Structural and dynamic differences between normal and transforming N-ras gene products: a 31P and isotope-edited 1H NMR study.
Structural and dynamic differences between normal and transforming N-ras gene products: a 31P and isotope-edited 1H NMR study.
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正常和转化 N-ras 基因产物之间的结构和动态差异：31P 和同位素编辑的 1H NMR 研究。
DOI：
10.1021/bi00450a035
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Campbell-Burk,S
中科院分区：
文献类型：
--
作者：
Campbell-Burk,S
马萨诸塞州沃尔瑟姆布兰德大学生物化学系，1989年3月29日收到；修订稿收到1989年7月21日摘要：[15N]甘氨酸被生物合成到正常细胞N-ras p21和一个第12位转化突变体中，以产生在鸟嘌呤核苷酸结合域的激活位置或附近含有多个位点特异性核磁共振探针的p21蛋白。我们先前已经指定了位于直接参与野生型p21蛋白中鸟苷二磷酸结合的环中的所有五个甘氨酸共振[Campbell-Burk，S.，Papastavros，.Z.，McCormick，F.和Redfield，A.G.(1989)Proc.纳特·阿卡德。SCI。美国86,817-820]，在本报告中，指定了p21突变体中相应的甘氨酸共振，并研究了正常和突变型p21-鸟苷二磷酸(p21-GDP)复合体之间的光谱差异。我们的联合{15}和31P核磁共振结果表明，天冬氨酸取代甘氨酸-12在突变点附近的磷酸结合结构域产生了扰动。虽然许多剩余的甘氨酸没有受到影响，但在GDP结合结构域之外也观察到了光谱差异。野生型p21-gdp中的五个活性中心甘氨酸中有两个与水具有非常慢的酰胺质子交换速率(k<；2.8X10™5 S“1)。活性中心甘氨酸位于暴露于溶剂的环中，因此它们明显的溶剂不可及性可能是由于甘氨酰胺质子与p21中结合的鸟二磷酸和/或其他邻近基团形成了强烈的氢键。
Department of Biochemistry, Brandéis University, Waltham, Massachusetts 02254 Received March 29, 1989; Revised Manuscript Received July 21, 1989 abstract:[15N] Glycine was biosynthetically incorporated into normal cellular N-ras p21 and a position 12 transforming mutant, in orderto produce p21 proteins containing several site-specific NMR probes at or near activating positions in the guanine nucleotide binding domain. We have previously assigned all five glycine resonances located in loops directly involved in binding of guanosine diphosphate in the wild-type p21 protein [Campbell-Burk, S., Papastavros,. Z., McCormick, F., & Redfield, A. G.(1989) Proc. Natl.Acad. Sci. USA 86, 817-820], In this report, the corresponding glycine resonances in the p21 mutant have been assigned, and spectral differences between normal and mutant p21-guanosine diphosphate (p21-GDP) complexes have been investigated. Our combined {15} and 31P NMR results show that substitution of aspartate for glycine-12produces perturbations in the phosphoryl binding domain, near the point of the mutation. Although many of the remaining glycines were unaffected, spectral differences were also observed outside the GDP binding domain. Two of the five active-site glycines in wild-type p21-GDP have very slow amide proton exchange rates with water (k< 2.8 X 10™ 5 s" 1). The active-site glycines are located in solvent-exposed loops, so their apparentsolvent inaccessibility may result from stronghydrogen bond formation between glycine amide protons and bound guanine diphosphate and/or other nearby groups in p21.