Weigelt Johan, van Dongen Maria, Uppenberg Jonas, Schultz Johan, Wikström Mats
Department of Structural Chemistry, Biovitrum, S-112 76 Stockholm, Sweden.
J Am Chem Soc. 2002 Mar 20;124(11):2446-7. doi: 10.1021/ja0178261.
A new method for site-selective screening by NMR is presented. The core of the new method is the dual amino acid sequence specific labeling technique. Amino acid X is labeled with (13)C and amino acid Y is labeled with (15)N. Provided only one XY pair occurs in the amino acid sequence, only one signal in the 1D carbonyl (13)C spectrum will display a splitting due to the (1)J(C'N) coupling. Using this labeling strategy it is possible to screen selectively for binding to a selected epitope without the need for sequence specific assignments. An HNCO spectrum (1D or 2D) can be used either directly as a screening experiment or indirectly to identify what signals to monitor in a 2D (1)H-(15)N correlation spectrum. Chemical shift perturbations upon addition of a potential ligand are easily detected even for large proteins due to the reduced spectral complexity resulting from the use of a selectively labeled sample. The new technique is demonstrated on the human adipocyte fatty acid binding protein FABP-4. Due to the reduced spectral complexity, the method should be applicable to larger proteins than are conventional methods.
本文介绍了一种通过核磁共振进行位点选择性筛选的新方法。该新方法的核心是双氨基酸序列特异性标记技术。氨基酸X用(13)C标记，氨基酸Y用(15)N标记。若氨基酸序列中仅出现一对XY，那么在一维羰基(13)C谱中只有一个信号会因(1)J(C'N)耦合而产生分裂。采用这种标记策略，无需进行序列特异性归属就能够选择性地筛选与选定表位的结合情况。一维或二维HNCO谱既可以直接用作筛选实验，也可以间接用于确定在二维(1)H-(15)N相关谱中要监测哪些信号。由于使用选择性标记样品降低了光谱复杂性，即使对于大蛋白，添加潜在配体时的化学位移扰动也很容易检测到。该新技术在人脂肪细胞脂肪酸结合蛋白FABP - 4上得到了验证。由于光谱复杂性降低，该方法应比传统方法适用于更大的蛋白。
