HIGH FREQUENCY DYNAMIC NUCLEAR POLARIZATION OF PROTEINS

蛋白质的高频动态核极化

• 批准号: 6279712
• 负责人: DENNIS A HALL
• 金额: $ 2.37万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6279712
• 关键词: biological products; biomedical equipment development; biomedical resource; proteins; technology /technique

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.

由于固体核磁共振固有的低灵敏度 在实验中，对生物固体的测量通常仅限于 相对较小的模型化合物。 动态的最新发展 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 未成对电子极化为核自旋，导致 高达三个数量级的潜在NMR信号增强。 高场（140 GHz EPR / 211 MHz NMR）DNP的最新进展 提出了在DNP下应用高分辨率、MAS 大生物的条件。 我们开发了一种水性溶剂 在该系统中，信号增强高达185倍， 在冷冻溶液中实现生物溶质。 系统 由在水/甘油中的氮氧自由基自旋标记4-氨基克里思组成 溶液 偏离氮氧EPR谱线中心的辐照 极化与原子团耦合的质子自旋。 自旋扩散 在质子自旋之间， 溶剂，然后交叉极化到低伽马核。 一个 在静态CP/MAS中获得约185的增强， 14 K下，在60：40甘油水溶液中的13 C-羰基甘氨酸。为了 实现高分辨率光谱，我们已经扩展了这个实验， 结合魔角旋转（MAS）。 因为两极分化 在远低于100 K的温度下，转移是最有效的（由于 增加的电子和核弛豫时间），DNP/MAS探针 在低温下建造的。 压缩氦气，冷却 通过液氦中的热交换器，被用来驱动 标准化学磁力转子。 传输线调谐是 为了避免内部电容器暴露在氦气中， 具有低击穿电压。 温度低至20 K，加速时 到5 KHz，实现了低温DNP设置。 我们有 在~ CPMAS光谱中获得了20的增强， 标记的L-精氨酸在克里思/水/甘油中在50 K。增强功能， 在25 K时已获得100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上 质子，这一步骤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 溶质质子的极化。 估计这些 与典型的质子T1弛豫速率相比， 高达几百kDa的蛋白质应该服从这一点 法 我们已经获得了大约50的增强， 15 N-Ala T4-溶菌酶的15 NCPMAS谱，所述溶菌酶是一种18.7kD的溶菌蛋白， 克里思/水/甘油溶液。