利用19f核磁共振研究GTP(γ F)-微管蛋白相互作用的核弛豫率。

O Monasterio
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引用次数: 0

摘要

为了研究GTP可交换性结合位点(E-site)与高亲和力金属结合位点之间的关系,我们合成了GTP类似物P3-fluoro P1-5′-鸟苷三磷酸(GTP(γ F))。我们的研究结果表明,这种类似物与小牛脑小管蛋白的可交换GTP结合位点结合。通过EPR光谱测定GTP(γ F)-Mn(II)配合物的解离常数和化学计量学分别为1.64 x 10(-4) M和1摩尔锰/摩尔核苷酸。利用高分辨率19F-NMR测定了二价金属离子高亲和力结合位点与交换性核苷酸结合位点之间的距离。研究了GTP(γ F)的31P-和19F-NMR谱,氟和γ -磷酸盐在耦合常数为936hz的双重态中分裂。用Weisenberg方法纯化的微管蛋白(Weisenberg, R.C, and Timashef, S.N. (1970) Biochemistry 9,4110 -4116)用秋水仙碱处理以稳定微管蛋白,加入GTP(γ F),并在头四个小时内测量254.1 MHz 19氟弛豫率。在秋水仙碱-微管蛋白- mn (II)(顺磁配合物)或与镁的三元配合物(反磁配合物)存在下测定纵向和横向弛豫率。温度依赖性弛豫数据分析表明,金属与可交换核苷酸结合位点在35℃时最大距离为6,在12℃时最大距离为8.1 a。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Nuclear relaxation rates study of GTP(gamma F)-tubulin interaction using 19F-nuclear magnetic resonance.

To study the relationship between the exchangeable GTP binding site (E-site) and the high affinity metal binding site we synthesized P3-fluoro P1-5'-guanosine tripaosphate (GTP(gamma F), an analog of GTP. Our results show that this analog binds to the exchangeable GTP binding site of calf brain tubulin. The values of the dissociation constant and the stoichiometry of the GTP(gamma F)-Mn(II) complex as determined by EPR spectroscopy were 1.64 x 10(-4) M and one mole of manganese per mole of nucleotide, respectively. The distance separating the high-affinity binding site for the divalent metal ion and the exchangeable nucleotide binding site was evaluated by using high-resolution 19F-NMR. The 31P- and 19F-NMR spectra of GTP(gamma F) were studied, both the fluorine and the gamma-phosphate were split in a doublet with a coupling constant of 936 Hz. Tubulin purified by the method of Weisenberg (Weisenberg, R.C., and Timashef, S.N. (1970) Biochemistry 9, 4110-4116) was treated with colchicine to stabilize it, GTP(gamma F) was added and the 254.1 MHz 19fluorine relaxation rates measured within the first four hours. Longitudinal and transversal relaxation rates were determined in the presence of colchicine-tubulin-Mn(II), (paramagnetic complex), or the ternary complex with magnesium (diamagnetic complex). The analysis of the temperature-dependent relaxation data indicates that the metal and the exchangeable nucleotide binding sites are separated by a maximal distance of 6 at 35 degrees C, to 8.1 A at 12 degrees C.

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