Identification of resonances from an oncogenic activating locus of human N-RAS-encoded p21 protein using isotope-edited NMR.
Identification of resonances from an oncogenic activating locus of human N-RAS-encoded p21 protein using isotope-edited NMR.
复制标题
使用同位素编辑 NMR 鉴定人类 N-RAS 编码的 p21 蛋白的致癌激活位点的共振。
DOI：
10.1073/pnas.86.3.817
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发表时间：
1989
影响因子：
11.1
通讯作者：
Redfield,AG
中科院分区：
文献类型：
--
作者：
Burk,SC;Papastavros,MZ;McCormick,F;Redfield,AG
大肠杆菌表达的人n - ras编码的p21蛋白是一个21 kda的蛋白，在14个甘氨酸酰胺位点上选择性地标记15N。二维质子- 15N相关光谱显示每个甘氨酸残基有一个峰。在核苷酸结合位点附近的残基上鉴定了五个甘氨酸共振，并提供了几个致癌基因激活位置的有用报告。这些共振中的三个被分配到残基10、15和115，来自一个样品的光谱，也被标记为[13C]缬氨酸。由于13C标签，这些共振表现出额外的分裂或拓宽，可以通过13C去耦来消除。另外两个峰通过一维编辑核Overhauser实验和与Asp-12突变体的光谱比较明确地确定为Gly-12和Gly-13。这些任务提供了p21关键结构域的几个位点特异性探针。
A sample of Escherichia coli-expressed human N-RAS-encoded p21, a 21-kDa protein, was selectively labeled with 15N at each of the 14 glycine amide positions. Two-dimensional proton-observe 15N correlation spectra showed one peak for each glycine residue. Five glycine resonances were identified with residues near the nucleotide binding site and provide useful reporters of several oncogene-activating positions. Three of these resonances were assigned to residues 10, 15, and 115 from the spectrum of a sample that was also labeled with [13C]valine. These resonances showed extra splitting or broadening due to the 13C label, which could be eliminated by 13C decoupling. Two other peaks were unambiguously identified as Gly-12 and Gly-13 using a one-dimensional edited nuclear Overhauser experiment and by spectral comparison with an Asp-12 mutant. These assignments have provided several site-specific probes of critical domains in p21.