Neri D, Szyperski T, Otting G, Senn H, Wüthrich K
Institut für Molekularbiologie und Biophysik, ETH-Hönggerberg, Zürich, Switzerland.
Biochemistry. 1989 Sep 19;28(19):7510-6. doi: 10.1021/bi00445a003.
Stereospecific 1H and 13C NMR assignments were made for the two diastereotopic methyl groups of the 14 valyl and leucyl residues in the DNA-binding domain 1-69 of the 434 repressor. These results were obtained with a novel method, biosynthetically directed fractional 13C labeling, which should be quite widely applicable for peptides and proteins. The method is based on the use of a mixture of fully 13C-labeled and unlabeled glucose as the sole carbon source for the biosynthetic production of the protein studied, knowledge of the independently established stereoselectivity of the pathways for valine and leucine biosynthesis, and analysis of the distribution of 13C labels in the valyl and leucyl residues of the product by two-dimensional heteronuclear NMR correlation experiments. Experience gained with the present project and a previous application of the same principles with the cyclic polypeptide cyclosporin A provides a basis for the selection of the optimal NMR experiments to be used in conjunction with biosynthetic fractional 13C labeling of proteins and peptides.
对434阻遏蛋白DNA结合结构域1 - 69中14个缬氨酰和亮氨酰残基的两个非对映异位甲基进行了立体专一性的1H和13C核磁共振归属。这些结果是通过一种新方法——生物合成定向分数13C标记获得的，该方法应广泛适用于肽和蛋白质。该方法基于使用完全13C标记和未标记的葡萄糖混合物作为所研究蛋白质生物合成生产的唯一碳源，已知缬氨酸和亮氨酸生物合成途径独立建立的立体选择性，以及通过二维异核核磁共振相关实验分析产物中缬氨酰和亮氨酰残基中13C标记的分布。本项目获得的经验以及先前将相同原理应用于环多肽环孢菌素A的情况为选择与蛋白质和肽的生物合成分数13C标记结合使用的最佳核磁共振实验提供了基础。
