The Swedish NMR Centre, University of Gothenburg, Box 465, 40530, Göteborg, Sweden.
J Biomol NMR. 2014 Feb;58(2):129-39. doi: 10.1007/s10858-013-9811-1. Epub 2014 Jan 17.
Time-resolved experiments demand high resolution both in spectral dimensions and in time of the studied kinetic process. The latter requirement traditionally prohibits applications of the multidimensional experiments, which, although capable of providing invaluable information about structure and dynamics and almost unlimited spectral resolution, require too lengthy data collection. Our work shows that the problem has a solution in using modern methods of NMR data collection and signal processing. A continuous fast pulsing three-dimensional experiment is acquired using non-uniform sampling during full time of the studied reaction. High sensitivity and time-resolution of a few minutes is achieved by simultaneous processing of the full data set with the multi-dimensional decomposition. The method is verified and illustrated in realistic simulations and by measuring deuterium exchange rates of amide protons in ubiquitin. We applied the method for characterizing kinetics of in vitro phosphorylation of two tyrosine residues in an intrinsically disordered cytosolic domain of the B cell receptor protein CD79b. Signals of many residues including tyrosines in both phosphorylated and unmodified forms of CD79b are found in a heavily crowded region of 2D ¹H-¹⁵N correlation spectrum and the significantly enhanced spectral resolution provided by the 3D time-resolved approach was essential for the quantitative site-specific analysis.
时间分辨实验在研究动力学过程的光谱维度和时间上都需要高分辨率。后一个要求传统上禁止多维实验的应用，尽管多维实验能够提供关于结构和动力学的宝贵信息，并且几乎具有无限的光谱分辨率，但需要太长的数据采集时间。我们的工作表明，使用现代 NMR 数据采集和信号处理方法可以解决这个问题。在研究反应的整个时间内，使用非均匀采样获取连续快速脉冲的三维实验。通过多维分解同时处理整个数据集，实现了几分钟的高灵敏度和时间分辨率。该方法在现实模拟和测量泛素酰胺质子的氘交换率中得到了验证和说明。我们应用该方法来表征 B 细胞受体蛋白 CD79b 胞质结构域中两个酪氨酸残基的体外磷酸化动力学。包括 CD79b 的磷酸化和未修饰形式中的酪氨酸在内的许多残基的信号在二维 ¹H-¹⁵N 相关谱的拥挤区域中被发现，并且 3D 时间分辨方法提供的显著增强的光谱分辨率对于定量的位点特异性分析是必不可少的。
