Faulhammer H G, Denninger G, Härtl P J, Azhayev A V, Schwoerer M, Sprinzl M
Biochim Biophys Acta. 1986 Oct 29;884(1):182-90. doi: 10.1016/0304-4165(86)90242-4.
New derivatives of GDP and GTP have been synthesized for the spectroscopic investigation of the interaction between guanosine nucleotides and guanosine nucleotide-binding proteins. The 3'-hydroxyl group in these nucleotides was replaced by a 3'-amino group, which was further derivatized by the introduction of a spin-label reporter group. The biological activity of 3'SL-GDP and 3'SL-GTP could be demonstrated by measuring the interaction of these spin-labelled derivatives with bacterial elongation factor Tu. The amino modification and spin labelling only slightly influenced the affinity of the guanosine nucleotides for EF-Tu from Escherichia coli or Thermus thermophilus. Electron paramagnetic resonance (EPR) measurements revealed a strong immobilization of the labelled nucleotides upon binding to T. thermophilus EF-Tu. Significant differences between the spectra of EF-Tu X 3'SL-GDP, EF-Tu X 3'SL-GTP and aminoacyl-tRNA X EF-Tu X 3'SL-GTP ternary complexes were observed. Our data demonstrate that spin-labelled guanosine nucleotides can be used as sensitive spectroscopic probes for the investigation of the local environment of the nucleotide-binding site during distinct functional states of a guanosine nucleotide-binding protein.
已合成GDP和GTP的新衍生物，用于对鸟苷核苷酸与鸟苷核苷酸结合蛋白之间的相互作用进行光谱研究。这些核苷酸中的3'-羟基被3'-氨基取代，通过引入自旋标记报告基团对其进一步衍生化。通过测量这些自旋标记衍生物与细菌延伸因子Tu的相互作用，可以证明3'SL-GDP和3'SL-GTP的生物活性。氨基修饰和自旋标记仅对鸟苷核苷酸与大肠杆菌或嗜热栖热菌的EF-Tu的亲和力有轻微影响。电子顺磁共振（EPR）测量显示，标记的核苷酸在与嗜热栖热菌EF-Tu结合时强烈固定。观察到EF-Tu X 3'SL-GDP、EF-Tu X 3'SL-GTP和氨酰-tRNA X EF-Tu X 3'SL-GTP三元复合物光谱之间的显著差异。我们的数据表明，自旋标记的鸟苷核苷酸可作为灵敏的光谱探针，用于研究鸟苷核苷酸结合蛋白在不同功能状态下核苷酸结合位点的局部环境。
