NMR SEQUENTIAL ASSIGNMENT OF ESCHERICHIA-COLI THIOREDOXIN UTILIZING RANDOM FRACTIONAL DEUTERIATION
NMR SEQUENTIAL ASSIGNMENT OF ESCHERICHIA-COLI THIOREDOXIN UTILIZING RANDOM FRACTIONAL DEUTERIATION
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DOI：
10.1021/bi00401a022
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发表时间：
1988-01-12
期刊：
影响因子：
2.9
通讯作者：
RICHARDS, FM
中科院分区：
文献类型：
--
作者：
LEMASTER, DM;RICHARDS, FM
所有非脯氨酸残基，除了N-末端二肽已被分配在108个残基的蛋白质大肠杆菌硫氧还蛋白。这些实验的核心是使用蛋白质样品，其中通过在部分氘化的碳源和培养基上的细菌生长，所有碳结合的氢位置被氘取代至75%。局部质子密度的稀释引起更窄的线宽，而灵敏度几乎没有损失。此外，被动或二次耦合到质子不直接参与相关实验的相干转移过程中在很大程度上被抑制，从而显着提高了侧链耦合的分辨率。通过掺入具有不同选择性α-氨基酸的几种氨基酸，已经获得了同时的多残基类型分配。和/或β-氘化成分数氘化背景。结合几个单一的残基类型的标记实验，这些选择性的标记产生了直接的残基类型分配的三分之二的蛋白质。除了改进的分辨率之外，酰胺至碳结合质子NOESY谱提供了等效的灵敏度，而酰胺至酰胺NOESY分配具有可能的3 α-NOE连接性中的2.35的最近邻NOE连接性的平均数。酰胺，β-酰胺和酰胺-酰胺连接性。
All non-proline residues except for the N-terminal dipeptide have been assigned in the 108-residue protein Escherichia coli thioredoxin. Central to these experiments has been the use of protein samples in which all carbon-bound hydrogen positions are substituted to 75% with deuterium by bacterial growth on partially deuteriated carbon sources and media. The dilution of the local proton density gives rise to narrower line widths with little loss in sensitivity. In addition, passive or secondary coupling to protons not directly involved in the coherence transfer process of correlation experiments is largely suppressed, thus significantly improving the resolution for side-chain couplings. Simultaneous multiresidue-type assignments have been obtained by incorporation of several amino acids with differing selective .alpha.- and/or .beta.-deuteriation into a fractionally deuteriated background. Combined with several single residue type labeling experiments, these selective labelings have yielded direct residue type assignments for two-thirds of the protein. In addition to improved resolution, the amide to carbon-bound proton NOESY spectra offered equivalent sensitivity while the amide to amide NOESY assignment has an average number of nearest-neighbor NOE connectivities of 2.35 out of the possible 3 .alpha.-amide, .beta.-amide, and amide-amide connectivities.