Leninger Maureen, Marsiglia William M, Jerschow Alexej, Traaseth Nathaniel J
Department of Chemistry, New York University, 100 Washington Square East, New York, NY, 10003, USA.
J Biomol NMR. 2018 May;71(1):19-30. doi: 10.1007/s10858-018-0186-1. Epub 2018 May 23.
Exchange between conformational states is required for biomolecular catalysis, allostery, and folding. A variety of NMR experiments have been developed to quantify motional regimes ranging from nanoseconds to seconds. In this work, we describe an approach to speed up the acquisition of chemical exchange saturation transfer (CEST) experiments that are commonly used to probe millisecond to second conformational exchange in proteins and nucleic acids. The standard approach is to obtain CEST datasets through the acquisition of a series of 2D correlation spectra where each experiment utilizes a single saturation frequency to H, N or C. These pseudo 3D datasets are time consuming to collect and are further lengthened by reduced signal to noise stemming from the long saturation pulse. In this article, we show how usage of a multiple frequency saturation pulse (i.e., MF-CEST) changes the nature of data collection from series to parallel, and thus decreases the total acquisition time by an integer factor corresponding to the number of frequencies in the pulse. We demonstrate the applicability of MF-CEST on a Src homology 2 (SH2) domain from phospholipase Cγ and the secondary active transport protein EmrE as model systems by collecting C methyl and N backbone datasets. MF-CEST can also be extended to additional sites within proteins and nucleic acids. The only notable drawback of MF-CEST as applied to backbone N experiments occurs when a large chemical shift difference between the major and minor populations is present (typically greater than ~ 8 ppm). In these cases, ambiguity may arise between the chemical shift of the minor population and the multiple frequency saturation pulse. Nevertheless, this drawback does not occur for methyl group MF-CEST experiments or in cases where somewhat smaller chemical shift differences occur are present.
生物分子催化、变构和折叠都需要构象状态之间的交换。已经开发了各种核磁共振实验来量化从纳秒到秒的运动状态。在这项工作中，我们描述了一种加速化学交换饱和转移（CEST）实验采集的方法，该实验通常用于探测蛋白质和核酸中毫秒到秒级的构象交换。标准方法是通过采集一系列二维相关谱来获得CEST数据集，其中每个实验利用单个饱和频率作用于氢、氮或碳。这些伪三维数据集收集起来很耗时，而且由于长饱和脉冲导致的信噪比降低，采集时间进一步延长。在本文中，我们展示了使用多频饱和脉冲（即MF-CEST）如何将数据采集的性质从串行变为并行，从而将总采集时间减少与脉冲中频率数量相对应的整数倍。我们通过收集碳甲基和氮骨架数据集，证明了MF-CEST在磷脂酶Cγ的Src同源2（SH2）结构域和次级主动转运蛋白EmrE作为模型系统中的适用性。MF-CEST还可以扩展到蛋白质和核酸中的其他位点。应用于骨架氮实验的MF-CEST唯一显著的缺点是，当主要和次要群体之间存在较大的化学位移差异时（通常大于约8 ppm）。在这些情况下，次要群体的化学位移和多频饱和脉冲之间可能会出现模糊性。然而，这种缺点在甲基基团MF-CEST实验中或存在较小化学位移差异的情况下不会出现。
