13C 13C CORRELATION SPECTROSCOPY OF U 13C ERYTHROMYCIN

U 13C 红霉素的 13C 13C 相关光谱

• 批准号: 6355115
• 负责人: CHAD M RIENSTRA
• 金额: $ 2.46万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6355115
• 关键词: biological products; biomedical resource; carbohydrates; proteins

In addition to the developments described below, we have recently demonstrated highly precise internuclear distance measurements with the CMR7 pulse sequence. Homonuclear couplings can be measured to a precision of better than 5 Hz with this approach, implying internuclear distance measurements of better than 0. 1-0.2 A precision out to 4-5 'A. 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 sequenc e 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-"Clabeled amino acids and "C-"C chemical shift correlation spectroscopy of the U-"C-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- "C, "N-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 13C chemical shift dimension, is provided by the U-"C-labeled macrolide antibiotic erythromycin 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 crystal s 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.

除了下文所述的发展外，我们最近还 展示了高度精确的核间距离测量， CMR 7脉冲序列。 可以将Homestructive耦合测量为 使用这种方法，精度优于5 Hz，这意味着 核间距离测量优于0。 1-0.2 A 精确到4-5 'A。 我们开发了一种脉冲序列 多自旋系统中有效的双量子偶极再耦合 在魔角旋转NMR下，基于Levitt的C7序列和 同事 对于双自旋系统，C7序列提供更高的 全极化转移双量子滤波 效率（-73%）比MELODRAMA序列（-52%），因为 消除了重新耦合的相互作用对转子相位的依赖性。 然而，在实验上，使用C7序列的DQF效率取决于 显著的错误，产生的交叉项之间 化学位移和射频（RF）场不均匀性。 许多 应用需要在多自旋中激发DQ相干性 系统，显示广泛的各向同性和各向异性 化学位移 此外，序列的实验可靠性 是在低成本条件下成功实施的关键 灵敏度和温度。 为了满足这些要求，我们 构建了一个脉冲序列， 独立于化学位移，通过结合各种Cn元素 对称性 去除了C7序列固有的误差项 通过复合MLEV型旋转;因此，我们参考新的 序列为CMR 7（MLEV-Refocusing with C7）。 我们有 证明了这种方法的实用性与双量子 U-“C”标记氨基酸的过滤和“C-“C化学位移 U-C标记抗生素红霉素的相关光谱 A.在73%的偏振转换下， 强度预计将接近3：1，在双自旋的情况下， 作为U-“C“，N-Gly，我们观察到优于2：1的相对 2D光谱中的强度。 然而，完整的理论DQF 在多自旋系统和类似系统中通常不能实现效率 在相关谱中观察到关于串扰的行为 强度。 然而，在许多情况下， 对角峰，并且在有利的情况下， 即使在多自旋限制下，强度也大于2：1。 一个 这些效果的说明性实例，以及 延伸至第二个13 C化学位移时观察到的分辨率 尺寸，由U-1C标记的大环内酯类抗生素提供 红霉素A（EA）。 EA通过与一种蛋白质结合来抑制蛋白质合成。 细菌核糖体 实际结构信息 由于缺乏红霉素-核糖体复合物， 可用晶体的数量和溶液NMR谱的分辨率差 这是由于核糖体缓慢的重新定向运动。 因此，在本发明中， 红霉素-核糖体复合物显示固态NMR谱 甚至在溶液中。 因此，为了方便结构 研究EA在其自由和复杂的形式，有必要 在固态下进行明确的化学位移分配。 我们 我用CMR 7方法完成了化学位移的归属 并正在进行基于CMR 7的进一步结构研究 法