GUANOSINE TRIPHOSPHATE BINDING OF RAS PROTEIN BY NMR AND CD SPECTROSCOPY

许多研究表明，在12位， 13、59或61或ras p21蛋白与一个基本的改变有关， 它们的生化特性包括它们转化细胞的能力。 本项目的主要目的是研究 非转化和转化rasp 21蛋白之间的差异， 以及加入GTP后的构象变化。 获得的结果 （1）含甘氨酸（Gly-肽）和 含缬氨酸（Val-肽）的34个氨基酸残基的肽 已经合成并纯化了p21蛋白的N-末端片段。 它们的结构经质谱和肽鉴定 测序 (2)值得注意的是，在所述氨基酸序列中的单个氨基酸取代是可能的。 N-末端片段产生溶解度的明显变化 特性. (3)在pH7.4的Tris缓冲液中，Gly-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， 甘氨酸肽显示α-螺旋结构的量增加（46%）。 (4)Val-肽在醋酸铵缓冲液（pH 7.4）中采用了更大的 相对于Gly肽的α螺旋结构量 Tris缓冲液。 (5)将GTP添加到Gly-肽诱导了更大的 其构象的变化量。 相反，在添加 核苷酸的Val-肽溶液，很少的整体构象 注意到了变化。 (6)当将Gly-肽加入到溶液中时， 包含GTP和SDS，所有三个P-31 NMR信号的线宽， α、β和γ被加宽（β和γ为10 Hz， 对于alpha）。 这一结果表明， GTP和Gly肽。 (7)平衡透析实验表明 Gly肽和Val肽与GTP的结合强度 与完整的p21蛋白的那些相当。 模型肽结合GTP ATP不加选择地 (8)p21蛋白的N-末端片段是 参与GTP的水解。

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.