Leibniz-Institut für Molekulare Pharmakologie, Robert-Roessle-Str. 10, 13125 Berlin, Germany.
J Am Chem Soc. 2010 Nov 3;132(43):15133-5. doi: 10.1021/ja106170h.
Biological magic angle spinning (MAS) solid-state nuclear magnetic resonance spectroscopy has developed rapidly over the past two decades. For the structure determination of a protein by solid-state NMR, routinely (13)C,(13)C distance restraints as well as dihedral restraints are employed. In protonated samples, this is achieved by growing the bacterium on a medium which contains [1,3]-(13)C glycerol or [2]-(13)C glycerol to dilute the (13)C spin system. Labeling schemes, which rely on heteronuclei, are insensitive both for detection and in terms of quantification of distances, since they are relying on low-γ nuclei. Proton detection can in principle provide a gain in sensitivity by a factor of 8 and 31, compared to the (13)C or (15)N detected version of the experiment. We report here a new labeling scheme, which enables (1)H-detection of aliphatic resonances with high resolution in MAS solid-state NMR spectroscopy. We prepared microcrystals of the SH3 domain of chicken α-spectrin with 5% protonation at nonexchangeable sites and obtained line widths on the order of 25 Hz for aliphatic (1)H resonances. We show further that (13)C resolved 3D-(1)H,(1)H correlation experiments yield access to long-range proton-proton distances in the protein.
生物魔角旋转（MAS）固态核磁共振波谱学在过去二十年中发展迅速。对于通过固态 NMR 确定蛋白质结构，通常使用（13）C，（13）C 距离约束以及二面角约束。在质子化样品中，通过在含有[1,3] - （13）C甘油或[2] - （13）C甘油的培养基中培养细菌来实现（13）C自旋系统的稀释。依赖于杂核的标记方案在检测和距离定量方面都不敏感，因为它们依赖于低γ核。与（13）C 或（15）N 检测实验版本相比，质子检测原则上可以提高 8 和 31 倍的灵敏度。我们在这里报告了一种新的标记方案，该方案可实现 MAS 固态 NMR 光谱中高分辨率的（1）H 检测脂肪族共振。我们制备了鸡α- spectrin SH3 结构域的微晶体，其在不可交换位置上有 5％的质子化，并获得了脂肪族（1）H 共振的约 25 Hz 的线宽。我们进一步表明，（13）C 分辨的 3D-（1）H，（1）H 相关实验可获得蛋白质中长程质子-质子距离。
