Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
J Am Chem Soc. 2010 Mar 10;132(9):2945-51. doi: 10.1021/ja907717b.
The primary limitation of solution state NMR with larger, highly dynamic, or paramagnetic systems originates from signal losses due to fast transverse relaxation. This is related to the high gyromagnetic ratio gamma of protons, which are usually detected. Thus, it is attractive to consider detection of nuclei with lower gamma, such as (13)C, for extending the size limits of NMR. Here, we present an approach for complete assignment of C(alpha) and N resonances in fast relaxing proteins using a C(alpha) detected 3D CANCA experiment for perdeuterated proteins. The CANCA experiment correlates alpha carbons with the sequentially adjacent and succeeding nitrogen and alpha carbons. This enables elongation of the chain of assigned residues simply by navigating along both nitrogen and carbon dimensions using a "stairway" assignment procedure. The simultaneous use of both C(alpha) and N sequential connectivities makes the experiment more robust than conventional 3D experiments, which rely solely on a single (13)C indirect dimension for sequential information. The 3D CANCA experiment, which is very useful for mainchain assignments of higher molecular weight proteins at high magnetic field, also provides an attractive alterative for smaller proteins. Two versions of the experiment are described for samples that are (13)C labeled either uniformly or at alternate positions for removing one-bond (13)C-(13)C couplings. To achieve both high resolution and sensitivity, extensive nonuniform sampling was employed. Adding longitudinal relaxation enhancement agents can allow for shorter recycling delays, decreased measuring time, or enhanced sensitivity.
溶液态 NMR 对于较大、高度动态或顺磁性系统的主要限制源于由于快速横向弛豫导致的信号损失。这与质子的高旋磁比γ有关，通常检测到的就是质子。因此，考虑检测具有较低γ值的核，如（13）C，以扩展 NMR 的尺寸限制是很有吸引力的。在这里，我们提出了一种使用完全氘代蛋白质的 C（α）检测 3D CANCA 实验对快速弛豫蛋白质的 C（α）和 N 共振进行完全分配的方法。CANCA 实验将α碳与顺序相邻的氮和α碳相关联。这使得通过使用“楼梯”分配程序沿着氮和碳维度导航，简单地延长了分配残基的链。同时使用 C（α）和 N 顺序连接使实验比仅依赖于单个（13）C 间接维度的传统 3D 实验更稳健。3D CANCA 实验对于在高磁场下对高分子量蛋白质的主链分配非常有用，对于较小的蛋白质也提供了一种有吸引力的替代方法。描述了两种实验版本，用于在（13）C 标记均匀或在交替位置的样品，以去除单键（13）C-（13）C 偶合。为了实现高分辨率和灵敏度，采用了广泛的非均匀采样。添加纵向弛豫增强剂可以允许缩短再循环延迟、减少测量时间或提高灵敏度。
