Department of Chemistry, Stanford University , 380 Roth Way, Stanford California 94305, United States.
J Phys Chem B. 2017 Oct 12;121(40):9331-9335. doi: 10.1021/acs.jpcb.7b06736. Epub 2017 Sep 29.
Solid-state NMR is a powerful tool for quantifying chemical composition and structure in complex assemblies and even whole cells. We employed N{P} REDOR NMR to obtain atomic-level distance propensities in intact N-labeled E. coli ribosomes. The experimental REDOR dephasing of shift-resolved lysyl amine nitrogens by phosphorus was comparable to that expected from a calculation of N-P distances involving the lysines included in the crystal structure coordinates. Among the nitrogen contributions to the REDOR spectra, the strongest dephasing emerged from the dipolar couplings to phosphorus involving nitrogen peaks ascribed primarily to rRNA, and the weakest dephasing arose from protein amide nitrogens. This approach is applicable to any macromolecular system and provides quantitative comparisons of distance proximities between shift-resolved nuclei of one type and heteronuclear dephasing spins. Enhanced molecular specificity could be achieved through the use of spectroscopic filters or specific labeling. Furthermore, ribosome C and N CPMAS spectra were compared with those of whole cells from which the ribosomes were isolated. Whole-cell signatures of ribosomes were identified and should be of value in comparing overall cellular ribosome content in whole-cell samples.
固态 NMR 是一种强大的工具，可用于量化复杂组装体甚至整个细胞中的化学组成和结构。我们采用 N{P} REDOR NMR 技术，在完整的 N 标记的大肠杆菌核糖体中获得原子级的距离倾向。通过磷对分辨的赖氨酸胺氮的实验 REDOR 退相与从涉及晶体结构坐标中包含的赖氨酸的 N-P 距离计算中预期的退相相当。在 REDOR 光谱的氮贡献中，与主要归因于 rRNA 的磷涉及氮峰的偶极耦合产生最强的退相，而与蛋白质酰胺氮产生最弱的退相。这种方法适用于任何大分子系统，并提供一种定量比较一种类型的分辨核与异核退相自旋之间的距离接近程度的方法。通过使用光谱滤波器或特定标记，可以实现增强的分子特异性。此外，比较了核糖体 C 和 N CPMAS 光谱与从其中分离出核糖体的整个细胞的光谱。鉴定了核糖体的全细胞特征，应该在比较全细胞样品中整个细胞核糖体含量方面具有价值。
