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: 3963537
• 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/3963537
• 关键词: chemical binding; circular dichroism; conformation; glycine; guanosine triphosphate; high performance liquid chromatography; nuclear magnetic resonance spectroscopy; oncoproteins

A number of studies indicate that a point mutation at either position 12, 13, 59 or 61 or 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 the conformational changes upon addition of GTP. Results obtained so far are as follows: (1) Both glycine-containing (Gly-peptide) and valine-containing (Val-peptide) 34 amino acid residue peptides of N-terminal segments of p21 proteins have been synthesized and purified. Their structures were confirmed by mass spectroscopy and peptide sequencing. (2) It is notable that a single amino acid substitution in the N-terminal segment produces a distinct change in the solubility properties. (3) In Tris buffer (pH 7.4), the Gly-peptide adopted a largely beta-sheet structure. However, in 40% trifluoroethanol (TFE), the Gly-peptide showed an increased amount of alpha-helical structure (46%). (4) The Val-peptide in ammonium acetate buffer (pH 7.4) adopted a greater amount of alpha-helical structure relative to that of the Gly-peptide in Tris buffer. (5) The addition of GTP to the Gly-peptide induces a larger amount of change in its conformation. In contrast, upon addition of nucleotides to the Val-peptide solution, little overall conformational change was noted. (6) When the Gly-peptide was added to the solution containing GTP and SDS, the line widths of all three P-31 NMR signals, alpha, beta, and gamma, were broadened (10 Hz for beta and gamma, and 5 Hz for alpha). The result implies that there is a complex formation between GTP and the Gly-peptide. (7) Equilibrium dialysis experiments indicate that the binding strength of the Gly-peptide and Val-peptide with GTP are comparable to those of intact p21 proteins. The model peptides bind GTP and ATP indiscriminately. (8) N-Terminal segments of p21 proteins are involved in the hydrolysis of GTP.

大量研究表明，12 位任一处的点突变， 13、59 或 61 或 ras p21 蛋白与 它们的生化特性包括转化细胞的能力。 该项目的主要目标是研究构象 非转化型和转化型 ras p21 蛋白之间的差异为 以及添加 GTP 后的构象变化。 获得的结果 到目前为止，如下：（1）含甘氨酸（Gly-peptide）和 含缬氨酸（Val-肽）的34个氨基酸残基的肽 p21 蛋白的 N 末端片段已合成并纯化。 它们的结构通过质谱和肽得到证实 测序。 (2) 值得注意的是，单氨基酸取代 N-末端片段产生明显的溶解度变化 特性。 (3) 在 Tris 缓冲液 (pH 7.4) 中，糖肽在很大程度上采用了 β-折叠结构。 然而，在 40% 三氟乙醇 (TFE) 中， 糖肽显示α-螺旋结构数量增加（46%）。 (4) 醋酸铵缓冲液(pH 7.4)中的Val肽采用了更大的 相对于甘肽的α螺旋结构的量 三羟甲基氨基甲烷缓冲液。 (5) 在Gly肽中添加GTP会诱导更大的 其构象的变化量。 相反，在添加 Val 肽溶液中的核苷酸，总体构象很少 注意到了变化。 (6) 当将糖肽添加到溶液中时 包含 GTP 和 SDS，所有三个 P-31 NMR 信号的线宽， alpha、beta 和 gamma 被拓宽（beta 和 gamma 为 10 Hz，5 Hz 对于阿尔法）。 结果表明，两者之间存在着复杂的结构。 GTP 和甘肽。 (7)平衡透析实验表明 甘肽和缬肽与 GTP 的结合强度为 与完整的 p21 蛋白相当。 模型肽结合 GTP 和 ATP 不加区别。 (8) p21 蛋白的 N 端片段是 参与GTP的水解。