HIGH FREQUENCY DYNAMIC NUCLEAR POLARIZATION OF PROTEINS

由于固态核磁共振固有的低灵敏度 实验中，对生物固体的测量通常仅限于 相对较小的模型化合物。动力学的最新发展 核极化(DNP)提供了更大的潜力 生物系统中的信噪比。DNP耦合高自旋 未配对的电子到核自旋的极化，导致 潜在的核磁共振信号增强高达三个数量级。 高场(140 GHz EPR/211 MHz核磁共振)DNP的最新进展 DNP在高分辨率下的应用，MAS 大型生物的条件。我们已经开发出一种水溶液 信号增强最高可达185倍的系统 实现了冰冻溶液中的生物溶质。系统 由氮氧自旋标记4-氨基TEMPO在水/甘油中组成 解决方案。氮氧化物电子顺磁共振谱线中心的辐照 极化质子自旋与自由基相耦合。自旋扩散 在质子自旋之间转移高极化贯穿整个 溶剂，然后交叉极化到低伽马原子核。一个 在静态CP/MAS中获得了大约185的增强 在14 K下，60：40甘油水中的‘3C-乙酰甘氨酸，以便 为了获得高分辨率的光谱，我们将这一实验扩展到 采用魔角旋转(MAS)。因为两极分化 传输效率在远低于100K的温度下(由于 增加的电子和核弛豫时间)，DNP/MAS探针 为低温而建造的。加压氦气，冷却 通过液氦中的热交换器，被用来驱动一个 标准的化学磁学转子。传输线调谐是 加入以避免内部电容器暴露在氦中，这 具有低击穿电压。温度低至20K，速度加快 利用低温DNP设置实现了到5 kHZ的输出。我们有 在均匀的~CPMAS谱中获得了20的增强 50K时在节奏/水/甘油中标记L-精氨酸 在25K时获得了100倍的系数。这个实验可以 很容易扩展到更大的生物溶质。因为 电子-质子极化转移步骤主要发生在溶剂中 质子，这一步受溶质大小的影响最小。 对溶质大小的唯一固有限制来自于 质子自旋扩散的效率，它提供了增强的 整个溶质中的质子的极化。对这些项目的估计 与典型的质子T1弛豫速率相比，速率表明 高达几百kDa的蛋白质应该对此顺从 技术。我们已经获得了大约50%的增强 5N-丙氨酸T4-溶菌酶的~5N-CPMAS谱，它是一种18.7kD的溶菌酶 节奏/水/甘油溶液。

Because of the inherently low sensitivity of solid-state NMR experiments, measurements on biological solids are often restricted to relatively small model compounds. Recent developments in Dynamic Nuclear Polarization (DNP) offer the potential of substantially larger signal/noise ratios in biological systems. DNP couples the high spin polarization of unpaired electrons to nuclear spins, resulting in a potential NMR signal enhancement of up to three orders of magnitude. Recent advances in high field (140 GHz EPR / 211 MHz NMR) DNP have been made in the application of DNP under high-resolution, MAS conditions for large biological. We have developed an aqueous solvent system in which signal enhancements of up to a factor of 185 can be achieved for biological solutes in frozen solution. The system consists of the nitroxide spin label 4-amino TEMPO in a water/glycerol solution. Irradiation off the center of the nitroxide EPR line polarizes the proton spins coupled to the radical. Spin diffusion among proton spins transfers the high polarization throughout the solvent, followed by cross-polarization to low-gamma nuclei. An enhancement of approximately 185 was obtained in the static CP/MAS of '3C-carbonyl glycine in 60:40 glycerol water at 14 K. In order to achieve high-resolution spectra, we have extended this experiment to incorporate magic-angle spinning (MAS). Because the polarization transfer is most efficient at temperatures well below 100 K (due to the increased electron and nuclear relaxation times), a DNP/MAS probe for low temperatures was constructed. Pressurized helium gas, cooled through a heat exchanger in liquid helium, was used to drive a standard Chemagnetics rotor. Transmission line tuning was incorporated to avoid exposure of internal capacitors to helium, which has a low breakdown voltage. Temperatures as low as 20 K at speeds up to 5 KHz were achieved with the low temperature DNP setup. We have obtained an enhancement of 20 in the ~ CPMAS spectrum of uniformly labeled L-arginine in TEMPO/water/glycerol at 50 K. Enhancements up to a factor of 100 have been obtained at 25 K. This experiment can be easily extended to larger biological solutes. Because the electron-proton polarization transfer step occurs primarily to solvent protons, this step is minimally affected by the size of the solute. The only inherent limit on the size of the solute arises from the efficiency of proton spin diffusion, which delivers the enhanced polarization throughout the protons of the solute. Estimates of these rates, compared with typical proton T1 relaxation rates, suggest that proteins up to several hundred kDa should be amenable to this technique. We have obtained an enhancement of approximately 50 in the '5N CPMAS spectrum of '5N-Ala T4-lysozyme, an 18.7 kD lytic protein in a TEMPO/water/glycerol solution.