifically, we have demonstrated this with [1-13C]pyruvate, but the protocol could be extended to other hyperpolarized substrates.

测量超极化 [1-13C]硫化的旋转-莱迪兹松弛磁场依赖性

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various ...

<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 ...

溶出动态核极化仪器用NMR实时酶反应速率测量

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High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR

测量超极化 [1-¹³C]硫化的旋转-莱迪兹松弛磁场依赖性

NMR ¹⁵N 弛豫实验，用于研究蛋白质皮秒至纳秒的结构动力学

NMR ¹⁵N 弛豫实验，用于研究蛋白质皮秒至纳秒的结构动力学