1-13C amino acid selective labeling in a 2H15N background for NMR studies of large proteins
1-13C amino acid selective labeling in a 2H15N background for NMR studies of large proteins
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DOI：
10.1007/s10858-007-9152-z
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发表时间：
2007-05-01
影响因子：
2.7
通讯作者：
Wagner, Gerhard
中科院分区：
文献类型：
--
作者：
Takeuchi, Koh;Ng, Elise;Wagner, Gerhard
通过残基类型的同位素标记（LBRT）长期以来一直是在其他方法（例如三重共振实验或NOESY方法）不能成功地产生完整归属的极限下进行共振归属的重要工具。虽然LBRT对于小蛋白质已经变得不那么重要，但它可以是完成最具挑战性的蛋白质系统分配的最后手段。在这里，我们提出了一种方法，其中LBRT是通过添加质子化的N-14氨基酸，C-13标记的羰基位置的介质中均匀的氘代和N-15标记。这与氘背景中的常规N-15 LBRT相比具有三个重要优点：（1）对于通过标记连接的氨基酸对，可以高灵敏度地观察到选择性的TROSY-HNCO交叉峰，并且13 C标记的氨基酸之后的残基的酰胺质子非常尖锐，因为其α位被氘代，（2）羰基位置上的C-13标记比α-氨基位置上的N-15更不易于扰乱，和（3）从H-1-N-15 TROSY中的大强度降低中可以容易地鉴定1-C-13标记的氨基酸的峰。一些不显著扰乱氮的残基的HSQC谱，如丙氨酸和酪氨酸。这种方法具有成本效益，并已成功应用于大于40 kDa的蛋白质。
Isotope labeling by residue type (LBRT) has long been an important tool for resonance assignments at the limit where other approaches, such as triple-resonance experiments or NOESY methods do not succeed in yielding complete assignments. While LBRT has become less important for small proteins it can be the method of last resort for completing assignments of the most challenging protein systems. Here we present an approach where LBRT is achieved by adding protonated N-14 amino acids that are C-13 labeled at the carbonyl position to a medium for uniform deuteration and N-15 labeling. This has three important benefits over conventional N-15 LBRT in a deuterated back ground: (1) selective TROSY-HNCO cross peaks can be observed with high sensitivity for amino-acid pairs connected by the labeling, and the amide proton of the residue following the 13C labeled amino acid is very sharp since its alpha position is deuterated, (2) the C-13 label at the carbonyl position is less prone to scrambling than the N-15 at the alpha-amino position, and (3) the peaks for the 1-C-13 labeled amino acids can be identified easily from the large intensity reduction in the H-1-N-15 TROSY-HSQC spectrum for some residues that do not significantly scramble nitrogens, such as alanine and tyrosine. This approach is cost effective and has been successfully applied to proteins larger than 40 kDa.