尽管NMR光谱通常用于研究生物分子的构象动力学,在交换比两状态更复杂的情况下,对数据的稳健分析受到挑战,例如,当一个\'可见\'主要构象在毫秒时间尺度上与两个\'不可见\'次要状态交换时。越来越清楚的是,最初开发用于研究经历缓慢相互转换的系统的化学交换饱和转移(CEST)NMR实验也对中快时间尺度生物分子构象交换敏感。在这里,我们通过研究人HYPA/FBP11(WTFF)的FF结构域的折叠(F)状态与两个之间的相互转化,研究了酰胺15NCEST实验在毫秒时间尺度上表征蛋白质三态交换的实用性。它的折叠中间体I1和I2。尽管15NCPMG实验与毫秒时间尺度上单个次要状态的F状态相互转换一致,15NCEST数据清楚地建立了F和一对次要状态之间的交换过程。通过分析在单个温度下记录的15NCEST数据无法获得独特的三态交换模型。然而,包括基于在多个温度下记录的CEST数据的简单两态分析的两个次要状态的化学位移的差异的相对符号,结果是一个鲁棒的三态模型,其中F,I1和I2状态在毫秒时间尺度上相互转换([公式:见文本]~550s-1,[公式:见文本]~1200s-1,[公式:见文本]~5000s-1),I1和I2稀疏分布在~0.15%和~0.35%,分别,15°C从CEST数据中提取最佳拟合交换参数不需要交换参数空间的计算要求高的网格搜索。CEST实验的实用性,因此,远远超出了NMR化学位移时间尺度上缓慢交换的构象异构体的研究,包括相互转换速率为数千/秒的系统。
Although NMR spectroscopy is routinely used to study the conformational dynamics of biomolecules, robust analyses of the data are challenged in cases where exchange is more complex than two-state, such as when a \'visible\' major conformer exchanges with two \'invisible\' minor states on the millisecond timescale. It is becoming increasingly clear that chemical exchange saturation transfer (CEST) NMR experiments that were initially developed to study systems undergoing slow interconversion are also sensitive to intermediate-fast timescale biomolecular conformational exchange. Here we investigate the utility of the amide 15N CEST experiment to characterise protein three-state exchange occurring on the millisecond timescale by studying the interconversion between the folded (F) state of the FF domain from human HYPA/FBP11 (WT FF) and two of its folding intermediates I1 and I2. Although 15N CPMG experiments are consistent with the F state interconverting with a single minor state on the millisecond timescale, 15N CEST data clearly establish an exchange process between F and a pair of minor states. A unique three-state exchange model cannot be obtained by analysis of 15N CEST data recorded at a single temperature. However, including the relative sign of the difference in the chemical shifts of the two minor states based on a simple two-state analysis of CEST data recorded at multiple temperatures, results in a robust three-state model in which the F, I1 and I2 states interconvert with each other on the millisecond timescale ( k e x , F I 1 ~ 550 s-1, k e x , F I 2 ~ 1200 s-1, k e x , I 1 I 2 ~ 5000 s-1), with I1 and I2 sparsely populated at ~ 0.15% and ~ 0.35%, respectively, at 15 °C. A computationally demanding grid-search of exchange parameter space is not required to extract the best-fit exchange parameters from the CEST data. The utility of the CEST experiment, thus, extends well beyond studies of conformers in slow exchange on the NMR chemical shift timescale, to include systems with interconversion rates on the order of thousands/second.