GUANOSINE TRIPHOSPHATE BINDING OF RAS PROTEIN BY NMR AND CD SPECTROSCOPY

通过 NMR 和 CD 光谱法观察三磷酸鸟苷与 RAS 蛋白的结合

• 批准号: 3939707
• 负责人: C-H NIU
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3939707
• 关键词: chemical binding; chemical structure function; circular dichroism; conformation; glycine; guanosine triphosphate; high performance liquid chromatography; magnesium; nuclear magnetic resonance spectroscopy; oncogenes; oncoproteins; phosphates; valine

A number of studies revealed that a point mutation at either position 12, 13, 59, or 61 of ras p21 proteins is associated with a fundamental change in their biochemical properties including their ability to transform cells. The main objective of this project is to study the conformational differences between non- transforming and transforming ras p21 proteins as well as their conformational changes upon binding to GTP. A few important observations concerning the conformational changes upon addition of GTP to synthetic N-terminal segments of ras p21 proteins appeared in the last report. Additional significant results are as follows: (1) Upon addition of either the glycine- containing (Gly-peptide) and valine-containing (Val-peptide) 34 amino acid residue peptides of the N-terminal segments of ras p21 proteins to the solution containing GTP or ATP, the line width of all three phosphorus-31 NMR resonance, alpha-, beta-, and gamma-phosphate, were broadened. Simultaneously, all three phosphate resonances shifted downfield upon binding with peptides. However, the degree of their shifts was somewhat different. Beta- and gamma-phosphate resonances shifted downfield noticeably, but the alpha-phosphate resonance was not shifted to any significant degree upon addition of either the Gly- peptide or Val-peptide. (2) It is known that magnesium ion plays an important role in binding guanine nucleotide to ras p21 proteins. Upon addition of magnesium ion to the mixture of the Gly-peptide with GTP, all of three phosphate resonances shifted further downfield without broadening their line widths. (3) The Gly-peptide, in contrast to the Val-peptide, catalyzes the hydrolysis of GTP.

多项研究表明，无论是哪种情况下的点突变 Ras p21蛋白的第12、13、59或61位与 它们的生化特性发生了根本的变化，包括 它们转化细胞的能力。这项工作的主要目标是 项目是研究非生物分子之间的构象差异 转化和转化ras p21蛋白以及它们的 与GTP结合时的构象变化。几个重要的问题 关于构象变化的观察 将GTP添加到合成的ras p21的N末端片段 蛋白质出现在上一份报告中。更重要的是 结果如下：(1)当添加甘氨酸时- 含(甘氨酸肽)和含缬氨酸(Val-肽)34 Ras N-末端片段的氨基酸残基 P21蛋白到含有GTP或ATP的溶液中，行 所有三个磷-31核磁共振的宽度，α，β， 和伽马磷酸盐，都被加宽了。同时，所有 三个磷酸盐共振在结合时向下场移动 多肽。然而，他们的转变程度有所不同 不一样。β-和伽马-磷酸盐的共振位移 前场很明显，但阿尔法-磷酸共振没有 在添加甘氨酸或甘氨酸的情况下， 多肽或Val-多肽。(2)众所周知，镁离子起着 鸟嘌呤核苷酸与ras p21结合的重要作用 蛋白质。在将镁离子添加到 甘氨酸肽与GTP，三个磷酸共振峰均移位 更远的前场，而不会加宽他们的线宽。(3) 与Val-肽不同，甘氨酸肽催化 GTP的水解性。