Departments of Molecular Genetics, Biochemistry & Chemistry, University of Toronto, Toronto, Ontario M5S 1A8 (Canada).
NMR Science, Campbell CA 95008 (USA).
Angew Chem Int Ed Engl. 2015 Sep 1;54(36):10507-11. doi: 10.1002/anie.201504070. Epub 2015 Jul 15.
Chemical exchange saturation transfer (CEST) NMR spectroscopy is a powerful tool for studies of slow timescale protein dynamics. Typical experiments are based on recording a large number of 2D data sets and quantifying peak intensities in each of the resulting planes. A weakness of the method is that peaks must be resolved in 2D spectra, limiting applications to relatively small proteins. Resolution is significantly improved in 3D spectra but recording uniformly sampled data is time-prohibitive. Here we describe non-uniformly sampled HNCO-based pseudo-4D CEST that provides excellent resolution in reasonable measurement times. Data analysis is done through fitting in the time domain, without the need of reconstructing the frequency dimensions, exploiting previously measured accurate peak positions in reference spectra. The methodology is demonstrated on several protein systems, including a nascent form of superoxide dismutase that is implicated in neurodegenerative disease.
化学交换饱和转移（CEST）NMR 光谱学是研究慢时间尺度蛋白质动力学的有力工具。典型的实验基于记录大量的 2D 数据集，并对每个结果平面中的峰强度进行量化。该方法的一个弱点是在 2D 光谱中必须解析峰，这限制了该方法应用于相对较小的蛋白质。在 3D 光谱中分辨率得到了显著提高，但记录均匀采样的数据在时间上是不允许的。在这里，我们描述了基于非均匀采样 HNCO 的伪 4D CEST，它在合理的测量时间内提供了出色的分辨率。数据分析是通过在时域中进行拟合来完成的，而无需重建频率维度，利用参考光谱中之前测量到的准确峰位置。该方法学在几个蛋白质系统中得到了验证，包括一种与神经退行性疾病有关的超氧化物歧化酶的新生形式。
