NMR studies of the conformational change in human N-p21ras produced by replacement of bound GDP with the GTP analog GTP gamma S.
NMR studies of the conformational change in human N-p21ras produced by replacement of bound GDP with the GTP analog GTP gamma S.
复制标题
通过用 GTP 类似物 GTP gamma S 取代结合的 GDP，对人 N-p21ras 中的构象变化进行 NMR 研究。
DOI：
10.1021/bi00157a007
复制
发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Redfield,AG
中科院分区：
文献类型：
--
作者：
Miller,AF;Papastavros,MZ;Redfield,AG
摘要：利用溶液中检测到的15n编辑NMR，研究了人类N-p21ras与gtpys结合形式之间的构象差异。观察了15n标记的甘氨酸和异亮氨酸的酰胺质子。共振分配给特别感兴趣的残基，甘氨酸-60和75以及异亮氨酸-21和36，除了[15N]甘氨酸和[15N]约亮氨酸外，还结合了各种13c标记的氨基酸，并用Co2+取代了Mg2+。当GTP7S取代p21ras活性位点的GDP时，14个甘氨酸酰胺共振中只有5个发生了重大变化，表明构象效应是相当局部的。反应性甘氨酸-10，-12，-13和-15在活性位点。Gly-75位于构象活性环和螺旋的远端，也对GTP7S取代GDP做出反应，而Gly-77则没有，支持Gly-75作为构象变化的旋转点的作用。异亮氨酸-36和-21以及第三种未确定的异亮氨酸的酰胺质子共振在GTP7S取代GDP后也发生了重大变化。因此，证实含有Ile-36的效应结合环参与了构象变化，含有Ile-21的螺旋也受到了影响。
Revised Manuscript Received July 7, 1992 abstract:-Detected 15N-edited NMR in solution was used to study the conformational differences between the GDP-and GTPYS-bound forms of human N-p21ras. The amide protons of 15N-labeled glycine and isoleucine were observed. Resonances were assigned to residues of particularinterest, glycines-60 and-75 and isoleucines-21 and-36, by incorporating various 13C-labeled amino acids in addition to [15N] glycine and [15N] iosleucine and by replacing Mg2+ by Co2+. When GTP7S replaced GDP in the active site of p21ras, only 5 of the 14 glycine amide resonances show major shifts, indicating that the conformational effects are fairly localized. Responsive glycines-10,-12,-13, and-15 are in the active site. Gly-75, located at the far end of a conformationally-active loopand helix, also responds to substitution of GTP7S for GDP, while Gly-77 does not, supporting a role for Gly-75 as a swivel point for the conformational change. The amide proton resonances of isoleucines-36 and-21 and a third unidentified isoleucine also undergo major shifts upon replacement of GDP by GTP7S. Thus, the effector-binding loop containing Ile-36 is confirmed to be involved in the conformational change, and the-helix containing Ile-21 is also shown to be affected.