GUANOSINE TRIPHOSPHATE BINDING OF RAS PROTEIN BY NMR AND CD SPECTROSCOPY

多项研究表明，任何一个位置12的点突变， 13、59或61或ras p21蛋白与根本性改变有关 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构象。 非转化型和转化型ras p21蛋白的差异 以及添加GTP后的构象变化。取得的成果 目前的研究结果如下：(1)含甘氨酸(甘氨酸肽)和 含缬氨酸(Val-肽)的34个氨基酸残基 合成并纯化了p21蛋白的N末端片段。 其结构经质谱学和多肽确证。 测序。(2)值得注意的是，蛋白质中的单一氨基酸取代 N-末端链段在溶解度上产生明显的变化 属性。(3)在Tris缓冲液(pH 7.4)中，甘氨酸肽主要以 β-Sheet结构。然而，在40%的三氟乙醇(TFE)中， 甘氨酸肽的α-螺旋结构增多(46%)。 (4)在醋酸铵缓冲液(pH 7.4)中，Val-多肽采用较大的 α-螺旋结构相对于甘氨酸肽的数量 Tris Buffer。(5)在甘氨酸肽中加入GTP可诱导更大的 其构象的变化量。相比之下，在添加 核苷酸到Val-多肽溶液，几乎没有总构象 注意到了变化。(6)当甘氨酸肽加入溶液中时 包含GTP和十二烷基硫酸钠，所有三个P-31核磁共振信号的线宽， Alpha、Beta和Gamma被加宽(Beta和Gamma分别为10赫兹和5赫兹 用于Alpha)。这一结果表明，在地球上存在着一种复杂的构造 GTP和甘氨酸肽。(7)平衡透析实验表明 Gly-肽和Val-肽与GTP的结合强度为 与完整的p21蛋白相当。结合GTP的模型多肽 和不分青红皂白的ATP。(8)p21蛋白N-末端片段为 参与GTP的水解酶。

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.