McIntosh L P, Griffey R H, Muchmore D C, Nielson C P, Redfield A G, Dahlquist F W
Proc Natl Acad Sci U S A. 1987 Mar;84(5):1244-8. doi: 10.1073/pnas.84.5.1244.
A strategy for resolution and assignment of single proton resonances in proteins of molecular mass up to at least 40 kDa is presented. This approach is based on 15N (or 13C) labeling of selected residues in a protein. The resonances from protons directly bonded to labeled atoms are detected in a two-dimensional 1H-15N (or 13C) spectrum. The nuclear Overhauser effects from isotopically tagged protons are selectively observed in one-dimensional isotope-directed measurements. Using this approach, we have observed approximately 160 resonances from 15N-bonded protons in the backbone and sidechains of uniformly 15N-labeled T4 lysozyme (molecular mass = 18.7 kDa). Partial proton-deuterium exchange can be used to simplify the 1H-15N spectrum of this protein. These resonances are identified by amino acid class using selective incorporation of 15N-labeled amino acids and are assigned to specific residues by mutational substitution, multiple 15N and 13C labeling, and isotope-directed nuclear Overhauser effect measurements. For example, using a phenyl[15N]alanine-labeled lysozyme variant containing two consecutive phenylalanine residues in an alpha-helical region, we observe an isotope-directed nuclear Overhauser effect from the amide proton of Phe-66 to that of Phe-67.
本文提出了一种用于解析和归属分子量至少达40 kDa蛋白质中单个质子共振峰的策略。该方法基于对蛋白质中选定残基进行15N（或13C）标记。在二维1H-15N（或13C）谱中检测直接与标记原子相连的质子的共振峰。在一维同位素导向测量中选择性地观察同位素标记质子的核Overhauser效应。使用这种方法，我们在均匀15N标记的T4溶菌酶（分子量 = 18.7 kDa）的主链和侧链中观察到了约160个来自与15N相连质子的共振峰。部分质子-氘交换可用于简化该蛋白质的1H-15N谱。通过选择性掺入15N标记的氨基酸，根据氨基酸类别鉴定这些共振峰，并通过突变取代、多重15N和13C标记以及同位素导向的核Overhauser效应测量将其归属到特定残基。例如，使用在α-螺旋区域含有两个连续苯丙氨酸残基的苯[15N]丙氨酸标记的溶菌酶变体，我们观察到了从Phe-66的酰胺质子到Phe-67的酰胺质子的同位素导向的核Overhauser效应。
