Directly observed 15N NMR spectra of uniformly enriched proteins.
Directly observed 15N NMR spectra of uniformly enriched proteins.
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直接观察均匀富集蛋白质的 15N NMR 谱。
DOI：
10.1021/bi00382a020
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Domingues,DJ
中科院分区：
文献类型：
--
作者：
Smith,GM;Yu,LP;Domingues,DJ
加州大学食品科学与技术系，Davis, California, 95616摘要：红红螺旋菌在15NH4C1培养基上生长，在15N条件下富集了细胞色素c2、细胞色素c′和核酮糖二磷酸羧化酶/加氧酶等蛋白。纯化后的蛋白均质化，用15N NMR对其进行了研究。测定了分子量在13000 ~ 114000之间的不同蛋白质组的纵向和横向弛豫时间以及核Overhauser效应。酰胺共振或刚性基团的这些参数值与蛋白质的分子量一致。精氨酸和赖氨酸的氨基端氨基和侧链氮原子的弛豫时间与更快速的运动一致。具有结合质子的氮原子通常被发现通过化学交换与质子解耦。可证明的* H-15N偶联被认为是交换受阻的迹象，或者是氢键相互作用，或者是基团无法接近溶剂。组氨酸侧链氮原子在质子化/去质子化过程中经历了很大的化学位移，经常被发现通过化学交换和互变异构作用而展宽到无法检测。本文还讨论了提高灵敏度和获得特定峰分配的策略。15N是自旋*/2的原子核，自然丰度为0.37%。由于其核磁共振化学位移对质子化状态、氢键和金属连接变化的异常大的响应（Roberts, 1980; Kanamori & Roberts, 1983; Mason, 1981），它被认为具有作为测量小分子、蛋白质和核酸中的这些相互作用的“报告基团”的巨大潜力[例如，参见Sogn等人（1973）、Morishima和Inubushi（1977）和Griffey等人（1983）]。这种潜力在历史上还没有被充分认识到，因为15N，除了其贫乏的自然丰度外，还有一个很小的负磁回比（），是质子的0.1013倍。核磁共振的灵敏度取决于3**，因此对于相同数量的自旋，其灵敏度仅为质子的10~ 3。因此，自然丰度下的15N信号约为相同浓度下质子信号的4 × 10~ 6。有几种方法可以用来克服这种严重的灵敏度不足，包括采用J耦合（Morris & Freeman, 1979）或偶极耦合（McArthur et al.）的不敏感核增强技术。
Department of Food Science and Technology, University of California, Davis, California 95616 Received October 27, 1986; Revised Manuscript Received December 15, 1986 abstract: The proteins cytochrome c2, cytochrome c', and ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum were enriched in 15N by growth of the organism on 15NH4C1. The proteins were purified to homogeneity and studied by 15N NMR. Longitudinal and transverse relaxation times as well as the nuclear Overhauser effects were determined for various groups of the proteins which vary in molecular weight from 13000 to 114000. The values of these parameters for the amide resonances or for groups thought to be rigid were consistent with the molecular weights of the proteins. Relaxation times of the amino-terminal-amino groups and the side chain nitrogen atoms of arginine and lysine were consistent with much more rapid motion. Nitrogen atoms having bound protons were generally found to be decoupled from the protonsby chemical exchange. Demonstrable* H-15N coupling was taken as an indication that exchange was hindered, either by hydrogen bonding interactions or by inaccessibility of the group to solvent. Histidine side chainnitrogen atoms, which experience a large chemical shift upon protonation/deprotonation, were often found to be broadened beyond detectability by chemical exchange and tautomerization. Strategies for improving sensitivity and for obtainingspecific peak assignments are also discussed.15N is a spin*/2 nucleus with a natural abundance of 0.37%. Because of the unusually large response of its NMR chemical shift to changes in protonation state, hydrogen bonding, and metal ligation (Roberts, 1980; Kanamori & Roberts, 1983; Mason, 1981), it is recognized to have great potential as a “reporter group” for measuring these interactions in small molecules, proteins, and nucleic acids [eg, see Sogn et al.(1973), Morishima and Inubushi (1977), and Griffey et al.(1983)]. This potential has historically not been fully realized because 15N, in addition to its meager natural abundance, has a small negative magnetogyric ratio () that is-0.1013 times that of a proton. NMR sensitivity, which depends on 3**, is thus only 10~ 3 that of protons for the same number of spins. The 15N signal at natural abundance is therefore about 4 X 10~ 6 that of protons at the same concentration. There are several approaches that might be used to overcome this serious lack of sensitivity, including insensitive nu-cleus enhancement techniques employing J coupling (Morris & Freeman, 1979) or dipolar coupling (McArthur et al.,