MULTI SPIN HOMONUCLEAR DOUBLE QUANTUM RECOUPLING FOR MAGIC ANGLE SPINNING NMR

用于魔角旋转核磁共振的多自旋同核双量子再耦合

• 批准号: 6279727
• 负责人: CHAD M RIENSTRA
• 金额: $ 0.24万
• 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/6279727
• 关键词: biomedical resource; carbohydrates; proteins; technology /technique

We have developed a pulse sequence for efficient double-quantum dipolar recoupling in multiple spin systems under magic-angle-spinning NMR, based on the C7 sequence of Levitt and co-workers. For two-spin systems, the C7 sequence offers higher overall polarization transfer and double-quantum filtration (DQF) efficiency ( 73%) than the MELODRAMA sequence ( 52%), because the dependence of the recoupled interaction on rotor phase is eliminated. Experimentally, however, DQF efficiency with the C7 sequence depends significantly on the errors that arise from cross terms between chemical shifts and radiofrequency (rf) field inhomogeneity. Many applications require the excitation of DQ coherence in multi-spin systems, which display a wide range of isotropic and anisotropic chemical shifts. Also, the experimental reliability of the sequence is crucial for successful implementation under conditions of low sensitivity and temperature. To meet these requirements, we have constructed a pulse sequence that recouples dipolar interactions independent of chemical shifts, by combining Cn elements of various symmetries. The error terms inherent to the C7 sequence are removed by composite MLEV-type rotations; therefore, we refer to the new sequence as CMR7 (Combined MLEV-Refocusing with C7). We have demonstrated the utility of this approach with double-quantum filtration of U-13C-labeled amino acids and '3C-13C chemical shift correlation spectroscopy of the U-13C-labeled antibiotic, erythromycin A. With 73% polarization transfer, the ratio of crosspeak to diagonal intensity is expected to be almost 3:1, and in two-spin cases such as U- 3C, 5N-Gly, we have observed better than 2:1 relative intensities in 2D spectra. However, the full theoretical DQF efficiency is usually not realized in multi-spin systems and similar behavior is observed in correlation spectra with respect to crosspeak intensities. Nevertheless, in many cases crosspeak intensities exceed the diagonal peaks, and in favorable instances the ratio of intensities is greater than 2:1, even in the multi-spin limit. An illustrative example of these effects, and the improvement in resolution observed upon extending to a second '3C chemical shift dimension, is provided by the U-3C-labeled macrolide antibiotic eiythromycin A (EA). EA inhibits protein synthesis by binding to a bacterial ribosome. Actual structural information on the erythromycin-ribosome complex has been inaccessible due to the paucity of available crystals and poor resolution of the solution NMR spectra due to the slow reorientational motion of the ribosome. As a result, the erythromycin-ribosome complex exhibits a solid-state NMR spectrum even in solution. Therefore, in order to facilitate structural studies of EA in both its free and complexed forms, it is necessary to make unambiguous chemical shifts assignments in the solid state. We have accomplished the chemical shift assignments using the CMR7 method and are pursuing further structural studies based upon the CMR7 method.

我们已经开发了一种脉冲序列，用于有效的双量子 魔角自旋下多自旋系统的偶极再耦合 NMR，基于Levitt及其同事的C7序列。 对于两个旋转 系统，C7序列提供更高的整体偏振转移 和双量子过滤（DQF）效率（73%）比 MELODRAMA序列（52%），因为重新耦合的依赖性 消除了转子相位上的相互作用。 然而，在实验上， C7序列的DQF效率显著取决于 由化学位移之间的交叉项引起的误差， 射频（RF）场不均匀性。 许多应用需要 多自旋系统中DQ相干性的激发， 宽范围的各向同性和各向异性化学位移。 还 序列的实验可靠性是成功的关键 在低灵敏度和温度条件下实现。 为了满足这些要求，我们构建了一个脉冲序列， 重偶偶极相互作用独立的化学位移， 组合各种对称性的Cn元素。 固有的误差项 通过复合MLEV型旋转移除C7序列; 因此，我们将新序列称为CMR 7（组合 MLEV-Refocusing with C7）。 我们已经证明了这一点的实用性 U-13 C标记氨基酸的双量子过滤方法 和13 C-13 C化学位移相关光谱 U-13 C标记抗生素，红霉素A。73%的极化 转移，交叉口与对角线强度的比率预计将 几乎是3：1，在双自旋的情况下，如U- 3C，5 N-Gly，我们有 在2D光谱中观察到优于2：1的相对强度。 然而，在这方面， 完全理论上的DQF效率通常不能实现， 在关联中观察到多自旋系统和类似的行为 相对于交叉口强度的光谱。 然而，在许多 在交叉说话强度超过对角峰的情况下， 在有利的情况下，强度比大于2：1，甚至 在多重旋转限制中。 这些效应的一个说明性例子， 以及在延伸到第二个时观察到的分辨率的改善 3C化学位移维度，由U-3C标记的 大环内酯类抗生素红霉素A（EA）。 EA抑制蛋白质 通过与细菌核糖体结合而合成。 实际结构 关于红霉素-核糖体复合物的信息 由于缺乏可用的晶体和差的分辨率， 溶液NMR谱由于缓慢的再取向运动的 核糖体 结果，红霉素-核糖体复合物表现出 固体核磁共振谱，甚至在溶液中。 因此为了 促进EA在其游离和络合物中的结构研究 形式，有必要作出明确的化学位移分配 在固体状态下。 我们已经完成了化学位移 任务使用CMR 7方法，并正在追求进一步的结构 基于CMR 7方法的研究。