Nagayama K, Yamazaki T, Yoshida M, Kanaya S, Nakamura H
Biometrology Lab, JEOL Ltd., Tokyo.
J Biochem. 1990 Aug;108(2):149-52. doi: 10.1093/oxfordjournals.jbchem.a123173.
A new strategy for the sequential assignment of backbone proton resonances in larger proteins involving a unique combination of four types of heteronuclear three-dimensional (3D) NMR spectroscopies is reported. This method relies on the uniform labeling of amide nitrogens with 15N and of alpha-carbons with 13C. Heteronuclear 1H-15N TOCSY-HMQC and NOESY-HMQC experiments can reveal connections between cross-peaks arising from the NHi-C alpha Hi-1 and NHi-C alpha Hi connectivities in the finger-print region in in general. They also specifically reveal the sequential amide-amide connectivities among the amide cross-peaks for the alpha-helices. Heteronuclear 1H-13C HMQC-TOCSY and HMQC-NOESY experiments can reveal connections between cross-peaks arising from the NHi-C alpha Hi and NHi+1-C alpha Hi connectivities in the finger-print region in general. The combination of the two sets of results reveals the complete unambiguous sequential connection of cross-peaks for the proton resonances in the peptide backbone. The application of the new strategy is reported for a protein, ribonuclease H, with a molecular weight of 17.6 kDa.
报道了一种用于确定较大蛋白质中主链质子共振序列归属的新策略，该策略涉及四种类型的异核三维（3D）核磁共振光谱的独特组合。此方法依赖于用¹⁵N均匀标记酰胺氮，用¹³C均匀标记α-碳原子。异核¹H-¹⁵N TOCSY-HMQC和NOESY-HMQC实验通常可揭示指纹区中由NHi-CαHi-1和NHi-CαHi连接性产生的交叉峰之间的联系。它们还能特别揭示α-螺旋中酰胺交叉峰之间的序列酰胺-酰胺连接性。异核¹H-¹³C HMQC-TOCSY和HMQC-NOESY实验通常可揭示指纹区中由NHi-CαHi和NHi+1-CαHi连接性产生的交叉峰之间的联系。两组结果的结合揭示了肽主链中质子共振交叉峰的完整、明确的序列连接。报道了该新策略在分子量为17.6 kDa的核糖核酸酶H蛋白上的应用。
